DNA Polymerase I: Difference between revisions
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<StructureSection load='1KLN_pymol.pdb' size='400' side='right' scene='Sandbox_dvoet/DNA_polymerase/Klenow-dna/4' caption='' > | |||
==Overview== | ==Overview== | ||
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==Structure of the Klenow fragment== | ==Structure of the Klenow fragment== | ||
Thomas Steitz determined the X-ray structure of Klenow fragment in complex with a 13-nucleotide (nt) primer strand and a 10-nt template strand<ref>PMID: 8469987</ref> (the primer strand is the strand that is synthesized by the polymerase as the complement of the template strand; the entire DNA is often referred to as primer−template DNA). <scene name='Sandbox_dvoet/DNA_polymerase/Klenow-dna/4'>Here</scene> Klenow fragment is shown in ribbon form colored in rainbow order from its N-terminus (''blue'') to its C-terminus (''red''). The DNA is drawn in stick form and colored according to atom type with template C cyan, primer C magenta, N blue, O red, and P orange and with an orange rod connecting successive P atoms in each strand. The 3' → 5' exonuclease active site at the N-terminal end of the protein is marked by a Zn<sup>2+</sup> ion (''gray sphere''). 'The arrangement of the polymerase's three domains is reminiscent of a right hand grasping a rod (the DNA) and hence, from N- to C-terminus, they are named “palm“, "fingers", and "thumb". The polymerase's active site is located in the palm domain near the cleft between the fingers and thumb domains. All DNA polymerases of known structure have a similar spatial arrangements of fingers, thumb, and palm domains, even though, in many cases, they have no recognizable sequence similarity with Pol I and the structure of their fingers, thumb, and palm domains bear no resemblance to those of Pol I. | Thomas Steitz determined the X-ray structure of Klenow fragment in complex with a 13-nucleotide (nt) primer strand and a 10-nt template strand<ref>PMID: 8469987</ref> (the primer strand is the strand that is synthesized by the polymerase as the complement of the template strand; the entire DNA is often referred to as primer−template DNA). <scene name='Sandbox_dvoet/DNA_polymerase/Klenow-dna/4'>Here</scene> Klenow fragment is shown in ribbon form colored in rainbow order from its N-terminus (''blue'') to its C-terminus (''red''). The DNA is drawn in stick form and colored according to atom type with template C cyan, primer C magenta, N blue, O red, and P orange and with an orange rod connecting successive P atoms in each strand. The 3' → 5' exonuclease active site at the N-terminal end of the protein is marked by a Zn<sup>2+</sup> ion (''gray sphere''). 'The arrangement of the polymerase's three domains is reminiscent of a right hand grasping a rod (the DNA) and hence, from N- to C-terminus, they are named “palm“, "fingers", and "thumb". The polymerase's active site is located in the palm domain near the cleft between the fingers and thumb domains. All DNA polymerases of known structure have a similar spatial arrangements of fingers, thumb, and palm domains, even though, in many cases, they have no recognizable sequence similarity with Pol I and the structure of their fingers, thumb, and palm domains bear no resemblance to those of Pol I. | ||
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==Structure of the whole ''Thermus aquaticus'' DNA polymerase I== | ==Structure of the whole ''Thermus aquaticus'' DNA polymerase I== | ||
As mentioned above, Pol I's primary and essential function is to excise the RNA primers from newly synthesized Okazaki fragments with its 5' → 3' exonuclease function and replace them with DNA using its polymerase function. This yields a double-stranded DNA (dsDNA) with a single strand nick between successive Okazaki fragments, a nick that is eventually sealed through the action of DNA ligase. | As mentioned above, Pol I's primary and essential function is to excise the RNA primers from newly synthesized Okazaki fragments with its 5' → 3' exonuclease function and replace them with DNA using its polymerase function. This yields a double-stranded DNA (dsDNA) with a single strand nick between successive Okazaki fragments, a nick that is eventually sealed through the action of DNA ligase. | ||
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, 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. | <scene name='Sandbox_dvoet/DNA_polymerase/Taq_pol_i/1'>Pol I from the thermophilic bacterium</scene> ''Thermus aquaticus'' (''Taq'', PDB entry [[1taq]]) 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, 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. | ||
</StructureSection> | |||
==Structures of Klentaq1 in its closed and open forms== | ==Structures of Klentaq1 in its closed and open forms== | ||
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A closeup of the active site region in the <scene name='Sandbox_dvoet/DNA_polymerase/Klentaq1-open_closeup/6' target='Open' >open conformation</scene> (''right'') reveals that the side chain of the conserved Tyr 671 (colored with C pink) is stacked on top of the template G that forms a base pair with the bound ddCTP, where it apparently participates in verifying that a Watson–Crick base pair has formed. In the <scene name='Sandbox_dvoet/DNA_polymerase/Klentaq1-closed_closeup/3' target='Closed' >closed conformation</scene> (''left''), Tyr 671, which is part of the fingers domain, has moved aside, presumably to permit the active site to form about the incoming dNTP (satisfy yourself that the Tyr 671 side chain is stacked on the template G in the open form but not in the closed form). | A closeup of the active site region in the <scene name='Sandbox_dvoet/DNA_polymerase/Klentaq1-open_closeup/6' target='Open' >open conformation</scene> (''right'') reveals that the side chain of the conserved Tyr 671 (colored with C pink) is stacked on top of the template G that forms a base pair with the bound ddCTP, where it apparently participates in verifying that a Watson–Crick base pair has formed. In the <scene name='Sandbox_dvoet/DNA_polymerase/Klentaq1-closed_closeup/3' target='Closed' >closed conformation</scene> (''left''), Tyr 671, which is part of the fingers domain, has moved aside, presumably to permit the active site to form about the incoming dNTP (satisfy yourself that the Tyr 671 side chain is stacked on the template G in the open form but not in the closed form). | ||
==3D structures of DNA polymerase== | |||
[[DNA polymerase]] | |||
==Additional Resources== | ==Additional Resources== | ||