Figure 1.
The structure of DNA polymerase I (PDB code 1TAU) bound to DNA (orange ribbons) is shown on the left depicting the 5′→3′ exonuclease (purple surface), 3′→5′ 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).
Table 1.
Summary of simulations performed on B. stearothermophilus DNA polymerase I in the binary complex.
Figure 2.
The dynamics of the fingers domain illustrated by A) the α-C distance between Pro699-Arg629 of DNA polymerase simulated with B) Desmond using the Charmm27 force field, C) Desmond using the Amber ff99Sb force field, and D) Amber using the Amber ff99SB force field.
The simulations are named according to their PDB codes and initial starting conformations where 1L3S (red) began in the open conformation, 3HP6 (green) began in the ajar conformation, and 1LV5 (blue) was started from the closed conformation. The black horizontal lines represent the distances corresponding to the three major observed conformations: open (O), closed (C), and the newly observed intermediate (I) state. The inset in B) displays a close-up of the distances from 0–5 ns (highlighted by the vertical orange dashed line) to more clearly depict the relatively quick opening of the simulation started from the 3HP6 ajar conformation.
Figure 3.
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.
Figure 4.
A histogram of the Pro699-Arg629 α-C distances for the 1.0 µs simulation (10,000 frames total) of PDB 1LV5 performed with Desmond using the Charmm27 force field.
The histogram clearly shows three distinct conformations were sampled: closed (C), open (O), and the newly observed intermediate (I) state.
Table 2.
An analysis of the percent of time that each DNA Polymerase simulation spent in the closed, intermediate, and open conformations.
Figure 5.
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.
Figure 6.
The O-helix distance as measured by the α-C distance between Arg629 and Pro699 depicting the opening of the fingers domain for the wild-type 1LV5 (blue) and R703A mutant (purple) simulated using Desmond and the Charmm27 force field.
The plot shows the mutant reaching the open conformation in <50 ns, while the wild-type does not open fully until ∼290 ns.
Figure 7.
An overlay of the DNA polymerase fingers subdomain for the 3HP6 crystal structure (blue ribbons) and the observed 1LV5 intermediate state (yellow ribbons) characterized by the Arg703-Glu562 salt bridge.
Although the end of the O-helix for both structures is near the same location, the α-helices in the fingers domain are clearly different resulting in the 4.3 Å RMSD between the 1LV5 intermediate and the 3HP6 crystal structure.
Figure 8.
The proposed pathway for opening and closing of DNA polymerase I in the presence and absence of dNTP.
In the binary complex (blue), the polymerase transitions through the intermediate observed in this study (EI•DNA), while the ternary complex (yellow) transition is a separate, partially-closed conformation (EPC•DNA•dNTP) on its way to the closed conformation. This pathway depicts the enzyme in two different “ajar” conformation (EI or EPC) determined by the presence or absence of dNTP in the active site.
Figure 9.
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.
Figure 10.
A close view of the exact orientations of the important dihedrals from the closed (1LV5) crystal structure for A) Asp680φ (purple) and Ile716φ (blue) and B) Gly711φ (pink) and Val713ψ (green).
The fingers domain is shown in an ice blue cartoon representation, while the O-helix is shown in yellow cartoon.
Figure 11.
The relative dihedral values as a function of simulation time for the backbone torsions determined to be key to the fingers domain of DNA polymerase transitioning from the closed to open conformations—A) Asp680φ, B) Gly711φ, C) Val713ψ, and D) Ile716φ.
Solid black lines indicate the values of each dihedral in the pertinent crystal structures, where 1L3S is in the open state and 3HP6 is the ajar conformation.
Figure 12.
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]).
Figure 13.
The distance between the side chain OH on Tyr714 and δ-C of Glu658 for the open (1L3S, red), ajar (3HP6, green), and closed (1LV5, blue) structured simulated using Desmond with the Charmm27 force field.
Tyr714 and Glu658 are not hydrogen bonded in the 1L3S PDB (corresponding to a distance of 6.3 Å) or during any of the 1L3S simulation. The two residues are hydrogen bonded in the initial 3HP6 and 1LV5 PDB structures (distance <4 Å), but the hydrogen bond is broken within first 5.0 ns of each simulation and does not reform. Note that this figure has been scaled to only the first 50 ns of simulation time to demonstrate the timing of the Tyr714-Glu658 hydrogen bond breaking.
Figure 14.
The relative degree of rotation of the N-β-glycosyl bond for the template nucleotide in the 1LV5 simulation performed with Desmond using Charmm27 force field.
The torsion corresponds to an angle of roughly −90° when the nucleotide is in the active site and then changes to ∼0° when the N-β-glycosyl bond rotates moving the template nucleotide out of the active site entirely. Representative conformations of the nucleotide are shown at 130 ns and 400 ns to show the rotation. The torsion being measured is defined in the bottom right of the figure.