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A model for transition of 5'-nuclease domain of DNA polymerase I from inert to active modes.

Xie P, Sayers JR - PLoS ONE (2011)

Bottom Line: By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data.We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5'-3' exonuclease activities.Moreover, predicted results for the latter model are presented.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Bacteria contain DNA polymerase I (PolI), a single polypeptide chain consisting of ∼930 residues, possessing DNA-dependent DNA polymerase, 3'-5' proofreading and 5'-3' exonuclease (also known as flap endonuclease) activities. PolI is particularly important in the processing of Okazaki fragments generated during lagging strand replication and must ultimately produce a double-stranded substrate with a nick suitable for DNA ligase to seal. PolI's activities must be highly coordinated both temporally and spatially otherwise uncontrolled 5'-nuclease activity could attack a nick and produce extended gaps leading to potentially lethal double-strand breaks. To investigate the mechanism of how PolI efficiently produces these nicks, we present theoretical studies on the dynamics of two possible scenarios or models. In one the flap DNA substrate can transit from the polymerase active site to the 5'-nuclease active site, with the relative position of the two active sites being kept fixed; while the other is that the 5'-nuclease domain can transit from the inactive mode, with the 5'-nuclease active site distant from the cleavage site on the DNA substrate, to the active mode, where the active site and substrate cleavage site are juxtaposed. The theoretical results based on the former scenario are inconsistent with the available experimental data that indicated that the majority of 5'-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus. By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data. We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5'-3' exonuclease activities. Moreover, predicted results for the latter model are presented.

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Schematic diagrams of polymerase I and flap DNA.(A) Polymerase I is composed of polymerase domain, which consists of finger, palm, thumb and 3′-5′ exonuclease subdomains, and 5′-nuclease domain. (B) Flap DNA with no single-stranded gap. (C) From left to right. Okazaki fragments consist of a few nucleotides of RNA primer (grey lines), which are then extended by DNA polymerases using deoxynucleside triphosphates to make the daughter DNA strand (dark arrows). Okazaki fragment synthesis gives rise to flap structures as follows when the 3′ end of a newly synthesized strand encounters the 5′ end of the RNA primer of the downstream Okazaki fragment. Strand displacement synthesis occurs (centre right) and the flap is then cleaved by FEN activity (red triangle) of Pol1 5′-nuclease domain. Normally a nick results which is sealed by DNA ligase. (D) Excessive or unregulated FEN EXO or GEN activity (shown by black and open triangles respectively) could give rise to extended single-stranded regions or even double strand breaks as shown.
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pone-0016213-g001: Schematic diagrams of polymerase I and flap DNA.(A) Polymerase I is composed of polymerase domain, which consists of finger, palm, thumb and 3′-5′ exonuclease subdomains, and 5′-nuclease domain. (B) Flap DNA with no single-stranded gap. (C) From left to right. Okazaki fragments consist of a few nucleotides of RNA primer (grey lines), which are then extended by DNA polymerases using deoxynucleside triphosphates to make the daughter DNA strand (dark arrows). Okazaki fragment synthesis gives rise to flap structures as follows when the 3′ end of a newly synthesized strand encounters the 5′ end of the RNA primer of the downstream Okazaki fragment. Strand displacement synthesis occurs (centre right) and the flap is then cleaved by FEN activity (red triangle) of Pol1 5′-nuclease domain. Normally a nick results which is sealed by DNA ligase. (D) Excessive or unregulated FEN EXO or GEN activity (shown by black and open triangles respectively) could give rise to extended single-stranded regions or even double strand breaks as shown.

Mentions: DNA polymerase I (PolI) is a well-characterized enzyme involved in DNA replication and repair [1]–[4]. The archetypal enzyme (Figure 1A) possesses three distinct biochemical activities, namely a DNA-dependent 5′-3′ DNA polymerase, a 3′-5′ proofreading exonuclease and a 5′-3′ exonuclease function [5], [6]. While polymerase and proofreading functions have been investigated extensively, the 5′-3′ exonuclease activity has been subject to less scrutiny. This activity has been variously described as a 5′-3′ exonuclease, a 5′ nuclease [7] and most commonly as a flap endonuclease or FEN activity [8] due to the biochemical and sequence homologies with their eukaryotic counterparts.


