DNA Polymerase I: Difference between revisions

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==Structure of ''E. coli'' DNA polymerase I==


<applet load='1KLN_pymol.pdb' size='450' frame='true' align='right' caption='Klenow–DNA closeup' scene='Sandbox_dvoet/DNA_polymerase/Klenow-dna-closeup/4' />
<applet load='1KLN_pymol.pdb' size='450' frame='true' align='right' caption='Klenow–DNA closeup' scene='Sandbox_dvoet/DNA_polymerase/Klenow-dna-closeup/4' />


The X-ray structure is that of an editing complex, that is, the 3' end of the primer strand, the end that is elongated by the polymerase, occupies the 3'→5' exonuclease active site. This is more clearly seen in a <scene name='Sandbox_dvoet/DNA_polymerase/Klenow-dna-closeup/3'>closeup of the DNA</scene> in which the the rods connecting successive P atoms have been removed for clarity. Note that the base pair closest to the polymerase active site, a G·C, has opened up to enable the 3' end of the primer strand to reach the exonuclease active site. Click here to <scene name='Sandbox_dvoet/DNA_polymerase/Dna-closeup/3'>hide the protein</scene>.
The X-ray structure is that of an editing complex, that is, the 3' end of the primer strand, the end that is elongated by the polymerase, occupies the 3'→5' exonuclease active site. This is more clearly seen in a <scene name='Sandbox_dvoet/DNA_polymerase/Klenow-dna-closeup/3'>closeup of the DNA</scene> in which the the rods connecting successive P atoms have been removed for clarity. Note that the base pair closest to the polymerase active site, a G·C, has opened up to enable the 3' end of the primer strand to reach the exonuclease active site. Click here to <scene name='Sandbox_dvoet/DNA_polymerase/Dna-closeup/3'>hide the protein</scene>.
==Structure of the whole ''Thermus aquaticus'' DNA polymerase I==


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==Structure of Klentaq1 in its closed and open forms==


<applet load='1taq.pdb' size='450' frame='true' align='right' caption='Thermus aquaticus Pol I' scene= 'Sandbox_dvoet/DNA_polymerase/Taq_pol_i/1'/>
<applet load='1taq.pdb' size='450' frame='true' align='right' caption='Thermus aquaticus Pol I' scene= 'Sandbox_dvoet/DNA_polymerase/Taq_pol_i/1'/>
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Pol I from the thermophilic bacterium ''Thermus aquaticus'' (''Taq'') is 51% identical in sequence with ''E. coli'' Pol I, although it lacks a 3' → 5' exonuclease function due to the absence of critical residues. The X-ray structure of the complete ''Taq'' Pol I (1taq), was also determined by Steitz<ref>PMID: 7792597</ref>. Here its C-terminal Klenow fragment portion is initially viewed as is that in the foregoing structure of Klenow·DNA and colored light green, whereas the N-terminal 5' → 3' exonuclease portion is colored in rainbow order from its N-terminus (''blue'') to its C-terminus (''red''). Note that there is only tenuous contact between the Klenow fragment and the 5' → 3' exonuclease. Hence, it is unclear how they coordinate their activities to yield a dsDNA molecule with a single nick.
Pol I from the thermophilic bacterium ''Thermus aquaticus'' (''Taq'') is 51% identical in sequence with ''E. coli'' Pol I, although it lacks a 3' → 5' exonuclease function due to the absence of critical residues. The X-ray structure of the complete ''Taq'' Pol I (1taq), was also determined by Steitz<ref>PMID: 7792597</ref>. Here its C-terminal Klenow fragment portion is initially viewed as is that in the foregoing structure of Klenow·DNA and colored light green, whereas the N-terminal 5' → 3' exonuclease portion is colored in rainbow order from its N-terminus (''blue'') to its C-terminus (''red''). Note that there is only tenuous contact between the Klenow fragment and the 5' → 3' exonuclease. Hence, it is unclear how they coordinate their activities to yield a dsDNA molecule with a single nick.


 
==Structure of Klentaq1 in its closed and open forms==


Pol I replicates DNA with high fidelity. How does it do so? Gabriel Waksman answered this question by crystallizing the C-terminal domain of ''Taq'' polymerase ('''Klentaq1''') with an 11-bp DNA that had a GGAAA-5' overhang at the 5' end of its template strand<ref>PMID: 9857206</ref>. The crystals were then soaked in solution containing 2',3'-dideoxy-CTP (ddCTP), which lacks a 3'-OH group, and hence terminates replication after its incorporation at the 3' end of the primer strand. The X-ray structure of these crystals (3ktq) revealed that a ddC residue had been covalently linked to the 3' end of the primer strand, where it formed a Watson–Crick base pair with the 3' G on the template overhang, thus demonstrating the Klentaq1 is enzymatically active in the crystal. In addition, a ddCTP molecule occupied the enzyme's active site, where it formed a Watson–Crick base pair with the template's next G.  
Pol I replicates DNA with high fidelity. How does it do so? Gabriel Waksman answered this question by crystallizing the C-terminal domain of ''Taq'' polymerase ('''Klentaq1''') with an 11-bp DNA that had a GGAAA-5' overhang at the 5' end of its template strand<ref>PMID: 9857206</ref>. The crystals were then soaked in solution containing 2',3'-dideoxy-CTP (ddCTP), which lacks a 3'-OH group, and hence terminates replication after its incorporation at the 3' end of the primer strand. The X-ray structure of these crystals (3ktq) revealed that a ddC residue had been covalently linked to the 3' end of the primer strand, where it formed a Watson–Crick base pair with the 3' G on the template overhang, thus demonstrating the Klentaq1 is enzymatically active in the crystal. In addition, a ddCTP molecule occupied the enzyme's active site, where it formed a Watson–Crick base pair with the template's next G.  
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