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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 relative degree of rotation of the N-β-glycosyl bond for the template nucleotide in the 1LV5 simulation performed with Desmond using Charmm27 force field.The torsion corresponds to an angle of roughly −90° when the nucleotide is in the active site and then changes to ∼0° when the N-β-glycosyl bond rotates moving the template nucleotide out of the active site entirely. Representative conformations of the nucleotide are shown at 130 ns and 400 ns to show the rotation. The torsion being measured is defined in the bottom right of the figure.
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pcbi-1003961-g014: The relative degree of rotation of the N-β-glycosyl bond for the template nucleotide in the 1LV5 simulation performed with Desmond using Charmm27 force field.The torsion corresponds to an angle of roughly −90° when the nucleotide is in the active site and then changes to ∼0° when the N-β-glycosyl bond rotates moving the template nucleotide out of the active site entirely. Representative conformations of the nucleotide are shown at 130 ns and 400 ns to show the rotation. The torsion being measured is defined in the bottom right of the figure.

Mentions: According to crystal structures, the position of Tyr714 in the active site changes substantially based on the state of DNA polymerase. In the ternary complex with the fingers domain closed (1LV5) or ajar (3HP6), Tyr714 is positioned next to the template base and hydrogen bonded to Glu658. In the binary state (1L3S), Tyr714 moves into the active site, taking the place of the template nucleotide. In all of the 1LV5 and 3HP6 simulations, after early disruption of the Tyr714-Glu568 hydrogen bond, Tyr714 becomes more mobile creating van der Waals contacts with the template base. Eventually, these clashes result in a ∼90° rotation of the N-β-glycosyl bond (Figure 14) of the nucleotide moving the nucleotide out of the active site entirely, while Tyr714 replaces the nucleotide in the active site and begins π-stacking with the n-1 base on the template strand, as the 1L3S (open) PDB structure suggested. For the 1LV5 Desmond simulation performed with the Charmm27 force field, this transition occurred at ∼300 ns and coincides with the opening of the fingers domain. By contrast, the same transition occurs after only 22 ns in the 3HP6 simulation. Although the transitions occurred later using the Amber force field, the relative timing between the N-β-glycosyl bond rotations for the 1LV5 and 3HP6 simulations remained consistent with the Charmm27 force field.


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

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

The relative degree of rotation of the N-β-glycosyl bond for the template nucleotide in the 1LV5 simulation performed with Desmond using Charmm27 force field.The torsion corresponds to an angle of roughly −90° when the nucleotide is in the active site and then changes to ∼0° when the N-β-glycosyl bond rotates moving the template nucleotide out of the active site entirely. Representative conformations of the nucleotide are shown at 130 ns and 400 ns to show the rotation. The torsion being measured is defined in the bottom right of the figure.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g014: The relative degree of rotation of the N-β-glycosyl bond for the template nucleotide in the 1LV5 simulation performed with Desmond using Charmm27 force field.The torsion corresponds to an angle of roughly −90° when the nucleotide is in the active site and then changes to ∼0° when the N-β-glycosyl bond rotates moving the template nucleotide out of the active site entirely. Representative conformations of the nucleotide are shown at 130 ns and 400 ns to show the rotation. The torsion being measured is defined in the bottom right of the figure.
Mentions: According to crystal structures, the position of Tyr714 in the active site changes substantially based on the state of DNA polymerase. In the ternary complex with the fingers domain closed (1LV5) or ajar (3HP6), Tyr714 is positioned next to the template base and hydrogen bonded to Glu658. In the binary state (1L3S), Tyr714 moves into the active site, taking the place of the template nucleotide. In all of the 1LV5 and 3HP6 simulations, after early disruption of the Tyr714-Glu568 hydrogen bond, Tyr714 becomes more mobile creating van der Waals contacts with the template base. Eventually, these clashes result in a ∼90° rotation of the N-β-glycosyl bond (Figure 14) of the nucleotide moving the nucleotide out of the active site entirely, while Tyr714 replaces the nucleotide in the active site and begins π-stacking with the n-1 base on the template strand, as the 1L3S (open) PDB structure suggested. For the 1LV5 Desmond simulation performed with the Charmm27 force field, this transition occurred at ∼300 ns and coincides with the opening of the fingers domain. By contrast, the same transition occurs after only 22 ns in the 3HP6 simulation. Although the transitions occurred later using the Amber force field, the relative timing between the N-β-glycosyl bond rotations for the 1LV5 and 3HP6 simulations remained consistent with the Charmm27 force field.

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