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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 dihedral values as a function of simulation time for the backbone torsions determined to be key to the fingers domain of DNA polymerase transitioning from the closed to open conformations—A) Asp680φ, B) Gly711φ, C) Val713ψ, and D) Ile716φ.Solid black lines indicate the values of each dihedral in the pertinent crystal structures, where 1L3S is in the open state and 3HP6 is the ajar conformation.
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pcbi-1003961-g011: The relative dihedral values as a function of simulation time for the backbone torsions determined to be key to the fingers domain of DNA polymerase transitioning from the closed to open conformations—A) Asp680φ, B) Gly711φ, C) Val713ψ, and D) Ile716φ.Solid black lines indicate the values of each dihedral in the pertinent crystal structures, where 1L3S is in the open state and 3HP6 is the ajar conformation.

Mentions: Close examination of the dihedral values as the simulation progresses (Figure 11) shows the ordering and impact of each dihedral. The transition from the closed state (Figure 9A) to the intermediate (Figure 9B) is initiated by the ∼30° rotation of the Asp680φ dihedral at ∼100 ns (Figure 11A), which results in a large-scale movement of the N-helix in the fingers domain. Subsequently, the Gly711φ and Val713ψ dihedrals rotate by ∼20° and ∼35° (Figure 11B–C), respectively, creating a bend in the O-helix (Figure 9D). In the Desmond/Charmm27 simulation the fingers domain transitions back into the closed conformation after ∼170 ns. Between 280–290 ns, the fingers domain undergoes two major dihedral rotations to complete the transition to the open conformation (Figure 9C). Once again, the process is initiated by the rotation about the Asp680φ dihedral (lowering the N-helix), followed shortly by a ∼60° rotation of the Ile716φ dihedral (Figure 11D). In this case, the rotation of the Asp680φ dihedral is enough to overcome the barrier necessary to rotate the Ile716φ dihedral and reach the fully open state.


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

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

The relative dihedral values as a function of simulation time for the backbone torsions determined to be key to the fingers domain of DNA polymerase transitioning from the closed to open conformations—A) Asp680φ, B) Gly711φ, C) Val713ψ, and D) Ile716φ.Solid black lines indicate the values of each dihedral in the pertinent crystal structures, where 1L3S is in the open state and 3HP6 is the ajar conformation.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g011: The relative dihedral values as a function of simulation time for the backbone torsions determined to be key to the fingers domain of DNA polymerase transitioning from the closed to open conformations—A) Asp680φ, B) Gly711φ, C) Val713ψ, and D) Ile716φ.Solid black lines indicate the values of each dihedral in the pertinent crystal structures, where 1L3S is in the open state and 3HP6 is the ajar conformation.
Mentions: Close examination of the dihedral values as the simulation progresses (Figure 11) shows the ordering and impact of each dihedral. The transition from the closed state (Figure 9A) to the intermediate (Figure 9B) is initiated by the ∼30° rotation of the Asp680φ dihedral at ∼100 ns (Figure 11A), which results in a large-scale movement of the N-helix in the fingers domain. Subsequently, the Gly711φ and Val713ψ dihedrals rotate by ∼20° and ∼35° (Figure 11B–C), respectively, creating a bend in the O-helix (Figure 9D). In the Desmond/Charmm27 simulation the fingers domain transitions back into the closed conformation after ∼170 ns. Between 280–290 ns, the fingers domain undergoes two major dihedral rotations to complete the transition to the open conformation (Figure 9C). Once again, the process is initiated by the rotation about the Asp680φ dihedral (lowering the N-helix), followed shortly by a ∼60° rotation of the Ile716φ dihedral (Figure 11D). In this case, the rotation of the Asp680φ dihedral is enough to overcome the barrier necessary to rotate the Ile716φ dihedral and reach the fully open state.

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