Proteopedia - User contributions [en]

Alpha Subunit of Thermus aquaticus DNA Polymerase III

2014-09-03T03:58:00Z

Catherine L Dornfeld:

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.swf</swf>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.swf</swf>

<Structure load='3e0d' size='400' frame='true' align='right' caption='Taq DNA Polymerase III Alpha Subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5' />
===Important Domains===

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<Structure load='3e0d' size='400' frame='true' align='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5' />
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.swf</swf>

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

==References==
<references />

Alpha Subunit of Thermus aquaticus DNA Polymerase III

2014-09-03T03:55:40Z

Catherine L Dornfeld:

Alpha Subunit of Thermus aquaticus DNA Polymerase III

2014-09-03T03:28:46Z

Catherine L Dornfeld:

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.swf</swf>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.swf</swf>

<Structure load='3e0d' size='400' frame='true' align='right' caption='Taq DNA Polymerase III Alpha Subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5' />
===Important Domains===

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.swf</swf>

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

==References==
<references />

Alpha Subunit of Thermus aquaticus DNA Polymerase III

2014-09-03T03:23:45Z

Catherine L Dornfeld: /* Challenge Questions */

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.swf</swf>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.swf</swf>

<StructureSection load='3e0d' size='400' side='right' caption='Taq DNA Polymerase III Alpha Subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
===Important Domains===

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.
</StructureSection>

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.swf</swf>

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
COMING SOON

==References==
<references />

Alpha Subunit of Thermus aquaticus DNA Polymerase III

2013-09-17T16:36:44Z

Catherine L Dornfeld:

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
COMING SOON

==Components of ''Taq'' DNA Polymerase III α-Subunit==
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.swf</swf>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.swf</swf>

<StructureSection load='3e0d' size='400' side='right' caption='Taq DNA Polymerase III Alpha Subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
===Important Domains===

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.
</StructureSection>

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.swf</swf>

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
COMING SOON

==References==
<references />

Alpha Subunit of Thermus aquaticus DNA Polymerase III

2013-09-16T22:08:15Z

Catherine L Dornfeld: /* Nucleotide Addition */

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
COMING SOON

==Components of ''Taq'' DNA Polymerase III α-Subunit==
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.swf</swf>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.swf</swf>

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/5' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
<swf width="564" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.swf</swf>

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
COMING SOON

==References==
<references />

Alpha Subunit of Thermus aquaticus DNA Polymerase III

2013-09-16T22:00:19Z

Catherine L Dornfeld: /* Components of ''Taq'' DNA Polymerase III α-Subunit */

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
COMING SOON

==Components of ''Taq'' DNA Polymerase III α-Subunit==
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.swf</swf>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.swf</swf>

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/5' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
COMING SOON

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-16T21:56:36Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
COMING SOON

==Components of ''Taq'' DNA Polymerase III α-Subunit==
===Videos Detailing Functional Core and Active Site===
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.swf</swf>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.swf</swf>

<StructureSection load='3e0d' size='400' side='right' caption='DNA Polymerase III Alpha Subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
===Important Domains===
The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.
</StructureSection>

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
===Video of Nucleotide Addition===
<swf width="564" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.swf</swf>

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
COMING SOON

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-16T21:43:44Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
COMING SOON

==Components of ''Taq'' DNA Polymerase III α-Subunit==
===Videos Detailing Functional Core and Active Site===
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.swf</swf>
<swf width="500" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.swf</swf>

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/5' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
===Video of Nucleotide Addition===
<swf width="564" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.swf</swf>

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
COMING SOON

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-16T21:32:47Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
COMING SOON

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

<swf width="450" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.swf</swf>
<swf width="450" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.swf</swf>

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/5' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]
<swf width="564" height="330">http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.swf</swf>

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
COMING SOON

==References==
<references />

User:Catherine L Dornfeld

2013-09-13T19:04:03Z

Catherine L Dornfeld:

==Biography==
Catherine graduated with a B.S. in microbiology and a B.A. in psychology from the University of Wisconsin-Milwaukee. She is a member of Phi Beta Kappa and Psi Chi. She is currently interning with Dr. Steven Forst at UW-Milwaukee and the MSOE Center for BioMolecular Modeling while working part-time in the education department at the Milwaukee Public Museum. She has assisted in Dr. Gyaneshwar Prasad's laboratory at UW-Milwaukee, which specializes in rhizobial symbiosis with legumes and cereals. She finds it both amusing and troubling that she knows more about prokaryotic RNA polymerase than she does her own eukaryotic self.

