Limits...
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.

Show MeSH

Related in: MedlinePlus

The O-helix distance as measured by the α-C distance between Arg629 and Pro699 depicting the opening of the fingers domain for the wild-type 1LV5 (blue) and R703A mutant (purple) simulated using Desmond and the Charmm27 force field.The plot shows the mutant reaching the open conformation in <50 ns, while the wild-type does not open fully until ∼290 ns.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256020&req=5

pcbi-1003961-g006: The O-helix distance as measured by the α-C distance between Arg629 and Pro699 depicting the opening of the fingers domain for the wild-type 1LV5 (blue) and R703A mutant (purple) simulated using Desmond and the Charmm27 force field.The plot shows the mutant reaching the open conformation in <50 ns, while the wild-type does not open fully until ∼290 ns.

Mentions: The intermediate state observed in the 1LV5 (closed) simulation is stabilized by a key salt bridge between an arginine residue in the O-helix and a glutamate residue in the thumb domain of DNA polymerase. To test whether the salt bridge constitutes a substantial obstacle for opening, we in silico mutated Arg703 to an alanine residue in 1LV5 and re-started the simulation under the same conditions and simulated for 500 ns. The fingers domain of the R703A mutant opened in <50 ns, while the wild-type required ∼290 ns to reach the same conformation (Figure 6). Given that the only difference between these two starting structures is the mutation from arginine to alanine at position 703, this result provides further evidence of the importance of the Arg703-Glu562 salt bridge intermediate along the opening pathway for the fingers domain of DNA polymerase. The arginine residue is highly conserved in bacterial DNA polymerase I enzymes. 28 out of 33 DNA polymerase I enzyme sequences from bacteria in UniProt contained an arginine at this location in the O-helix, including ones from Escherichia coli and Thermus aquaticus (Taq), which have been structurally characterized. Arg703 is also known to be important for polymerase activity in bacteria [46]. Mutation studies of the corresponding arginine in Taq DNA polymerase I showed a clear loss of polymerase function when mutated, although the role of the arginine residue was not described [46]. Our simulations support those mutagenesis studies, indicating the importance of this arginine to the polymerase and additionally illustrate its role in forming a key intermediate during the opening of the fingers domain.


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

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

The O-helix distance as measured by the α-C distance between Arg629 and Pro699 depicting the opening of the fingers domain for the wild-type 1LV5 (blue) and R703A mutant (purple) simulated using Desmond and the Charmm27 force field.The plot shows the mutant reaching the open conformation in <50 ns, while the wild-type does not open fully until ∼290 ns.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g006: The O-helix distance as measured by the α-C distance between Arg629 and Pro699 depicting the opening of the fingers domain for the wild-type 1LV5 (blue) and R703A mutant (purple) simulated using Desmond and the Charmm27 force field.The plot shows the mutant reaching the open conformation in <50 ns, while the wild-type does not open fully until ∼290 ns.
Mentions: The intermediate state observed in the 1LV5 (closed) simulation is stabilized by a key salt bridge between an arginine residue in the O-helix and a glutamate residue in the thumb domain of DNA polymerase. To test whether the salt bridge constitutes a substantial obstacle for opening, we in silico mutated Arg703 to an alanine residue in 1LV5 and re-started the simulation under the same conditions and simulated for 500 ns. The fingers domain of the R703A mutant opened in <50 ns, while the wild-type required ∼290 ns to reach the same conformation (Figure 6). Given that the only difference between these two starting structures is the mutation from arginine to alanine at position 703, this result provides further evidence of the importance of the Arg703-Glu562 salt bridge intermediate along the opening pathway for the fingers domain of DNA polymerase. The arginine residue is highly conserved in bacterial DNA polymerase I enzymes. 28 out of 33 DNA polymerase I enzyme sequences from bacteria in UniProt contained an arginine at this location in the O-helix, including ones from Escherichia coli and Thermus aquaticus (Taq), which have been structurally characterized. Arg703 is also known to be important for polymerase activity in bacteria [46]. Mutation studies of the corresponding arginine in Taq DNA polymerase I showed a clear loss of polymerase function when mutated, although the role of the arginine residue was not described [46]. Our simulations support those mutagenesis studies, indicating the importance of this arginine to the polymerase and additionally illustrate its role in forming a key intermediate during the opening of the fingers domain.

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