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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 structure of DNA polymerase I (PDB code 1TAU) bound to DNA (orange ribbons) is shown on the left depicting the 5β€²β†’3β€² exonuclease (purple surface), 3β€²β†’5β€² exonuclease (yellow surface), and polymerase (white surface) domains (left).The inset on the right shows a close-up of the mobile fingers subdomain (light green) of Bacillus stearothermophilus DNA polymerase I, with the open (red), ajar (blue), and closed (yellow) conformations of the O-helix shown in relation to the dNTP substrate (sticks) and Mg2+ ion (pink).
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pcbi-1003961-g001: The structure of DNA polymerase I (PDB code 1TAU) bound to DNA (orange ribbons) is shown on the left depicting the 5β€²β†’3β€² exonuclease (purple surface), 3β€²β†’5β€² exonuclease (yellow surface), and polymerase (white surface) domains (left).The inset on the right shows a close-up of the mobile fingers subdomain (light green) of Bacillus stearothermophilus DNA polymerase I, with the open (red), ajar (blue), and closed (yellow) conformations of the O-helix shown in relation to the dNTP substrate (sticks) and Mg2+ ion (pink).

Mentions: DNA polymerase I consists of 5β€²β†’3β€² exonuclease, 3β€²β†’5β€² exonuclease, and polymerase domains (Figure 1). The Klenow fragment of DNA polymerase I is an N-terminal deletion of the dispensible 5β€²β†’3β€² exonuclease domain [2]. Within the Klenow fragment, the polymerase domain resembles the shape of a human hand with a thumb subdomain that grasps the DNA, a palm subdomain that contains the active site, and a mobile fingers subdomain involved in dNTP binding [3], [4]. The thumb (residues 496–595), palm (residue 617–655 and 830–869), and fingers (residues 656–818) subdomains of DNA polymerase were named based on their positioning around the bound DNA as observed in crystal structures. The fingers domain consists of multiple Ξ±-helices highlighted by the O-helix that directly interacts with the dNTP substrate upon binding. X-ray crystallography and solution kinetics studies have observed the fingers subdomain in three distinct conformations (Figure 1), which are dependent on the presence or absence of a dNTP in the active site [5], [6], [7], [8]. The fingers subdomain primarily resides in an β€œopen” conformation with no dNTP bound (binary state) to the polymerase. Upon binding of a dNTP (ternary state) that forms a proper Watson-Crick base pair with the template strand, the fingers domain enters a β€œclosed” conformation that helps position the substrate in the active site during catalysis [5], [9]. And recently a third β€œajar” conformation was discovered that places the fingers domain in a semi-open state when a dNTP binds that forms a mismatch with the template strand [10].


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

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

The structure of DNA polymerase I (PDB code 1TAU) bound to DNA (orange ribbons) is shown on the left depicting the 5β€²β†’3β€² exonuclease (purple surface), 3β€²β†’5β€² exonuclease (yellow surface), and polymerase (white surface) domains (left).The inset on the right shows a close-up of the mobile fingers subdomain (light green) of Bacillus stearothermophilus DNA polymerase I, with the open (red), ajar (blue), and closed (yellow) conformations of the O-helix shown in relation to the dNTP substrate (sticks) and Mg2+ ion (pink).
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003961-g001: The structure of DNA polymerase I (PDB code 1TAU) bound to DNA (orange ribbons) is shown on the left depicting the 5β€²β†’3β€² exonuclease (purple surface), 3β€²β†’5β€² exonuclease (yellow surface), and polymerase (white surface) domains (left).The inset on the right shows a close-up of the mobile fingers subdomain (light green) of Bacillus stearothermophilus DNA polymerase I, with the open (red), ajar (blue), and closed (yellow) conformations of the O-helix shown in relation to the dNTP substrate (sticks) and Mg2+ ion (pink).
Mentions: DNA polymerase I consists of 5β€²β†’3β€² exonuclease, 3β€²β†’5β€² exonuclease, and polymerase domains (Figure 1). The Klenow fragment of DNA polymerase I is an N-terminal deletion of the dispensible 5β€²β†’3β€² exonuclease domain [2]. Within the Klenow fragment, the polymerase domain resembles the shape of a human hand with a thumb subdomain that grasps the DNA, a palm subdomain that contains the active site, and a mobile fingers subdomain involved in dNTP binding [3], [4]. The thumb (residues 496–595), palm (residue 617–655 and 830–869), and fingers (residues 656–818) subdomains of DNA polymerase were named based on their positioning around the bound DNA as observed in crystal structures. The fingers domain consists of multiple Ξ±-helices highlighted by the O-helix that directly interacts with the dNTP substrate upon binding. X-ray crystallography and solution kinetics studies have observed the fingers subdomain in three distinct conformations (Figure 1), which are dependent on the presence or absence of a dNTP in the active site [5], [6], [7], [8]. The fingers subdomain primarily resides in an β€œopen” conformation with no dNTP bound (binary state) to the polymerase. Upon binding of a dNTP (ternary state) that forms a proper Watson-Crick base pair with the template strand, the fingers domain enters a β€œclosed” conformation that helps position the substrate in the active site during catalysis [5], [9]. And recently a third β€œajar” conformation was discovered that places the fingers domain in a semi-open state when a dNTP binds that forms a mismatch with the template strand [10].

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