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Molecular dynamics study of the opening mechanism for DNA polymerase I.

Miller BR, Parish CA, Wu EY - PLoS Comput. Biol. (2014)

Bottom Line: The dynamics of this process are crucial to the overall effectiveness of catalysis.All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies.In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Richmond, Richmond, Virginia, United States of America; Department of Chemistry, University of Richmond, Richmond, Virginia, United States of America.

ABSTRACT
During DNA replication, DNA polymerases follow an induced fit mechanism in order to rapidly distinguish between correct and incorrect dNTP substrates. The dynamics of this process are crucial to the overall effectiveness of catalysis. Although X-ray crystal structures of DNA polymerase I with substrate dNTPs have revealed key structural states along the catalytic pathway, solution fluorescence studies indicate that those key states are populated in the absence of substrate. Herein, we report the first atomistic simulations showing the conformational changes between the closed, open, and ajar conformations of DNA polymerase I in the binary (enzyme:DNA) state to better understand its dynamics. We have applied long time-scale, unbiased molecular dynamics to investigate the opening process of the fingers domain in the absence of substrate for B. stearothermophilis DNA polymerase in silico. These simulations are biologically and/or physiologically relevant as they shed light on the transitions between states in this important enzyme. All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies. Furthermore, we have detailed the key stages in the opening process starting from the open and ajar crystal structures, including the observation of a previously unknown key intermediate structure. Four backbone dihedrals were identified as important during the opening process, and their movements provide insight into the recognition of dNTP substrate molecules by the polymerase binary state. In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics.

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The proposed pathway for opening and closing of DNA polymerase I in the presence and absence of dNTP.In the binary complex (blue), the polymerase transitions through the intermediate observed in this study (EI•DNA), while the ternary complex (yellow) transition is a separate, partially-closed conformation (EPC•DNA•dNTP) on its way to the closed conformation. This pathway depicts the enzyme in two different “ajar” conformation (EI or EPC) determined by the presence or absence of dNTP in the active site.
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pcbi-1003961-g008: The proposed pathway for opening and closing of DNA polymerase I in the presence and absence of dNTP.In the binary complex (blue), the polymerase transitions through the intermediate observed in this study (EI•DNA), while the ternary complex (yellow) transition is a separate, partially-closed conformation (EPC•DNA•dNTP) on its way to the closed conformation. This pathway depicts the enzyme in two different “ajar” conformation (EI or EPC) determined by the presence or absence of dNTP in the active site.

Mentions: As a final note on the intermediate state, although the fingers domain is clearly between the closed and open conformations, this newly observed state is not identical to the ajar state observed in the 3HP6 PDB structure. The simulated intermediate has a heavy atom root-mean-square deviation (RMSD) of 4.3 Å from the 3HP6 crystal structure. The largest structural differences between the intermediate and the 3HP6 crystal structure arise in the fingers subdomain with Arg703 and the thumb region of the polymerase where Glu562 resides (Figure 7). The Arg703-Glu562 salt bridge is not present in the 3HP6 crystal structure nor does it ever exist in any of the simulations starting from the 3HP6 ajar conformation. The 3HP6 crystal structure was generated by trapping DNA polymerase with a non-Watson Crick dNTP paired to the template strand in the active site, while our simulations are performed in the absence of a dNTP molecule to mimic the dynamics of the protein after elongation of the DNA primer strand has occurred. This means that, experimentally, the two “ajar” conformations reside on two different potential energy surfaces where the 3HP6 ajar state is only observed in the presence of a dNTP in the active site, while the proposed intermediate state is present only in the absence of dNTP (Figure 8). This is contrary to the literature reported prior to this study that assumed the polymerase conformation observed in the 3HP6 crystal structure was identical to the conformation of polymerase observed in the absence of dNTP. The single-molecule FRET experiments [13] that previously reported the presence of open, ajar, and closed conformations in the binary state probably observed the intermediate proposed in this study instead of the ajar state documented from X-ray crystallography that likely only occurs with a bound mismatch dNTP. Thus, the hypothesis for this new intermediate structure from MD is consistent with solution studies that show an intermediate state between the open and closed conformations in the absence of dNTP. The exact purpose of the intermediate is not fully understood yet, but it is clear that the presence of the intermediate slows the transition from the closed to the open conformation in the binary complex. Based on the similarity between binary and ternary pathways to the closed conformation (Figure 8), we speculate that the intermediate may also play a role in the closing of the fingers domain during dNTP binding, possibly providing an energetic barrier to opening that aids the enzyme during substrate recognition.