==CREST Projects==
*[[Alpha Subunit of Thermus aquaticus DNA Polymerase III]]
*[[Beta-Prime Subunit of Bacterial RNA Polymerase]]
*[[User:Catherine L Dornfeld/Templates]]
*[[User:Catherine L Dornfeld/Sandbox 1]]

Alpha Subunit of Thermus aquaticus DNA Polymerase III

2013-09-13T19:00:27Z

Catherine L Dornfeld: New page: ==Introduction== ===Role of DNA Polymerase in Replication=== The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DN...

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
COMING SOON

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/5' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
COMING SOON

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-13T18:52:45Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
COMING SOON

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/5' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
COMING SOON

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-12T20:39:13Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
'''COMING SOON'''

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/5' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/4'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/3'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/3'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/3'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/3'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/4'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/3'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/3'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/3'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/4'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/5'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/3'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/4'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/3'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/3'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/4'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/3'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-12T07:58:42Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
'''COMING SOON'''

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/4' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/3'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/2'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/2'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/2'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/2'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/3'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/2'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/2'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/2'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/3'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/4'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/2'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/3'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/2'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/2'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/3'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/2'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-12T06:34:53Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
'''COMING SOON'''

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/2'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/2'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/3'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/2'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/2'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/2'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/1'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/1'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/2'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/1'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-12T02:55:09Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
'''COMING SOON'''

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/2'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/2'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/1'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/1'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/2'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/1'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-12T01:17:55Z

Catherine L Dornfeld: /* Introduction */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

===Challenge Questions===
'''COMING SOON'''

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/1'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/1'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/1'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/2'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/1'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-12T01:17:00Z

Catherine L Dornfeld: /* Nucleotide Addition */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/1'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/1'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP <ref name='bailey' /><ref name='wing' />. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/1'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/2'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/1'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-12T01:15:00Z

Catherine L Dornfeld: /* Nucleotide Addition */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/1'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/1'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP in order to facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/1'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/2'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/1'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-12T01:14:06Z

Catherine L Dornfeld: /* Important Domains */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/1'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>

[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/1'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP in order to facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/1'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/2'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/1'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-12T01:13:11Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/1'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in gray and turquoise) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>

[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Highly conserved arginine residues <scene name='46/461391/Dnapol_positioning/1'>(Taq452, Taq458, Taq766 and Taq767)</scene> interact with the triphosphate of the incoming dNTP in order to facilitate base pairing between the primer base and dNTP through orientation of the nucleotide<ref name='bailey' /><ref name='wing' />.
* The incoming dNTP is also coordinated by <scene name='46/461391/Dnapol_gs767/1'> the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/2'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/1'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Model Researchers and Designers===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-11T03:36:24Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/1'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in gray and turquoise) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its <scene name='46/461391/Dnapol_hhh/1'>helix-hairpin-helix (HhH) motif </scene> (Taq892-910, in turquoise) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq848, Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* Residues from the thumb domain (Taq698 and Taq813-821) form a <scene name='46/461391/Dnapol_dntppocket/1'>nucleotide binding pocket</scene> for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Arginine residues <scene name='46/461391/Dnapol_positioning/1'>(Taq452, Taq458 and Taq766)</scene> interact with the triphosphate of the incoming dNTP, orienting the nucleotide so that the incoming dNTP base enters in the "base-forward" position, near the primer base<ref name='bailey' /><ref name='wing' />. These arginine residues are highly conserved<ref name='bailey' />.
* The incoming dNTP is coordinated by <scene name='46/461391/Dnapol_gs767/1'>Arginine 767 and the GS motif </scene>(Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between <scene name='46/461391/Dnapol_lys616/2'>Lysine 616 and the 3' primer terminal phosphate</scene><ref name='wing' />.
* Ribonucleotides and incorrectly-matched dNTPs may exit between the <scene name='46/461391/Dnapol_ntpexit/1'>fingers and thumb domains</scene><ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the <scene name='46/461391/Dnapol_metalion/1'>second metal ion</scene> is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-11T02:25:44Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/1'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in gray and turquoise) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its <scene name='46/461391/Dnapol_hhh/1'>helix-hairpin-helix (HhH) motif </scene> (Taq892-910, in turquoise) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq848, Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so that the incoming dNTP base enters in the "base-forward" position, near the primer base<ref name='wing' />. Arginine 766 also helps position the incoming dNTP<ref name='bailey' />. These arginine residues are highly conserved<ref name='bailey' />.
* The incoming dNTP is coordinated by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Ribonucleotides and incorrectly-matched dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-11T02:20:52Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/1'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in gray and turquoise) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its <scene name='46/461391/Dnapol_hhh/1'>helix-hairpin-helix (HhH) motif </scene> (Taq892-910, in turquoise) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq848, Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a <scene name='46/461391/Dnapol_dntpentry/1'>groove formed by the PHP, fingers and palm domains</scene><ref name='wing' />.
* <scene name='46/461391/Dnapol_his817tyr821/1'>Histidine 817 and Tyrosine 821</scene> evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so that the incoming dNTP base enters in the "base-forward" position, near the primer base<ref name='wing' />.
* The incoming dNTP is coordinated by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Ribonucleotides and incorrectly-matched dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-11T01:58:18Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates <scene name='46/461391/Dnapol_aspartates/1'>(Taq463, Taq465 and Taq618)</scene><ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in gray and turquoise) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its <scene name='46/461391/Dnapol_hhh/1'>helix-hairpin-helix (HhH) motif </scene> (Taq892-910, in turquoise) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq848, Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop <scene name='46/461391/Dnapol_phpdna/2'>(Taq 232-241)</scene><ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so that the incoming dNTP base enters in the "base-forward" position, near the primer base<ref name='wing' />.
* The incoming dNTP is coordinated by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Ribonucleotides and incorrectly-matched dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T23:50:06Z

