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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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Effect of temperature.(A) Calculated results of the mean transition time Tm of the 5′-nuclease domain to the active mode as a function of the temperature. (B) Calculated results of the mean dissociation time Td of the flap DNA substrate from the polymerase domain as a function of the temperature.
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pone-0016213-g008: Effect of temperature.(A) Calculated results of the mean transition time Tm of the 5′-nuclease domain to the active mode as a function of the temperature. (B) Calculated results of the mean dissociation time Td of the flap DNA substrate from the polymerase domain as a function of the temperature.

Mentions: In the above, we have fixed temperature T = 298 K (25°C). To see the effect of the variation of temperature on the results, we change the temperature in our calculations. The value of viscosity η as a function of the temperature is taken from the experimental data (see Table S1 and [43]). The calculated results of the mean time Tm for the 5′-nuclease domain to transit to the active mode as a function of the temperature for d = 2 nm are shown in Figure 8A. It is seen that, as the temperature increases, the mean transition time Tm decreases, which results from both the decrease of the viscosity η and the increase of the noise strength. On the other hand, the statistical results of the mean time Td for the flap DNA with a nick to dissociate from the polymerase domain as a function of the temperature are shown in Figure 8b, where we take U0 = 16 kBT that is consistent with the available experimental data [40] (see above). As expected, the mean dissociation time Td decreases as the temperature increases. By comparing Figure 8A with Figure 8B, it is seen that, at a given temperature, Td is much larger than Tm, implying that, at any temperature in the range of 10–50°C, the 5′-nucleolytic processing event is most probably carried out by the same PolI molecule that has just extended the upstream primer terminus.


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

Xie P, Sayers JR - PLoS ONE (2011)

Effect of temperature.(A) Calculated results of the mean transition time Tm of the 5′-nuclease domain to the active mode as a function of the temperature. (B) Calculated results of the mean dissociation time Td of the flap DNA substrate from the polymerase domain as a function of the temperature.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016213-g008: Effect of temperature.(A) Calculated results of the mean transition time Tm of the 5′-nuclease domain to the active mode as a function of the temperature. (B) Calculated results of the mean dissociation time Td of the flap DNA substrate from the polymerase domain as a function of the temperature.
Mentions: In the above, we have fixed temperature T = 298 K (25°C). To see the effect of the variation of temperature on the results, we change the temperature in our calculations. The value of viscosity η as a function of the temperature is taken from the experimental data (see Table S1 and [43]). The calculated results of the mean time Tm for the 5′-nuclease domain to transit to the active mode as a function of the temperature for d = 2 nm are shown in Figure 8A. It is seen that, as the temperature increases, the mean transition time Tm decreases, which results from both the decrease of the viscosity η and the increase of the noise strength. On the other hand, the statistical results of the mean time Td for the flap DNA with a nick to dissociate from the polymerase domain as a function of the temperature are shown in Figure 8b, where we take U0 = 16 kBT that is consistent with the available experimental data [40] (see above). As expected, the mean dissociation time Td decreases as the temperature increases. By comparing Figure 8A with Figure 8B, it is seen that, at a given temperature, Td is much larger than Tm, implying that, at any temperature in the range of 10–50°C, the 5′-nucleolytic processing event is most probably carried out by the same PolI molecule that has just extended the upstream primer terminus.

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