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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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Related in: MedlinePlus

The distance between the side chain OH on Tyr714 and δ-C of Glu658 for the open (1L3S, red), ajar (3HP6, green), and closed (1LV5, blue) structured simulated using Desmond with the Charmm27 force field.Tyr714 and Glu658 are not hydrogen bonded in the 1L3S PDB (corresponding to a distance of 6.3 Å) or during any of the 1L3S simulation. The two residues are hydrogen bonded in the initial 3HP6 and 1LV5 PDB structures (distance <4 Å), but the hydrogen bond is broken within first 5.0 ns of each simulation and does not reform. Note that this figure has been scaled to only the first 50 ns of simulation time to demonstrate the timing of the Tyr714-Glu658 hydrogen bond breaking.
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pcbi-1003961-g013: The distance between the side chain OH on Tyr714 and δ-C of Glu658 for the open (1L3S, red), ajar (3HP6, green), and closed (1LV5, blue) structured simulated using Desmond with the Charmm27 force field.Tyr714 and Glu658 are not hydrogen bonded in the 1L3S PDB (corresponding to a distance of 6.3 Å) or during any of the 1L3S simulation. The two residues are hydrogen bonded in the initial 3HP6 and 1LV5 PDB structures (distance <4 Å), but the hydrogen bond is broken within first 5.0 ns of each simulation and does not reform. Note that this figure has been scaled to only the first 50 ns of simulation time to demonstrate the timing of the Tyr714-Glu658 hydrogen bond breaking.

Mentions: The Tyr714/Glu658 motif of DNA polymerase plays a vital role in dNTP binding and stability of the ternary complex due to its position in the active site. The 1LV5 and 3HP6 crystal structure suggests a stabilizing hydrogen bond between the side chains of Tyr714 and Glu658 for the ternary complex in the closed and ajar conformations respectively, while no hydrogen bond is expected for the open, binary state based on the 1L3S crystal structure. Upon removing the dNTP in the 1LV5 and 3HP6 structures, the Tyr714-Glu658 hydrogen bond is broken quickly (<5 ns, see Figure 13), suggesting this is the initial step toward opening of the fingers domain. Furthermore, recent studies have suggested this hydrogen bond plays only a minor role in stability of the ternary complex [13], so it is not surprising the hydrogen bond is not present for long.


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

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

The distance between the side chain OH on Tyr714 and δ-C of Glu658 for the open (1L3S, red), ajar (3HP6, green), and closed (1LV5, blue) structured simulated using Desmond with the Charmm27 force field.Tyr714 and Glu658 are not hydrogen bonded in the 1L3S PDB (corresponding to a distance of 6.3 Å) or during any of the 1L3S simulation. The two residues are hydrogen bonded in the initial 3HP6 and 1LV5 PDB structures (distance <4 Å), but the hydrogen bond is broken within first 5.0 ns of each simulation and does not reform. Note that this figure has been scaled to only the first 50 ns of simulation time to demonstrate the timing of the Tyr714-Glu658 hydrogen bond breaking.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g013: The distance between the side chain OH on Tyr714 and δ-C of Glu658 for the open (1L3S, red), ajar (3HP6, green), and closed (1LV5, blue) structured simulated using Desmond with the Charmm27 force field.Tyr714 and Glu658 are not hydrogen bonded in the 1L3S PDB (corresponding to a distance of 6.3 Å) or during any of the 1L3S simulation. The two residues are hydrogen bonded in the initial 3HP6 and 1LV5 PDB structures (distance <4 Å), but the hydrogen bond is broken within first 5.0 ns of each simulation and does not reform. Note that this figure has been scaled to only the first 50 ns of simulation time to demonstrate the timing of the Tyr714-Glu658 hydrogen bond breaking.
Mentions: The Tyr714/Glu658 motif of DNA polymerase plays a vital role in dNTP binding and stability of the ternary complex due to its position in the active site. The 1LV5 and 3HP6 crystal structure suggests a stabilizing hydrogen bond between the side chains of Tyr714 and Glu658 for the ternary complex in the closed and ajar conformations respectively, while no hydrogen bond is expected for the open, binary state based on the 1L3S crystal structure. Upon removing the dNTP in the 1LV5 and 3HP6 structures, the Tyr714-Glu658 hydrogen bond is broken quickly (<5 ns, see Figure 13), suggesting this is the initial step toward opening of the fingers domain. Furthermore, recent studies have suggested this hydrogen bond plays only a minor role in stability of the ternary complex [13], so it is not surprising the hydrogen bond is not present for long.

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