Catherine L Dornfeld: /* Components of ''Taq'' DNA Polymerase III α-Subunit */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/2'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates (Taq463, Taq465 and Taq618)<ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in gray and turquoise) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its <scene name='46/461391/Dnapol_hhh/1'>helix-hairpin-helix (HhH) motif </scene> (Taq892-910, in turquoise) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq848, Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so that the incoming dNTP base enters in the "base-forward" position, near the primer base<ref name='wing' />.
* The incoming dNTP is coordinated by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Ribonucleotides and incorrectly-matched dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T23:46:16Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* <scene name='46/461391/Dnapol_palm/1'>Palm</scene> (residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates (Taq463, Taq465 and Taq618)<ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* <scene name='46/461391/Dnapol_fingers/1'>Fingers</scene> (residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* <scene name='46/461391/Dnapol_thumb/1'>Thumb</scene> (residues 493-574, in gray and turquoise) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_betabinding/1'>β-Binding Domain</scene> (residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* <scene name='46/461391/Dnapol_ctd/1'>C-Terminal Domain (CTD)</scene> (residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* <scene name='46/461391/Dnapol_php/1'>Polymerase and Histidinol Phosphatase (PHP) Domain</scene> (residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* <scene name='46/461391/Dnapol_dntp/1'>Incoming Deoxyribonucleotide (dNTP)</scene> (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* <scene name='46/461391/Dnapol_aspartates/1'>Catalytic Aspartates</scene> (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its <scene name='46/461391/Dnapol_hhh/1'>helix-hairpin-helix (HhH) motif </scene> (Taq892-910, in turquoise) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues <scene name='46/461391/Dnapol_backboneresidues/1'>(Taq848, Taq895, Taq932 and Taq933)</scene> on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so that the incoming dNTP base enters in the "base-forward" position, near the primer base<ref name='wing' />.
* The incoming dNTP is coordinated by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Ribonucleotides and incorrectly-matched dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T22:43:37Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Dnapol_overview/3' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates (Taq463, Taq465 and Taq618)<ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histidinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapol_overview/3'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so that the incoming dNTP base enters in the "base-forward" position, near the primer base<ref name='wing' />.
* The incoming dNTP is coordinated by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Ribonucleotides and incorrectly-matched dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T19:49:06Z

Catherine L Dornfeld: /* Nucleotide Addition */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates (Taq463, Taq465 and Taq618)<ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histidinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter the complex through a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 evaluate sugar groups on incoming nucleotides and select for dNTPs<ref name='bailey' />.
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so that the incoming dNTP base enters in the "base-forward" position, near the primer base<ref name='wing' />.
* The incoming dNTP is coordinated by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Ribonucleotides and incorrectly-matched dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T19:39:53Z

Catherine L Dornfeld: /* Components of ''Taq'' DNA Polymerase III α-Subunit */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates (Taq463, Taq465 and Taq618)<ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histidinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs). Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented. '''(REFERENCE?)'''
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so the incoming dNTP base enters near the primer base<ref name='wing' />.
* The incoming dNTP is held in position by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrectly-matched NTPs and dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T19:39:11Z

Catherine L Dornfeld: /* Important Domains */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates (Taq463, Taq465 and Taq618)<ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='bailey'>PMID: 16959569</ref>. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histodinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs). Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented. '''(REFERENCE?)'''
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so the incoming dNTP base enters near the primer base<ref name='wing' />.
* The incoming dNTP is held in position by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrectly-matched NTPs and dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T05:44:58Z

Catherine L Dornfeld: /* Nucleotide Addition */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates (Taq463, Taq465 and Taq618)<ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histodinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs). Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented. '''(REFERENCE?)'''
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so the incoming dNTP base enters near the primer base<ref name='wing' />.
* The incoming dNTP is held in position by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrectly-matched NTPs and dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T05:44:36Z

Catherine L Dornfeld: /* Important Domains */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.

* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates (Taq463, Taq465 and Taq618)<ref name='wing' />. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. '''(CHECK REFERENCE)'''
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histodinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs). Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented. (REFERENCE?)
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so the incoming dNTP base enters near the primer base<ref name='wing' />.
* The incoming dNTP is held in position by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrectly-matched NTPs and dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T05:34:40Z

Catherine L Dornfeld: /* Nucleotide Addition */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved catalytic aspartates<ref name='wing' />. The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histodinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs). Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented. (REFERENCE?)
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so the incoming dNTP base enters near the primer base<ref name='wing' />.
* The incoming dNTP is held in position by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrectly-matched NTPs and dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T05:34:15Z

Catherine L Dornfeld: /* Nucleotide Addition */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved catalytic aspartates<ref name='wing' />. The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histodinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs). Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented. (REFERENCE?)
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar groups of the incoming dNTPs<ref name='wing' />.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so the incoming base enters near the primer base<ref name='wing' />.
* The incoming dNTP is held in position by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrectly-matched NTPs and dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T05:33:29Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved catalytic aspartates<ref name='wing' />. The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histodinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates (residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. The PHP domain may contact the DNA at a loop (Taq 232-241)<ref name='wing' />.

Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains<ref name='wing' />.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs). Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented. (REFERENCE?)
* Residues from the thumb domain (Taq698 and Taq813-821) form a nucleotide binding pocket for the base and sugar moieties of the incoming dNTPs<ref name='wing' />.
* Arginine residues (Taq452 and Taq458) interact with the triphosphate of the incoming dNTP, orienting the nucleotide so the incoming base enters near the primer base<ref name='wing' />.
* The incoming dNTP is held in position by Arginine 767 and the GS motif (Taq G425-S426) via interactions with the γ-phosphate of the dNTP<ref name='wing' />. The GS motif is absolutely conserved<ref name='aravind'>PMID: 10075991</ref>.
* The 3' end of the primer strand is positioned by formation of a salt bridge between Lysine 616 and the 3' primer terminal phosphate<ref name='wing' />.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrectly-matched NTPs and dNTPs may exit between the fingers and thumb domains<ref name='wing' />. Arginine residues block the exit through the entry channel<ref name='wing' />.

===Catalysis===
* All polymerases employ a two metal ion mechanism for catalysis<ref name='steitzsmerdon'>PMID: 7528445</ref>.
* One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the other is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP<ref name='wing' />.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T04:39:51Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved catalytic aspartates<ref name='wing' />. The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA<ref name='wing' />.
* β-Binding Domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-Terminal Domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and Histodinol Phosphatase (PHP) Domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming Deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.
* Catalytic Aspartates - These aspartate residues are highly conserved and essential to catalysis <ref name='wing' /><ref name='pritchard'>PMID: 9887268 </ref>.

===DNA Interactions & Conformational Changes===
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)<ref name='wing' />. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)<ref name='wing' />. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues (Taq848, Taq895, Taq932 and Taq933) on the negatively-charged backbone<ref name='wing' />. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T03:46:24Z

Catherine L Dornfeld:

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition and elongation of the growing DNA strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved catalytic aspartates<ref name='wing' />. The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA<ref name='wing' />.
* β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined<ref name='stano'>PMID: 16604084</ref>.
* Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T03:40:25Z

Catherine L Dornfeld: /* Important Domains */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition and elongation of the growing DNA strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template <ref name='steitz'>PMID: 10364165</ref>.These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved catalytic aspartates<ref name='wing' />. The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA<ref name='wing' />.
* β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove<ref name='wing' />.
* Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined<ref name='wing' />.
* Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T03:22:37Z

Catherine L Dornfeld: /* Thermophilic DNA Polymerase Alpha-Subunit */

=Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition and elongation of the growing DNA strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved catalytic aspartates<ref name='wing' />. The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates)<ref name='wing' />.
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA<ref name='wing' />.
* β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />.
* C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove<ref name='wing' />.
* Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined<ref name='wing' />.
* Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />.

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />.

==Nucleotide Addition==
<StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T03:10:27Z

Catherine L Dornfeld: /* Prokaryotic DNA Polymerases */

=Thermophilic DNA Polymerase Alpha-Subunit=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition and elongation of the growing DNA strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex). Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='350' frame='true' align='right' caption='DNA pol III alpha-subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. These domains form binding pockets for both DNA and incoming dNTPs. They are also involved in correct dNTP selection, position and addition along with DNA interactions.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including the three conserved catalytic aspartates found in all nucleotidyl transferases (CHECK INFO). The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates).
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis. The fingers domain also forms part of the active site.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA.
* β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA.
* C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove.
* Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined.
* Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain.

