Sandbox Reserved 1750: Difference between revisions

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The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.

== Recognition of the PAM sequence ==

For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through ~~bidentate~~ hydrogen bonds ~~(scene of bonds)~~ as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.

For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.

== Recognition of the sgRNA scaffold ==

The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.

== Endonuclease Activity of Cas9 ==

Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity ~~(Scene)~~. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.

Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.

</structuresection>

==References==

Sandbox Reserved 1750: Difference between revisions

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 The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.
 == Recognition of the PAM sequence ==
-For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.
+For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.
 == Recognition of the sgRNA scaffold ==
 The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.
 == Endonuclease Activity of Cas9 ==
-Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.
+Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.
 </structuresection>
 ==References==
 <references />