A model for transition of 5'-nuclease domain of DNA polymerase I from inert to active modes.

Xie P, Sayers JR - PLoS ONE (2011)

Schematic diagrams of polymerase I and flap DNA.(A) Polymerase I is composed of polymerase domain, which consists of finger, palm, thumb and 3′-5′ exonuclease subdomains, and 5′-nuclease domain. (B) Flap DNA with no single-stranded gap. (C) From left to right. Okazaki fragments consist of a few nucleotides of RNA primer (grey lines), which are then extended by DNA polymerases using deoxynucleside triphosphates to make the daughter DNA strand (dark arrows). Okazaki fragment synthesis gives rise to flap structures as follows when the 3′ end of a newly synthesized strand encounters the 5′ end of the RNA primer of the downstream Okazaki fragment. Strand displacement synthesis occurs (centre right) and the flap is then cleaved by FEN activity (red triangle) of Pol1 5′-nuclease domain. Normally a nick results which is sealed by DNA ligase. (D) Excessive or unregulated FEN EXO or GEN activity (shown by black and open triangles respectively) could give rise to extended single-stranded regions or even double strand breaks as shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3021548&req=5

pone-0016213-g001: Schematic diagrams of polymerase I and flap DNA.(A) Polymerase I is composed of polymerase domain, which consists of finger, palm, thumb and 3′-5′ exonuclease subdomains, and 5′-nuclease domain. (B) Flap DNA with no single-stranded gap. (C) From left to right. Okazaki fragments consist of a few nucleotides of RNA primer (grey lines), which are then extended by DNA polymerases using deoxynucleside triphosphates to make the daughter DNA strand (dark arrows). Okazaki fragment synthesis gives rise to flap structures as follows when the 3′ end of a newly synthesized strand encounters the 5′ end of the RNA primer of the downstream Okazaki fragment. Strand displacement synthesis occurs (centre right) and the flap is then cleaved by FEN activity (red triangle) of Pol1 5′-nuclease domain. Normally a nick results which is sealed by DNA ligase. (D) Excessive or unregulated FEN EXO or GEN activity (shown by black and open triangles respectively) could give rise to extended single-stranded regions or even double strand breaks as shown.
Mentions: DNA polymerase I (PolI) is a well-characterized enzyme involved in DNA replication and repair [1]–[4]. The archetypal enzyme (Figure 1A) possesses three distinct biochemical activities, namely a DNA-dependent 5′-3′ DNA polymerase, a 3′-5′ proofreading exonuclease and a 5′-3′ exonuclease function [5], [6]. While polymerase and proofreading functions have been investigated extensively, the 5′-3′ exonuclease activity has been subject to less scrutiny. This activity has been variously described as a 5′-3′ exonuclease, a 5′ nuclease [7] and most commonly as a flap endonuclease or FEN activity [8] due to the biochemical and sequence homologies with their eukaryotic counterparts.

Bottom Line: By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data.We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5'-3' exonuclease activities.Moreover, predicted results for the latter model are presented.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Bacteria contain DNA polymerase I (PolI), a single polypeptide chain consisting of ∼930 residues, possessing DNA-dependent DNA polymerase, 3'-5' proofreading and 5'-3' exonuclease (also known as flap endonuclease) activities. PolI is particularly important in the processing of Okazaki fragments generated during lagging strand replication and must ultimately produce a double-stranded substrate with a nick suitable for DNA ligase to seal. PolI's activities must be highly coordinated both temporally and spatially otherwise uncontrolled 5'-nuclease activity could attack a nick and produce extended gaps leading to potentially lethal double-strand breaks. To investigate the mechanism of how PolI efficiently produces these nicks, we present theoretical studies on the dynamics of two possible scenarios or models. In one the flap DNA substrate can transit from the polymerase active site to the 5'-nuclease active site, with the relative position of the two active sites being kept fixed; while the other is that the 5'-nuclease domain can transit from the inactive mode, with the 5'-nuclease active site distant from the cleavage site on the DNA substrate, to the active mode, where the active site and substrate cleavage site are juxtaposed. The theoretical results based on the former scenario are inconsistent with the available experimental data that indicated that the majority of 5'-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus. By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data. We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5'-3' exonuclease activities. Moreover, predicted results for the latter model are presented.

Show MeSH