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

==Nucleotide Addition==
<StructureSection load='3e0d' size='350' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapoloverview/2'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

/////////

==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine==
<Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' />

'''Abstract'''

==RNA Polymerase Elongation Complex==
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur<ref name='loading' />.

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix<ref name='loading' />. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript <ref name='loading' />. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides <ref name='loading' />.

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T03:09:51Z

Catherine L Dornfeld: /* Nucleotide Addition */

=Thermophilic DNA Polymerase Alpha-Subunit=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition and elongation of the growing DNA strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex). Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='350' frame='true' align='right' caption='DNA pol III alpha-subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. These domains form binding pockets for both DNA and incoming dNTPs. They are also involved in correct dNTP selection, position and addition along with DNA interactions.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including the three conserved catalytic aspartates found in all nucleotidyl transferases (CHECK INFO). The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates).
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis. The fingers domain also forms part of the active site.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA.
* β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA.
* C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove.
* Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined.
* Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain.

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

==Nucleotide Addition==
<StructureSection load='3e0d' size='350' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapoloverview/2'>
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition]

===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

/////////

==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine==
<Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' />

'''Abstract'''

==RNA Polymerase Elongation Complex==
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur<ref name='loading' />.

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix<ref name='loading' />. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript <ref name='loading' />. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides <ref name='loading' />.

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T03:08:02Z

Catherine L Dornfeld: /* Components of ''Taq'' DNA Polymerase III α-Subunit */

=Thermophilic DNA Polymerase Alpha-Subunit=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition and elongation of the growing DNA strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex). Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video1.html Video of DNA Polymerase Functional Core] 
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video2.html Video of DNA Polymerase Active Site]

===Important Domains===
<Structure load='3e0d' size='350' frame='true' align='right' caption='DNA pol III alpha-subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb domains of DNA polymerase III are highly conserved. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. These domains form binding pockets for both DNA and incoming dNTPs. They are also involved in correct dNTP selection, position and addition along with DNA interactions.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including the three conserved catalytic aspartates found in all nucleotidyl transferases (CHECK INFO). The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates).
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis. The fingers domain also forms part of the active site.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA.
* β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA.
* C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove.
* Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined.
* Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain.

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

==Nucleotide Addition==
<StructureSection load='3e0d' size='350' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapoloverview/2'>
===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

VIDEO 3

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

/////////

==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine==
<Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' />

'''Abstract'''

==RNA Polymerase Elongation Complex==
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur<ref name='loading' />.

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix<ref name='loading' />. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript <ref name='loading' />. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides <ref name='loading' />.

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T02:59:46Z

Catherine L Dornfeld:

=Thermophilic DNA Polymerase Alpha-Subunit=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with multiple subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition and elongation of the growing DNA strand.

===Prokaryotic DNA Polymerases===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex). Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
VIDEO 1
VIDEO 2

===Important Domains===
<Structure load='3e0d' size='350' frame='true' align='right' caption='DNA pol III alpha-subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb are highly conserved domains. They are nicknamed as such because the structures resemble a closed hand. They form the nucleotide binding pocket and are involved in nucleotide positioning and sugar selection, nucleotide addition, and DNA interactions.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including the three conserved catalytic aspartates found in all nucleotidyl transferases (CHECK INFO). The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates).
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis. The fingers domain also forms part of the active site.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA.
* β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA.
* C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove.
* Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined.
* Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain.

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

==Nucleotide Addition==
<StructureSection load='3e0d' size='350' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapoloverview/2'>
===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

VIDEO 3

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

/////////

==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine==
<Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' />

'''Abstract'''

==RNA Polymerase Elongation Complex==
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur<ref name='loading' />.

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix<ref name='loading' />. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript <ref name='loading' />. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides <ref name='loading' />.

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-10T02:38:15Z

Catherine L Dornfeld: /* Introduction */

=Thermophilic DNA Polymerase Alpha-Subunit=

==Introduction==
===Role of DNA Polymerase in Replication===
The primary function of DNA polymerase is to replicate the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.

The replisome is an enzymatic complex with several subunits (see [http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg Replisome Diagram]). The core DNA polymerase subunits are α, ε, and θ, which are bound to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition and elongation of the nascent DNA strand.

===DNA Polymerase III in Bacteria===
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones, which must be accounted for during replication. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far.

Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. The crystallized structure of the α-subunit of Taq DNA polymerase III was the first to contain DNA, DNA polymerase, and an incoming dNTP, referred to as the ternary complex. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
VIDEO 1
VIDEO 2

===Important Domains===
<Structure load='3e0d' size='350' frame='true' align='right' caption='DNA pol III alpha-subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb are highly conserved domains. They are nicknamed as such because the structures resemble a closed hand. They form the nucleotide binding pocket and are involved in nucleotide positioning and sugar selection, nucleotide addition, and DNA interactions.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including the three conserved catalytic aspartates found in all nucleotidyl transferases (CHECK INFO). The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates).
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis. The fingers domain also forms part of the active site.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA.
* β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA.
* C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove.
* Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined.
* Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain.

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

==Nucleotide Addition==
<StructureSection load='3e0d' size='350' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapoloverview/2'>
===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

VIDEO 3

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

/////////

==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine==
<Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' />

'''Abstract'''

==RNA Polymerase Elongation Complex==
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur<ref name='loading' />.

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix<ref name='loading' />. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript <ref name='loading' />. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides <ref name='loading' />.

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-09T22:50:05Z

Catherine L Dornfeld: /* Important Domains */

=Thermophilic DNA Polymerase Alpha-Subunit=

==Introduction==
===Role of DNA Polymerase in Replication===
HOW MUCH DETAIL TO INCLUDE?

A primary function of DNA polymerase is to replicate, or copy, the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of connected deoxynucleotides (dNTPs) running anti-parallel to each other in a double helix. Deoxynucleotides consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The nitrogen bases are adenine (A), cytosine (C), guanine (G), and thymine (T). The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds. Overall, the backbone has a negative charge. The nitrogen bases of the dNTPs extend into the helix and form pairs that are stabilized by hydrogen bonds. Due to the structure of the nitrogen bases, these hydrogen bonds always form so that adenine pairs with thymine and cytosine pairs with guanine (Watson-Crick base pairs). When the base pairs are formed, double-stranded DNA results.

The replisome is an enzymatic complex with several subunits. There are the core polymerase subunits (α, ε, and θ), the β-clamp, and the clamp loader complex. This tutorial will focus on the α-subunit because it contains the catalytic site for dNTP addition and elongation of the nascent DNA strand.

[[Image:http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg]]

===DNA Polymerase III in Bacteria===
[http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the primary replicative polymerase in bacteria.

The crystallized structure of ''Thermus aquaticus'' (Taq) DNA polymerase III α subunit was the first to contain the ternary complex (polymerase, DNA and incoming dNTP). Taq DNA pol III is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic non-replicative repair polymerase.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
VIDEO 1
VIDEO 2

===Important Domains===
<Structure load='3e0d' size='350' frame='true' align='right' caption='DNA pol III alpha-subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb are highly conserved domains. They are nicknamed as such because the structures resemble a closed hand. They form the nucleotide binding pocket and are involved in nucleotide positioning and sugar selection, nucleotide addition, and DNA interactions.
* Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including the three conserved catalytic aspartates found in all nucleotidyl transferases (CHECK INFO). The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates).
* Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis. The fingers domain also forms part of the active site.
* Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA.
* β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA.
* C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove.
* Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined.
* Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain.

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

==Nucleotide Addition==
<StructureSection load='3e0d' size='350' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapoloverview/2'>
===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

VIDEO 3

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

/////////

==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine==
<Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' />

'''Abstract'''

==RNA Polymerase Elongation Complex==
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur<ref name='loading' />.

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix<ref name='loading' />. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript <ref name='loading' />. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides <ref name='loading' />.

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-09T21:40:13Z

Catherine L Dornfeld: /* Components of ''Taq'' DNA Polymerase III α-Subunit */

=Thermophilic DNA Polymerase Alpha-Subunit=

==Introduction==
===Role of DNA Polymerase in Replication===
HOW MUCH DETAIL TO INCLUDE?

A primary function of DNA polymerase is to replicate, or copy, the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of connected deoxynucleotides (dNTPs) running anti-parallel to each other in a double helix. Deoxynucleotides consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The nitrogen bases are adenine (A), cytosine (C), guanine (G), and thymine (T). The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds. Overall, the backbone has a negative charge. The nitrogen bases of the dNTPs extend into the helix and form pairs that are stabilized by hydrogen bonds. Due to the structure of the nitrogen bases, these hydrogen bonds always form so that adenine pairs with thymine and cytosine pairs with guanine (Watson-Crick base pairs). When the base pairs are formed, double-stranded DNA results.

The replisome is an enzymatic complex with several subunits. There are the core polymerase subunits (α, ε, and θ), the β-clamp, and the clamp loader complex. This tutorial will focus on the α-subunit because it contains the catalytic site for dNTP addition and elongation of the nascent DNA strand.

[[Image:http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg]]

===DNA Polymerase III in Bacteria===
[http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the primary replicative polymerase in bacteria.

