In silico evidence for DNA polymerase-beta's substrate-induced conformational change

Abstract

Structural information for mammalian DNA pol-beta combined with molecular and essential dynamics studies have provided atomistically detailed views of functionally important conformational rearrangements that occur during DNA repair and replication. This conformational closing before the chemical reaction is explored in this work as a function of the bound substrate. Anchors for our study are available in crystallographic structures of the DNA pol-beta in "open" (polymerase bound to gapped DNA) and "closed" (polymerase bound to gapped DNA and substrate, dCTP) forms; these different states have long been used to deduce that a large-scale conformational change may help the polymerase choose the correct nucleotide, and hence monitor DNA synthesis fidelity, through an "induced-fit" mechanism. However, the existence of open states with bound substrate and closed states without substrates suggest that substrate-induced conformational closing may be more subtle. Our dynamics simulations of two pol-beta/DNA systems (with/without substrates at the active site) reveal the large-scale closing motions of the thumb and 8-kDa subdomains in the presence of the correct substrate--leading to nearly perfect rearrangement of residues in the active site for the subsequent chemical step of nucleotidyl transfer--in contrast to an opening trend when the substrate is absent, leading to complete disassembly of the active site residues. These studies thus provide in silico evidence for the substrate-induced conformational rearrangements, as widely assumed based on a variety of crystallographic open and closed complexes. Further details gleaned from essential dynamics analyses clarify functionally relevant global motions of the polymerase-beta/DNA complex as required to prepare the system for the chemical reaction of nucleotide extension.

FIGURE 1

General pathway for nucleotide insertion by DNA pol-β (a) and corresponding crystal open (b) and closed (c) conformations of pol-β/DNA complex. E, DNA polymerase; dNTP, 2′-deoxyribonucleoside 5′-triphosphate; PP_i, pyrophosphate; DNA_n/DNA_n+1, DNA before/after nucleotide incorporation to DNA primer. T6 is the template residue (G) corresponding to the incoming dCTP.

FIGURE 2

C_α traces of superimposed pol-β/DNA complex with dCTP (a1) and without dCTP (b1) for the intermediate starting structure (yellow), crystal closed (red), and crystal open (green) and the trajectory final structures (blue). Notable are the residue motions in the thumb subdomain and the 8-kDa domain. The positions of α-helix N in the simulated systems are compared to the crystal structures and shown from two points of view in pabels a2 and a3, and panels b2 and b3.

FIGURE 3

Evolution of the root-mean-square deviations (RMSD) of the C_α residues in α-helix N of the thumb subdomain in the simulated structure with respect to the crystal open (green) and crystal closed structures (red); (a) simulated closing of pol-β with substrate and (b) simulated opening of pol-β without substrate in the binding site after removing the shift distances.

FIGURE 4

Radius of gyration (R_g) for all C_α atoms (a) simulated closing of pol-β with substrate (b) simulated opening of pol-β without substrate in the binding site.

FIGURE 5

Positions of key residues Tyr-296, Arg-258, Asp-192, and Phe-272 in the 10-ns simulated (blue), crystal closed (red), crystal open (green), and starting intermediate (yellow) structures; (a) trajectory with substrate and (b) without substrate.

FIGURE 6

Coordination sphere of catalytic (Mg²⁺) and nucleotide (Mg²⁺) binding magnesium ions in the pol-β/DNA complex with bound dCTP substrate after 10 ns. All the distances within 2 Å are depicted by white dotted lines. WAT2 is the crystallographically observed water. The dCTP:Pα-P10:O3′ distance crucial for nucleotidyl transfer reaction is shown in a green dotted line.

FIGURE 7

Results of the essential dynamics analysis of pol-β with substrate. (a) Contribution of each C_α atom to the motions along the first five normalized eigenvectors. (b) Time evolution of projection of these eigenvectors on the dynamics trajectory. (c) Corresponding probability distributions together with fitted Gaussian distributions.

FIGURE 8

Results of the essential dynamics analysis of pol-β without substrate. (a) Contribution of each C_α atom to the motions along the first five normalized eigenvectors. (b) Time evolution of projection of these eigenvectors on the dynamics trajectory. (c) Corresponding probability distributions together with fitted Gaussian distributions.

FIGURE 9

Twenty-five frames taken at equally spaced intervals from the motions along the first three eigenvectors of (a) pol-β/DNA with dCTP and (b) pol-β/DNA without substrate in the binding site. Frames correspond to displacements between the minimum and maximum displacement corresponding to eigenvalues. Different subdomains of polymerase are color coded: thumb (yellow), palm (green), fingers (light blue), and 8-kDa (mauve). The loop regions showing persistent movement are labeled as L1 (residues 200–210), L2 (residues 242–250), and L3 (residues 302–310).