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

Comparison of two different methods for measuring the opening/closing of the O-helix on DNA Polymerase I.A) The α-C distance between Arg629 and Pro699 shown in this manuscript compared to B) the angle between the α-C of Arg629, Gly711, and Asn700 used by Golosov et al. to determine the conformation of the O-helix and C) a plot of the RMSD of the fingers domain as a function of time in reference to the original crystal structure used to start each simulation. The distance, angle, and RMSD measurements are directly comparable, validating our use of the Arg629-Pro699 distance.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g003: Comparison of two different methods for measuring the opening/closing of the O-helix on DNA Polymerase I.A) The α-C distance between Arg629 and Pro699 shown in this manuscript compared to B) the angle between the α-C of Arg629, Gly711, and Asn700 used by Golosov et al. to determine the conformation of the O-helix and C) a plot of the RMSD of the fingers domain as a function of time in reference to the original crystal structure used to start each simulation. The distance, angle, and RMSD measurements are directly comparable, validating our use of the Arg629-Pro699 distance.

Mentions: To describe the conformation of the fingers domain at any given time, we measured the distance between the α-carbons of Pro699 at the end of the O-helix and Arg629 residue in the thumb domain of DNA polymerase (See Figure 2A). This single distance is able to successfully capture the movements of the fingers domain as well as an angle used in a publication by Golosov et al. [20] and a plot of the RMSD of the fingers domain as a function of time in reference to the original crystal structure used to start each simulation (Figure 3). The plot of the Pro699-Arg629 distance (Figure 2B) illustrates the dynamics of the fingers domain for each simulation using the Desmond MD package with the Charmm27 force field (See Figure 2C and 2D for corresponding plots with the Amber ff99SB force field). The 1L3S (open) simulation remains in the open conformation for the entire 500 ns trajectory. 3HP6 (ajar) begins in the ajar conformation, but very quickly (<5 ns) opens to a distance corresponding to the open conformation. Meanwhile, the fingers domain for the 1LV5 (closed) simulation is initially closed for more than 100 ns, but partially opens into a conformation similar to but distinct from the ajar shortly at ∼125 ns. The polymerase remains in this intermediate state for ∼50 ns before it returns the closed state for ∼100 ns duration and finally fully opens at ∼290 ns where it persists for the remainder of the simulation. The ability of the DNA polymerase fingers domain to clearly sample all three conformations (Figure 4 and Table 2) coincides with experimental evidence that suggests each state is thermodynamically accessible in the binary state [13].


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

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

Comparison of two different methods for measuring the opening/closing of the O-helix on DNA Polymerase I.A) The α-C distance between Arg629 and Pro699 shown in this manuscript compared to B) the angle between the α-C of Arg629, Gly711, and Asn700 used by Golosov et al. to determine the conformation of the O-helix and C) a plot of the RMSD of the fingers domain as a function of time in reference to the original crystal structure used to start each simulation. The distance, angle, and RMSD measurements are directly comparable, validating our use of the Arg629-Pro699 distance.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g003: Comparison of two different methods for measuring the opening/closing of the O-helix on DNA Polymerase I.A) The α-C distance between Arg629 and Pro699 shown in this manuscript compared to B) the angle between the α-C of Arg629, Gly711, and Asn700 used by Golosov et al. to determine the conformation of the O-helix and C) a plot of the RMSD of the fingers domain as a function of time in reference to the original crystal structure used to start each simulation. The distance, angle, and RMSD measurements are directly comparable, validating our use of the Arg629-Pro699 distance.
Mentions: To describe the conformation of the fingers domain at any given time, we measured the distance between the α-carbons of Pro699 at the end of the O-helix and Arg629 residue in the thumb domain of DNA polymerase (See Figure 2A). This single distance is able to successfully capture the movements of the fingers domain as well as an angle used in a publication by Golosov et al. [20] and a plot of the RMSD of the fingers domain as a function of time in reference to the original crystal structure used to start each simulation (Figure 3). The plot of the Pro699-Arg629 distance (Figure 2B) illustrates the dynamics of the fingers domain for each simulation using the Desmond MD package with the Charmm27 force field (See Figure 2C and 2D for corresponding plots with the Amber ff99SB force field). The 1L3S (open) simulation remains in the open conformation for the entire 500 ns trajectory. 3HP6 (ajar) begins in the ajar conformation, but very quickly (<5 ns) opens to a distance corresponding to the open conformation. Meanwhile, the fingers domain for the 1LV5 (closed) simulation is initially closed for more than 100 ns, but partially opens into a conformation similar to but distinct from the ajar shortly at ∼125 ns. The polymerase remains in this intermediate state for ∼50 ns before it returns the closed state for ∼100 ns duration and finally fully opens at ∼290 ns where it persists for the remainder of the simulation. The ability of the DNA polymerase fingers domain to clearly sample all three conformations (Figure 4 and Table 2) coincides with experimental evidence that suggests each state is thermodynamically accessible in the binary state [13].

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