The crystallized structure of ''Thermus aquaticus'' (Taq) DNA polymerase III α subunit was the first to contain the ternary complex (polymerase, DNA and incoming dNTP). Taq DNA pol III is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic non-replicative repair polymerase.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
VIDEO 1
VIDEO 2

===Important Domains===
<Structure load='3e0d' size='350' frame='true' align='right' caption='DNA pol III alpha-subunit' scene='46/461391/Simple_scene/7' />

The fingers, palm and thumb are highly conserved domains. They are nicknamed as such because the structures resemble a closed hand. They form the nucleotide binding pocket and are involved in nucleotide positioning and sugar selection, nucleotide addition, and DNA interactions.
* Fingers - selects for deoxyribonucleotides, positions incoming dNTPs, and forms part of the active site
* Palm - contains the catalytic site for nucleotide addition and aids incoming nucleotide positioning
* Thumb - forms part of the DNA exit channel and interacts with DNA
* β-binding domain - binds β-sliding clamp and positions double-stranded DNA
* C-terminal domain (CTD) - contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove
* Polymerase and histodinol phosphatase (PHP) domain - forms part of the DNA exit channel and has potential proofreading function via zinc-ion dependent exonuclease activity

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

==Nucleotide Addition==
<StructureSection load='3e0d' size='350' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapoloverview/2'>
===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

VIDEO 3

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

/////////

==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine==
<Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' />

'''Abstract'''

==RNA Polymerase Elongation Complex==
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur<ref name='loading' />.

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix<ref name='loading' />. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript <ref name='loading' />. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides <ref name='loading' />.

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-09T20:37:10Z

Catherine L Dornfeld: /* Components of ''Taq'' DNA Polymerase III α-Subunit */

=Thermophilic DNA Polymerase Alpha-Subunit=

==Introduction==
===Role of DNA Polymerase in Replication===
HOW MUCH DETAIL TO INCLUDE?

A primary function of DNA polymerase is to replicate, or copy, the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of connected deoxynucleotides (dNTPs) running anti-parallel to each other in a double helix. Deoxynucleotides consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The nitrogen bases are adenine (A), cytosine (C), guanine (G), and thymine (T). The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds. Overall, the backbone has a negative charge. The nitrogen bases of the dNTPs extend into the helix and form pairs that are stabilized by hydrogen bonds. Due to the structure of the nitrogen bases, these hydrogen bonds always form so that adenine pairs with thymine and cytosine pairs with guanine (Watson-Crick base pairs). When the base pairs are formed, double-stranded DNA results.

The replisome is an enzymatic complex with several subunits. There are the core polymerase subunits (α, ε, and θ), the β-clamp, and the clamp loader complex. This tutorial will focus on the α-subunit because it contains the catalytic site for dNTP addition and elongation of the nascent DNA strand.

[[Image:http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg]]

===DNA Polymerase III in Bacteria===
[http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the primary replicative polymerase in bacteria.

The crystallized structure of ''Thermus aquaticus'' (Taq) DNA polymerase III α subunit was the first to contain the ternary complex (polymerase, DNA and incoming dNTP). Taq DNA pol III is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic non-replicative repair polymerase.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
VIDEO 1
VIDEO 2

===Important Domains===
<Structure load='3e0d' size='350' frame='true' align='right' caption='DNA pol III alpha-subunit' scene='' />

The fingers, palm and thumb are highly conserved domains. They are nicknamed as such because the structures resemble a closed hand. They form the nucleotide binding pocket and are involved in nucleotide positioning and sugar selection, nucleotide addition, and DNA interactions.
* Fingers - selects for deoxyribonucleotides, positions incoming dNTPs, and forms part of the active site
* Palm - contains the catalytic site for nucleotide addition and aids incoming nucleotide positioning
* Thumb - forms part of the DNA exit channel and interacts with DNA
* β-binding domain - binds β-sliding clamp and positions double-stranded DNA
* C-terminal domain (CTD) - contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove
* Polymerase and histodinol phosphatase (PHP) domain - forms part of the DNA exit channel and has potential proofreading function via zinc-ion dependent exonuclease activity

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

==Nucleotide Addition==
<StructureSection load='3e0d' size='350' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapoloverview/2'>
===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

VIDEO 3

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

/////////

==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine==
<Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' />

'''Abstract'''

==RNA Polymerase Elongation Complex==
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur<ref name='loading' />.

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix<ref name='loading' />. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript <ref name='loading' />. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides <ref name='loading' />.