Molecular dynamics study of the opening mechanism for DNA polymerase I.

Miller BR, Parish CA, Wu EY - PLoS Comput. Biol. (2014)

The proposed pathway for opening and closing of DNA polymerase I in the presence and absence of dNTP.In the binary complex (blue), the polymerase transitions through the intermediate observed in this study (EI•DNA), while the ternary complex (yellow) transition is a separate, partially-closed conformation (EPC•DNA•dNTP) on its way to the closed conformation. This pathway depicts the enzyme in two different “ajar” conformation (EI or EPC) determined by the presence or absence of dNTP in the active site.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256020&req=5

pcbi-1003961-g008: The proposed pathway for opening and closing of DNA polymerase I in the presence and absence of dNTP.In the binary complex (blue), the polymerase transitions through the intermediate observed in this study (EI•DNA), while the ternary complex (yellow) transition is a separate, partially-closed conformation (EPC•DNA•dNTP) on its way to the closed conformation. This pathway depicts the enzyme in two different “ajar” conformation (EI or EPC) determined by the presence or absence of dNTP in the active site.
Mentions: As a final note on the intermediate state, although the fingers domain is clearly between the closed and open conformations, this newly observed state is not identical to the ajar state observed in the 3HP6 PDB structure. The simulated intermediate has a heavy atom root-mean-square deviation (RMSD) of 4.3 Å from the 3HP6 crystal structure. The largest structural differences between the intermediate and the 3HP6 crystal structure arise in the fingers subdomain with Arg703 and the thumb region of the polymerase where Glu562 resides (Figure 7). The Arg703-Glu562 salt bridge is not present in the 3HP6 crystal structure nor does it ever exist in any of the simulations starting from the 3HP6 ajar conformation. The 3HP6 crystal structure was generated by trapping DNA polymerase with a non-Watson Crick dNTP paired to the template strand in the active site, while our simulations are performed in the absence of a dNTP molecule to mimic the dynamics of the protein after elongation of the DNA primer strand has occurred. This means that, experimentally, the two “ajar” conformations reside on two different potential energy surfaces where the 3HP6 ajar state is only observed in the presence of a dNTP in the active site, while the proposed intermediate state is present only in the absence of dNTP (Figure 8). This is contrary to the literature reported prior to this study that assumed the polymerase conformation observed in the 3HP6 crystal structure was identical to the conformation of polymerase observed in the absence of dNTP. The single-molecule FRET experiments [13] that previously reported the presence of open, ajar, and closed conformations in the binary state probably observed the intermediate proposed in this study instead of the ajar state documented from X-ray crystallography that likely only occurs with a bound mismatch dNTP. Thus, the hypothesis for this new intermediate structure from MD is consistent with solution studies that show an intermediate state between the open and closed conformations in the absence of dNTP. The exact purpose of the intermediate is not fully understood yet, but it is clear that the presence of the intermediate slows the transition from the closed to the open conformation in the binary complex. Based on the similarity between binary and ternary pathways to the closed conformation (Figure 8), we speculate that the intermediate may also play a role in the closing of the fingers domain during dNTP binding, possibly providing an energetic barrier to opening that aids the enzyme during substrate recognition.

Bottom Line: The dynamics of this process are crucial to the overall effectiveness of catalysis.All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies.In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Richmond, Richmond, Virginia, United States of America; Department of Chemistry, University of Richmond, Richmond, Virginia, United States of America.

ABSTRACT
During DNA replication, DNA polymerases follow an induced fit mechanism in order to rapidly distinguish between correct and incorrect dNTP substrates. The dynamics of this process are crucial to the overall effectiveness of catalysis. Although X-ray crystal structures of DNA polymerase I with substrate dNTPs have revealed key structural states along the catalytic pathway, solution fluorescence studies indicate that those key states are populated in the absence of substrate. Herein, we report the first atomistic simulations showing the conformational changes between the closed, open, and ajar conformations of DNA polymerase I in the binary (enzyme:DNA) state to better understand its dynamics. We have applied long time-scale, unbiased molecular dynamics to investigate the opening process of the fingers domain in the absence of substrate for B. stearothermophilis DNA polymerase in silico. These simulations are biologically and/or physiologically relevant as they shed light on the transitions between states in this important enzyme. All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies. Furthermore, we have detailed the key stages in the opening process starting from the open and ajar crystal structures, including the observation of a previously unknown key intermediate structure. Four backbone dihedrals were identified as important during the opening process, and their movements provide insight into the recognition of dNTP substrate molecules by the polymerase binary state. In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics.

Show MeSH
Related in: MedlinePlus