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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

A depiction of the residues with backbone dihedrals—Asp680φ (purple), Gly711φ (pink), Val713ψ (green), and Ile716φ (blue)—identified as important in the finger domain opening process of DNA polymerase.The fingers domain is shown in an ice blue cartoon representation, while the O-helix is shown in yellow cartoon. The times within the black arrows between panels indicate the transition times between the conformations. A) Conformation of the fingers domain in the 1LV5 crystal structure (closed) prior to running MD. B) Representative conformation of the intermediate state observed from ∼100–170 ns (139 ns shown) of simulation time. The red arrow indicates the large-scale motion of the N-helix due to a rotation about the Asp680φ dihedral. C) Representative conformation of the open state observed from ∼290–1000 ns (500 ns shown) of MD caused by a rotation of the Asp680φ and Ile716φ dihedrals. D) A side view of the intermediate state at 139 ns depicting the bend in the O-helix caused by rotations of the Gly711φ and Val713ψ dihedrals.
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pcbi-1003961-g009: A depiction of the residues with backbone dihedrals—Asp680φ (purple), Gly711φ (pink), Val713ψ (green), and Ile716φ (blue)—identified as important in the finger domain opening process of DNA polymerase.The fingers domain is shown in an ice blue cartoon representation, while the O-helix is shown in yellow cartoon. The times within the black arrows between panels indicate the transition times between the conformations. A) Conformation of the fingers domain in the 1LV5 crystal structure (closed) prior to running MD. B) Representative conformation of the intermediate state observed from ∼100–170 ns (139 ns shown) of simulation time. The red arrow indicates the large-scale motion of the N-helix due to a rotation about the Asp680φ dihedral. C) Representative conformation of the open state observed from ∼290–1000 ns (500 ns shown) of MD caused by a rotation of the Asp680φ and Ile716φ dihedrals. D) A side view of the intermediate state at 139 ns depicting the bend in the O-helix caused by rotations of the Gly711φ and Val713ψ dihedrals.

Mentions: Each backbone dihedral in the fingers subdomain of the simulation started from the closed conformation (1LV5) was compared to the corresponding open (1L3S), ajar (3HP6), and closed (1LV5) crystal structure values. This investigation revealed four specific backbone torsions important for opening of the fingers domain—Asp680φ, Gly711φ, Val713ψ, and Ile716φ (Figure 9). These dihedrals were identified because each dihedral rotation corresponds to a significant change in the structure of the fingers domain involved in converting between the open, ajar, and closed conformations. The original rotation of each dihedral in the closed (1LV5) crystal structure is shown in Figure 10. In the open crystal structure (1L3S) the Asp680φ, Gly711φ, Val713ψ, and Ile716φ dihedrals have values of −74.5°, −61.3°, −33.8°, and −141.7°, respectively. According to the ajar crystal structure, the Gly711φ and Val713ψ dihedrals rotate by ∼11° and ∼7°, respectively, during an ajar-to-open transition. Meanwhile, the Asp680φ and Ile716φ values undergo significant (∼25° and ∼60°, respectively) transitions themselves between the closed (1LV5) and open (1L3S) crystal structures. These changes were all observed during our simulation that began in the closed and transitioned to the fully open conformation.


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

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

A depiction of the residues with backbone dihedrals—Asp680φ (purple), Gly711φ (pink), Val713ψ (green), and Ile716φ (blue)—identified as important in the finger domain opening process of DNA polymerase.The fingers domain is shown in an ice blue cartoon representation, while the O-helix is shown in yellow cartoon. The times within the black arrows between panels indicate the transition times between the conformations. A) Conformation of the fingers domain in the 1LV5 crystal structure (closed) prior to running MD. B) Representative conformation of the intermediate state observed from ∼100–170 ns (139 ns shown) of simulation time. The red arrow indicates the large-scale motion of the N-helix due to a rotation about the Asp680φ dihedral. C) Representative conformation of the open state observed from ∼290–1000 ns (500 ns shown) of MD caused by a rotation of the Asp680φ and Ile716φ dihedrals. D) A side view of the intermediate state at 139 ns depicting the bend in the O-helix caused by rotations of the Gly711φ and Val713ψ dihedrals.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g009: A depiction of the residues with backbone dihedrals—Asp680φ (purple), Gly711φ (pink), Val713ψ (green), and Ile716φ (blue)—identified as important in the finger domain opening process of DNA polymerase.The fingers domain is shown in an ice blue cartoon representation, while the O-helix is shown in yellow cartoon. The times within the black arrows between panels indicate the transition times between the conformations. A) Conformation of the fingers domain in the 1LV5 crystal structure (closed) prior to running MD. B) Representative conformation of the intermediate state observed from ∼100–170 ns (139 ns shown) of simulation time. The red arrow indicates the large-scale motion of the N-helix due to a rotation about the Asp680φ dihedral. C) Representative conformation of the open state observed from ∼290–1000 ns (500 ns shown) of MD caused by a rotation of the Asp680φ and Ile716φ dihedrals. D) A side view of the intermediate state at 139 ns depicting the bend in the O-helix caused by rotations of the Gly711φ and Val713ψ dihedrals.
Mentions: Each backbone dihedral in the fingers subdomain of the simulation started from the closed conformation (1LV5) was compared to the corresponding open (1L3S), ajar (3HP6), and closed (1LV5) crystal structure values. This investigation revealed four specific backbone torsions important for opening of the fingers domain—Asp680φ, Gly711φ, Val713ψ, and Ile716φ (Figure 9). These dihedrals were identified because each dihedral rotation corresponds to a significant change in the structure of the fingers domain involved in converting between the open, ajar, and closed conformations. The original rotation of each dihedral in the closed (1LV5) crystal structure is shown in Figure 10. In the open crystal structure (1L3S) the Asp680φ, Gly711φ, Val713ψ, and Ile716φ dihedrals have values of −74.5°, −61.3°, −33.8°, and −141.7°, respectively. According to the ajar crystal structure, the Gly711φ and Val713ψ dihedrals rotate by ∼11° and ∼7°, respectively, during an ajar-to-open transition. Meanwhile, the Asp680φ and Ile716φ values undergo significant (∼25° and ∼60°, respectively) transitions themselves between the closed (1LV5) and open (1L3S) crystal structures. These changes were all observed during our simulation that began in the closed and transitioned to the fully open conformation.

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