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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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Calculated results on dynamics of flap DNA transition from PolI's polymerase active site to 5′-nuclease active site or dissociation into solution.(A) Probability, Pn, for the DNA to transit to the 5′-nuclease active site versus the interaction strength U0. (B) Mean time, Td, for the DNA to detach from the polymerase or to transfer to the 5′-nuclease active site versus the interaction strength U0. Filled dots represent results with potentials given by Eqs. (2)–(7), with forms of U (x, 0, 0), U (0, y, 0) and U (0, 0, z) shown in Figure S2; while unfilled triangles represent results with forms of potentials U (x, 0, 0), U (0, y, 0) and U (0, 0, z) shown in Figure S7.
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pone-0016213-g003: Calculated results on dynamics of flap DNA transition from PolI's polymerase active site to 5′-nuclease active site or dissociation into solution.(A) Probability, Pn, for the DNA to transit to the 5′-nuclease active site versus the interaction strength U0. (B) Mean time, Td, for the DNA to detach from the polymerase or to transfer to the 5′-nuclease active site versus the interaction strength U0. Filled dots represent results with potentials given by Eqs. (2)–(7), with forms of U (x, 0, 0), U (0, y, 0) and U (0, 0, z) shown in Figure S2; while unfilled triangles represent results with forms of potentials U (x, 0, 0), U (0, y, 0) and U (0, 0, z) shown in Figure S7.

Mentions: To study the transition dynamics of the flap DNA substrate, we solved Eqs. (8)–(10) numerically by using the stochastic Runge-Kutta method [32], [33]. The method has been proved suitable for simulation of stochastic dynamics in physical, chemical and biological systems [32]–[35]. In our simulation, we take A = 0.5 nm as is consistent with the Debye length in the order of 1 nm in solution (see Figure S2). In Figures S3, S4, S5, S6 we show some typical results for the trace of the flap DNA substrate relative to the polymerase, where Figure S3 corresponds to the situation where the DNA is transferred from the polymerase active site at (x, y, z)  =  (0, 0, 0) to the 5′-nuclease active site at (x, y, z)  =  (7 nm, 0, 0), while Figsure S4–S6 correspond to the case where the DNA detaches from the polymerase or dissociates into solution. Note that the different traces shown in Figures S3, S4, S5, S6 correspond to different thermal noise realizations. The statistical results of the probability, Pn, for the DNA to transfer to the 5′-nuclease active site versus the interaction strength U0 are shown in Figure 3A (denoted by dots). The corresponding probability for the DNA to dissociate into solution is thus given by 1 – Pn. The statistical results of the mean time, Td, for the DNA to detach from the polymerase (i.e., to move to position satisfying ≥10 nm) or to move to the 5′-nuclease active site at (x, y, z)  =  (7 nm, 0, 0) versus U0 are shown in Figure 3B. To see the effect of the potential forms, in Figure 3A. We also show some results (denoted by triangles) resulting from use of another form of the potential U(x, y, z) such as that plotted in Figure S7. It is seen that different potential forms only give slightly different statistical results.


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

Xie P, Sayers JR - PLoS ONE (2011)

Calculated results on dynamics of flap DNA transition from PolI's polymerase active site to 5′-nuclease active site or dissociation into solution.(A) Probability, Pn, for the DNA to transit to the 5′-nuclease active site versus the interaction strength U0. (B) Mean time, Td, for the DNA to detach from the polymerase or to transfer to the 5′-nuclease active site versus the interaction strength U0. Filled dots represent results with potentials given by Eqs. (2)–(7), with forms of U (x, 0, 0), U (0, y, 0) and U (0, 0, z) shown in Figure S2; while unfilled triangles represent results with forms of potentials U (x, 0, 0), U (0, y, 0) and U (0, 0, z) shown in Figure S7.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3021548&req=5

pone-0016213-g003: Calculated results on dynamics of flap DNA transition from PolI's polymerase active site to 5′-nuclease active site or dissociation into solution.(A) Probability, Pn, for the DNA to transit to the 5′-nuclease active site versus the interaction strength U0. (B) Mean time, Td, for the DNA to detach from the polymerase or to transfer to the 5′-nuclease active site versus the interaction strength U0. Filled dots represent results with potentials given by Eqs. (2)–(7), with forms of U (x, 0, 0), U (0, y, 0) and U (0, 0, z) shown in Figure S2; while unfilled triangles represent results with forms of potentials U (x, 0, 0), U (0, y, 0) and U (0, 0, z) shown in Figure S7.
Mentions: To study the transition dynamics of the flap DNA substrate, we solved Eqs. (8)–(10) numerically by using the stochastic Runge-Kutta method [32], [33]. The method has been proved suitable for simulation of stochastic dynamics in physical, chemical and biological systems [32]–[35]. In our simulation, we take A = 0.5 nm as is consistent with the Debye length in the order of 1 nm in solution (see Figure S2). In Figures S3, S4, S5, S6 we show some typical results for the trace of the flap DNA substrate relative to the polymerase, where Figure S3 corresponds to the situation where the DNA is transferred from the polymerase active site at (x, y, z)  =  (0, 0, 0) to the 5′-nuclease active site at (x, y, z)  =  (7 nm, 0, 0), while Figsure S4–S6 correspond to the case where the DNA detaches from the polymerase or dissociates into solution. Note that the different traces shown in Figures S3, S4, S5, S6 correspond to different thermal noise realizations. The statistical results of the probability, Pn, for the DNA to transfer to the 5′-nuclease active site versus the interaction strength U0 are shown in Figure 3A (denoted by dots). The corresponding probability for the DNA to dissociate into solution is thus given by 1 – Pn. The statistical results of the mean time, Td, for the DNA to detach from the polymerase (i.e., to move to position satisfying ≥10 nm) or to move to the 5′-nuclease active site at (x, y, z)  =  (7 nm, 0, 0) versus U0 are shown in Figure 3B. To see the effect of the potential forms, in Figure 3A. We also show some results (denoted by triangles) resulting from use of another form of the potential U(x, y, z) such as that plotted in Figure S7. It is seen that different potential forms only give slightly different statistical results.

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
Related in: MedlinePlus