Limits...
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.

Show MeSH

Related in: MedlinePlus

The proposed opening mechanism for the fingers domain for DNA polymerase I.The secondary structure of the relevant polymerase residues including the O-helix are shown in yellow ribbons, while the DNA is shown in orange. The key event in each image is circled. A) The X-ray crystal structure of PDB 1LV5. B). The intermediate state showing the breaking of the Tyr714-Glu658 hydrogen bond, and the formation of the salt bridge between Arg703 and Glu562. C) Depiction of the breaking of the Arg703-Glu562 salt bridge, which is quickly followed by D) the rotation of the N-β-glycosyl bond of the template nucleotide allowing Tyr714 to replace the base in the active site, and resulting in the fully open conformation of DNA polymerase I. Simulation times and O-helix distances correspond to the 1LV5 simulation performed using Desmond and the Charmm27 force field.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256020&req=5

pcbi-1003961-g005: The proposed opening mechanism for the fingers domain for DNA polymerase I.The secondary structure of the relevant polymerase residues including the O-helix are shown in yellow ribbons, while the DNA is shown in orange. The key event in each image is circled. A) The X-ray crystal structure of PDB 1LV5. B). The intermediate state showing the breaking of the Tyr714-Glu658 hydrogen bond, and the formation of the salt bridge between Arg703 and Glu562. C) Depiction of the breaking of the Arg703-Glu562 salt bridge, which is quickly followed by D) the rotation of the N-β-glycosyl bond of the template nucleotide allowing Tyr714 to replace the base in the active site, and resulting in the fully open conformation of DNA polymerase I. Simulation times and O-helix distances correspond to the 1LV5 simulation performed using Desmond and the Charmm27 force field.

Mentions: The detailed motions of DNA polymerase during the transition from closed to open observed with Charmm27 are shown in Figure 5. The 1LV5 (closed) crystal structure showed the existence of a hydrogen bond between the side chains of Tyr714 and Glu658 in the ternary (enzyme-DNA-dNTP) state (Figure 5A), but after removing the dNTP and simulating the closed structure this hydrogen bond is quickly broken (Figure 5B). This allows Tyr714 to move toward the template DNA base and causes the O-helix to open slightly (∼1.5 Å). The O-helix is held in this intermediate position by a salt bridge between Arg703 and Glu562 of the thumb subdomain, while Tyr714 and the guanine in the DNA template continue to compete for the insertion site (Figure 5B). Shortly after the Arg703-Glu562 salt bridge interaction is broken (Figure 5C) the O-helix opens further, pulling Tyr714 into the insertion site, inducing a rotation of the N-β-glycosyl bond of the template nucleotide, and moving the template base out of the active site (Figure 5D). The precise ordering of the last two steps is not fully established because different force fields yielded different results. The Charmm27 force field predicted the salt bridge to break prior to the N-β-glycosyl bond rotation, while the two Amber ff99SB force field simulations suggested the opposite ordering. However, in all three simulations the steps succeeding the intermediate conformation (Figure 5B) appear closely correlated implying that they may occur nearly simultaneously.


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

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

The proposed opening mechanism for the fingers domain for DNA polymerase I.The secondary structure of the relevant polymerase residues including the O-helix are shown in yellow ribbons, while the DNA is shown in orange. The key event in each image is circled. A) The X-ray crystal structure of PDB 1LV5. B). The intermediate state showing the breaking of the Tyr714-Glu658 hydrogen bond, and the formation of the salt bridge between Arg703 and Glu562. C) Depiction of the breaking of the Arg703-Glu562 salt bridge, which is quickly followed by D) the rotation of the N-β-glycosyl bond of the template nucleotide allowing Tyr714 to replace the base in the active site, and resulting in the fully open conformation of DNA polymerase I. Simulation times and O-helix distances correspond to the 1LV5 simulation performed using Desmond and the Charmm27 force field.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g005: The proposed opening mechanism for the fingers domain for DNA polymerase I.The secondary structure of the relevant polymerase residues including the O-helix are shown in yellow ribbons, while the DNA is shown in orange. The key event in each image is circled. A) The X-ray crystal structure of PDB 1LV5. B). The intermediate state showing the breaking of the Tyr714-Glu658 hydrogen bond, and the formation of the salt bridge between Arg703 and Glu562. C) Depiction of the breaking of the Arg703-Glu562 salt bridge, which is quickly followed by D) the rotation of the N-β-glycosyl bond of the template nucleotide allowing Tyr714 to replace the base in the active site, and resulting in the fully open conformation of DNA polymerase I. Simulation times and O-helix distances correspond to the 1LV5 simulation performed using Desmond and the Charmm27 force field.
Mentions: The detailed motions of DNA polymerase during the transition from closed to open observed with Charmm27 are shown in Figure 5. The 1LV5 (closed) crystal structure showed the existence of a hydrogen bond between the side chains of Tyr714 and Glu658 in the ternary (enzyme-DNA-dNTP) state (Figure 5A), but after removing the dNTP and simulating the closed structure this hydrogen bond is quickly broken (Figure 5B). This allows Tyr714 to move toward the template DNA base and causes the O-helix to open slightly (∼1.5 Å). The O-helix is held in this intermediate position by a salt bridge between Arg703 and Glu562 of the thumb subdomain, while Tyr714 and the guanine in the DNA template continue to compete for the insertion site (Figure 5B). Shortly after the Arg703-Glu562 salt bridge interaction is broken (Figure 5C) the O-helix opens further, pulling Tyr714 into the insertion site, inducing a rotation of the N-β-glycosyl bond of the template nucleotide, and moving the template base out of the active site (Figure 5D). The precise ordering of the last two steps is not fully established because different force fields yielded different results. The Charmm27 force field predicted the salt bridge to break prior to the N-β-glycosyl bond rotation, while the two Amber ff99SB force field simulations suggested the opposite ordering. However, in all three simulations the steps succeeding the intermediate conformation (Figure 5B) appear closely correlated implying that they may occur nearly simultaneously.

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