History of DNA polymerase β X-ray crystallography

Abstract

DNA polymerase β (Pol β) is an essential mammalian enzyme involved in the repair of DNA damage during the base excision repair (BER) pathway. In hopes of faithfully restoring the coding potential to damaged DNA during BER, Pol β first uses a lyase activity to remove the 5'-deoxyribose phosphate moiety from a nicked BER intermediate, followed by a DNA synthesis activity to insert a nucleotide triphosphate into the resultant 1-nucleotide gapped DNA substrate. This DNA synthesis activity of Pol β has served as a model to characterize the molecular steps of the nucleotidyl transferase mechanism used by mammalian DNA polymerases during DNA synthesis. This is in part because Pol β has been extremely amenable to X-ray crystallography, with the first crystal structure of apoenzyme rat Pol β published in 1994 by Dr. Samuel Wilson and colleagues. Since this first structure, the Wilson lab and colleagues have published an astounding 267 structures of Pol β that represent different liganded states, conformations, variants, and reaction intermediates. While many labs have made significant contributions to our understanding of Pol β, the focus of this article is on the long history of the contributions from the Wilson lab. We have chosen to highlight select seminal Pol β structures with emphasis on the overarching contributions each structure has made to the field.

Figure 1.

A timeline of the seminal Pol β structures published by Wilson and colleagues [–19].

Figure 2.

Structural snapshots of rat Pol β. A The extended structure of apoenzyme rat Pol β (PDB 1BPD) is shown with the 8 kDa lyase domain in green and the 31 kDa polymerase domain in blue. Domains, subdomains, and the flexible hinge are indicated. B The active site of the rat Pol β ternary complex (Pol β:DNA:ddCTP, PDB 2BPF) is shown. Key active site features are indicated.

Figure 3.

Structures of human Pol β during 1-nt gap-filling support an induced-fit model for fidelity. A An overlay of the open binary complex of Pol β bound to gapped DNA (green, PDB 1BPX), a closed ternary complex of Pol β bound to gapped DNA and ddCTP (cyan, PDB 1BPY), and an open Pol β binary product complex bound to nicked DNA (yellow, PDB 1BPZ). α-helix N, and the 90° kink of the DNA are indicated. B A focused-view of the closed ternary structure (cyan protein, gray/orange DNA) showing rotation of α-helix N into a closed conformation to contact the nascent base pair and assemble the active site upon nucleotide binding. The position of α-helix N and Asp192 in the open binary conformation are indicated (green). C The structure of an A:C mismatched base pair is shown with a magenta surface representation for the mismatched base pairs (magenta protein and gray/orange DNA, PDB 1TV9). The position of α-helix N in the open binary (green) and closed ternary (cyan) conformations are indicated for comparison.

Figure 4.

Non-hydrolyzable analog dUMPNPP ternary substrate complex of a pre-catalytic complex with ideal metal octahedral geometry (PDB 2FMS). The protein is represented in cyan, and DNA in gray/orange. Mg²⁺ octahedral geometry is indicated by dashed lines. The water molecules (blue spheres), Mg²⁺ ions (red spheres), and catalytic triad are indicated. An arrow is shown between the attacking 3’-OH of the primer terminus and Pα of dUMPNPP.

Figure 5.

Pre-catalytic complexes showing the various strategies used by Pol β to accommodate the oxidized DNA lesion, 8-oxoG. A 8-oxoG(anti) in the templating position opposite dCTP (PDB 3RJI) is shown overlaid with the non-damaged guanine(anti) opposite dCTP (black, PDB 2FMP). B 8-oxoG(syn) in the templating position opposite dATP is shown (PDB 3RJF). C An incoming 8-oxodGTP(anti) opposite cytosine is shown with the additional metal near Pα indicated by Me²⁺ (PDB 4UBC). D An incoming 8-oxodGTP(syn) opposite adenine is shown (PDB code 4UAW). Metals are shown in red, Pol β in yellow, and DNA in gray/orange, except where noted.