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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

Bacillus DNA polymerase-DNA complexes before and after processive DNA synthesis in the crystal.The fingers subdomain is shown before (1L3S.pdb, cyan), and after the incorporation of 1 (1L3T.pdb, green), 2 (1L3U.pdb, magenta), 3 (1L5U.pdb, gray), and 6 (1L3V.pdb, yellow) nucleotides into the DNA (crystal structures from [7]).
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pcbi-1003961-g012: Bacillus DNA polymerase-DNA complexes before and after processive DNA synthesis in the crystal.The fingers subdomain is shown before (1L3S.pdb, cyan), and after the incorporation of 1 (1L3T.pdb, green), 2 (1L3U.pdb, magenta), 3 (1L5U.pdb, gray), and 6 (1L3V.pdb, yellow) nucleotides into the DNA (crystal structures from [7]).

Mentions: The dihedrals from the simulations appear to correlate well with the values from the existing crystal structures for each state (Figure 9). Of interest, though, is the observation that although the fingers domain appears fully open after 290 ns, the Gly711φ, Val713ψ, and Ile716φ all make substantial (≥20°) transitions between 600 and 725 ns producing structures in excellent agreement with the experimental values. The ∼60° rotation about the Ile716φ dihedral actually coincides with the movement of the template DNA base flipping out of the pre-insertion site and back into the active site (where it resided in the 1LV5 closed crystal structure). The rotations by Gly711φ and Val713ψ correspond to a rotation of the Tyr710χ1 dihedral so the tyrosine side chain is positioned for better π-stacking with the nucleotide of the template DNA base. The dynamical nature of this region of the O-helix is consistent with structural heterogeneity in crystal structures of open, binary complexes of Bacillus DNA polymerase before and after catalyzing DNA synthesis [7]. While the overall structure of the enzyme remains the same, the structure of the loop between the O and O1 helices (residues 714–717) flips back and forth between two states after each step of processive DNA synthesis in the crystal (Figure 12), suggesting this region near Val713 and Ile716 is flexible. Although the final orientation of Tyr710 and the template DNA base is not consistent with the original 1L3S open crystal structure, this movement hints at the fundamental dynamics of the DNA polymerase active site while in the open state. Based on these simulations, we can conclude that the template base entering the active site is energetically accessible while polymerase is in the open state. Currently, it is unknown where the template base recognizes an incoming dNTP, although it has been hypothesized the preliminary interactions occur outside the active site [47]. These simulations suggest that the template DNA base could enter the active site prior to dNTP binding and recognize the incoming base while already in the active site instead of outside the active site.


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

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

Bacillus DNA polymerase-DNA complexes before and after processive DNA synthesis in the crystal.The fingers subdomain is shown before (1L3S.pdb, cyan), and after the incorporation of 1 (1L3T.pdb, green), 2 (1L3U.pdb, magenta), 3 (1L5U.pdb, gray), and 6 (1L3V.pdb, yellow) nucleotides into the DNA (crystal structures from [7]).
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g012: Bacillus DNA polymerase-DNA complexes before and after processive DNA synthesis in the crystal.The fingers subdomain is shown before (1L3S.pdb, cyan), and after the incorporation of 1 (1L3T.pdb, green), 2 (1L3U.pdb, magenta), 3 (1L5U.pdb, gray), and 6 (1L3V.pdb, yellow) nucleotides into the DNA (crystal structures from [7]).
Mentions: The dihedrals from the simulations appear to correlate well with the values from the existing crystal structures for each state (Figure 9). Of interest, though, is the observation that although the fingers domain appears fully open after 290 ns, the Gly711φ, Val713ψ, and Ile716φ all make substantial (≥20°) transitions between 600 and 725 ns producing structures in excellent agreement with the experimental values. The ∼60° rotation about the Ile716φ dihedral actually coincides with the movement of the template DNA base flipping out of the pre-insertion site and back into the active site (where it resided in the 1LV5 closed crystal structure). The rotations by Gly711φ and Val713ψ correspond to a rotation of the Tyr710χ1 dihedral so the tyrosine side chain is positioned for better π-stacking with the nucleotide of the template DNA base. The dynamical nature of this region of the O-helix is consistent with structural heterogeneity in crystal structures of open, binary complexes of Bacillus DNA polymerase before and after catalyzing DNA synthesis [7]. While the overall structure of the enzyme remains the same, the structure of the loop between the O and O1 helices (residues 714–717) flips back and forth between two states after each step of processive DNA synthesis in the crystal (Figure 12), suggesting this region near Val713 and Ile716 is flexible. Although the final orientation of Tyr710 and the template DNA base is not consistent with the original 1L3S open crystal structure, this movement hints at the fundamental dynamics of the DNA polymerase active site while in the open state. Based on these simulations, we can conclude that the template base entering the active site is energetically accessible while polymerase is in the open state. Currently, it is unknown where the template base recognizes an incoming dNTP, although it has been hypothesized the preliminary interactions occur outside the active site [47]. These simulations suggest that the template DNA base could enter the active site prior to dNTP binding and recognize the incoming base while already in the active site instead of outside the active site.

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