==References==
<references />

User:Catherine L Dornfeld/Sandbox 1

2013-09-09T20:36:16Z

Catherine L Dornfeld: /* Important Domains */

=Thermophilic DNA Polymerase Alpha-Subunit=

==Introduction==
===Role of DNA Polymerase in Replication===
HOW MUCH DETAIL TO INCLUDE?

A primary function of DNA polymerase is to replicate, or copy, the DNA of an organism. [http://en.wikipedia.org/wiki/DNA_replication DNA replication] occurs just prior to cell division is necessary for growth and reproduction of a living organism (see [[DNA Replication, Transcription and Translation]]). [[DNA]] consists of two strands of connected deoxynucleotides (dNTPs) running anti-parallel to each other in a double helix. Deoxynucleotides consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The nitrogen bases are adenine (A), cytosine (C), guanine (G), and thymine (T). The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds. Overall, the backbone has a negative charge. The nitrogen bases of the dNTPs extend into the helix and form pairs that are stabilized by hydrogen bonds. Due to the structure of the nitrogen bases, these hydrogen bonds always form so that adenine pairs with thymine and cytosine pairs with guanine (Watson-Crick base pairs). When the base pairs are formed, double-stranded DNA results.

The replisome is an enzymatic complex with several subunits. There are the core polymerase subunits (α, ε, and θ), the β-clamp, and the clamp loader complex. This tutorial will focus on the α-subunit because it contains the catalytic site for dNTP addition and elongation of the nascent DNA strand.

[[Image:http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg]]

===DNA Polymerase III in Bacteria===
[http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the primary replicative polymerase in bacteria.

The crystallized structure of ''Thermus aquaticus'' (Taq) DNA polymerase III α subunit was the first to contain the ternary complex (polymerase, DNA and incoming dNTP). Taq DNA pol III is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic non-replicative repair polymerase.

===Learning Objectives===
* How does the α subunit of DNA polymerase III contact DNA?
* Which domains select for deoxyribonucleotides?
* Which domains form the catalytic site?
* Which domains form the DNA exit channel?

==Components of ''Taq'' DNA Polymerase III α-Subunit==
VIDEO 1
VIDEO 2

===Important Domains===
<Structure load='3e0d' size='500' frame='true' align='right' caption='Insert caption here' scene='46/461391/Dnapoloverview/2' />

The fingers, palm and thumb are highly conserved domains. They are nicknamed as such because the structures resemble a closed hand. They form the nucleotide binding pocket and are involved in nucleotide positioning and sugar selection, nucleotide addition, and DNA interactions.
* Fingers - selects for deoxyribonucleotides, positions incoming dNTPs, and forms part of the active site
* Palm - contains the catalytic site for nucleotide addition and aids incoming nucleotide positioning
* Thumb - forms part of the DNA exit channel and interacts with DNA
* β-binding domain - binds β-sliding clamp and positions double-stranded DNA
* C-terminal domain (CTD) - contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove
* Polymerase and histodinol phosphatase (PHP) domain - forms part of the DNA exit channel and has potential proofreading function via zinc-ion dependent exonuclease activity

===DNA Interactions & Conformational Changes===
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

==Nucleotide Addition==
<StructureSection load='3e0d' size='350' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Dnapoloverview/2'>
===Deoxyribonucleotide Selection and Positioning===
* Nucleotides enter in a groove formed by the PHP, fingers and palm domains.
* Arginine residues interact with the triphosphate of the nucleotides, orienting nucleotides so bases enter first.
* Incoming nucleotides are held in position by the G-S motif (G425-S426). This motif is absolutely conserved.
* Arginine 767 interacts with the γ-phosphate of the incoming nucleotide.
* The 3' end of the nascent DNA strand is positioned by Lysine 616 by forming a salt bridge with the previous phosphate.
* Histidine 817 and Tyrosine 821 select for deoxyribonucleotides (dNTPs).
* Incorrect selection of ribonucleotide, whether sugar group or base, results in misalignment. Catalysis is prevented.
* Deoxyribonucleotides (dNTPs) are selected over ribonucleotides (NTPs), and the conjugate dNTP for the template dNTP must be selected. Incorrect NTPs and dNTPs may exit between the fingers and thumb domains. Arginine residues block exit through the entry channel.

===Catalysis===
* Divalent magnesium ions coordinate conserved aspartate residues in the catalytic site and α-phosphate (?) of incoming dNTP.
* The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the previously added dNTP and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates leaves.

VIDEO 3

</StructureSection>

==2012 UW-Milwaukee CREST Team==
===Team Members===
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

===Abstract===
ABSTRACT

/////////

==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine==
<Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' />

'''Abstract'''

==RNA Polymerase Elongation Complex==
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur<ref name='loading' />.

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix<ref name='loading' />. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript <ref name='loading' />. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides <ref name='loading' />.

==References==
<references />