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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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Predicted results of the mean transition time Tm versus the external load Fload acting on the 5′-nuclease domain.
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pone-0016213-g009: Predicted results of the mean transition time Tm versus the external load Fload acting on the 5′-nuclease domain.

Mentions: Next, we present our predicted results for the effect of the external force on the transition of the 5′-nuclease domain from the inactive to active modes. Consider a load, Fload, acting on the residues indicated by the blue dot (Figure S11) along the x direction. Then, the right-hand sides of Eqs. (17), (18) and (19) should be added by terms , , , respectively. The calculated results of the mean transition time Tm versus Fload at T = 298 K (25°C) are shown in Figure 9. It is seen that Tm increases significantly with the increase of the external load. It is interesting to note that, only at Fload>2.5 pN, Tm>600 µs that is much larger than Td = 133 µs at U0 = 16 kBT (see above). This implies that the flap DNA substrate becomes dissociated from the polymerase before the 5′-nuclease domain transits to the active mode. Thus, the polymerase molecule that has just extended the upstream primer terminus cannot perform the activity of cleaving the downstream 5′-flap, i.e., the polymerase cannot simultaneously perform the two activities.


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

Xie P, Sayers JR - PLoS ONE (2011)

Predicted results of the mean transition time Tm versus the external load Fload acting on the 5′-nuclease domain.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016213-g009: Predicted results of the mean transition time Tm versus the external load Fload acting on the 5′-nuclease domain.
Mentions: Next, we present our predicted results for the effect of the external force on the transition of the 5′-nuclease domain from the inactive to active modes. Consider a load, Fload, acting on the residues indicated by the blue dot (Figure S11) along the x direction. Then, the right-hand sides of Eqs. (17), (18) and (19) should be added by terms , , , respectively. The calculated results of the mean transition time Tm versus Fload at T = 298 K (25°C) are shown in Figure 9. It is seen that Tm increases significantly with the increase of the external load. It is interesting to note that, only at Fload>2.5 pN, Tm>600 µs that is much larger than Td = 133 µs at U0 = 16 kBT (see above). This implies that the flap DNA substrate becomes dissociated from the polymerase before the 5′-nuclease domain transits to the active mode. Thus, the polymerase molecule that has just extended the upstream primer terminus cannot perform the activity of cleaving the downstream 5′-flap, i.e., the polymerase cannot simultaneously perform the two activities.

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