Deaminated purine bypass by DNA polymerase η

Genomic DNA of living cells is frequently exposed to a variety of endogenous or exogenous factors that can lead to its damage. The damaged DNA is an obstacle for the replication machinery and it usually results in a replication block due to the inability of the classical high-fidelity DNA polymerases to incorporate nucleotide opposite to the DNA lesion. This block can cause replication fork collapse leading to DNA breaks, chromosomal rearrangements and ultimately to a cell death. To maintain genomic stability, cells have evolved different mechanisms to either repair, or tolerate and bypass the site of DNA damage. The latter mechanism involves the temporary replacement of the replicative polymerase by one or more specialized translesion synthesis polymerases that are able to replicate through the damaged bases, typically in an error-prone manner [1]. In eukaryotes, the DNA polymerases that mediate the translesion synthesis are members of the so-called Y-family that include polymerase eta, kappa, iota and REV1. They exhibit different specificity and fidelity in the lesion bypass owing to the differences in their active site architectures. The Y-family polymerases share similar organization consisting of an N-terminal catalytic core followed by a flexible intrinsically disordered region and a set of regulatory protein–protein interaction modules toward the C-termini. Architecturally the catalytic core is reminiscent to the classical polymerases in which the palm domain harbours the residues crucial for catalysis, whereas the thumb and finger domains grasp the DNA and make contacts with the primer and the template, respectively. The structural similarity between the classical replicative and the Y-family lesion-bypass polymerases is, however, limited to the palm domains that superpose quite well with their functional residues at equivalent conserved positions. A distinct feature of the Y-family is the presence of a little finger domain, also known as polymerase associated domain (PAD), that is unique to this family of polymerases. This domain makes additional contacts with the DNA and is implicated in the ternary complex stability.

The most intriguing and probably best-studied member of the Y-family is the human polymerase eta (hPolη). This enzyme has the unique ability to efficiently replicate through UV-induced cis-syn thymine-thymine (T-T) dimers in an error-free manner by inserting AA opposite TT. The deficiency in this polymerase is associated with a human disorder xeroderma pigmentosum, which increases the risk of developing skin and other internal cancers. hPolη also facilitates the translesion synthesis of cancer drug adducts, such as cisplatin and phenanthriplatin, that can lead to impaired response to cancer treatment and development of cancer chemoresistance. In addition to the above, the enzyme has been implicated in the bypass of various mutagenic DNA lesions, including O6-methyl-2′-deoxyguanosine (O6-MeG), 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), 8,5′-cyclo-2′-deoxyadenosine (cdA), abasic sites (AP) etc. Over the past decade, the crystal structures of the hPolη replicative bypass of these lesions have been determined, revealing the intimate mechanistic details of the polymerase function [2–4]. Two new crystal structures reported in the recent issue of Biochemical Journal by Jung et al. [5] expand the broader work on human Polη substrates and provide the first structural insights into the mutagenic bypass of deaminated purine lesions. These structures show the ternary complex of the polymerase with a template DNA containing either xanthine (XT) or hypoxanthine (HX) lesion in the active site and a non-hydrolyzable dCMPNPP (dCTP*) as an incoming nucleotide. In both structures, the templating XT/HX is positioned between the finger and little finger domains, whereas the incoming dCTP* is nested between the palm and finger domains. The Polη-XT:dCTP* and Polη-HX:dCTP* complexes reveal significant similarity to the Polη-dG:dCTP* complex with undamaged DNA and to other hPolη structures inserting dCTP* opposite a damaged base, suggesting that the polymerase and its substrates undergo little conformational changes (Figure 1). Some key elements such as the side-chain conformation of the active site residues, the binding of the metals, the position of the primer 3′-OH and the 5′ α-phosphate of the incoming dCTP* are essentially the same in these structures. An exception makes the side chain of Arg61 that displays some conformational flexibility. This residue is located at the tip of the finger domain and its dynamics and importance for the polymerase activity has been extensively discussed previously [2–4]. In both, Polη-XT:dCTP* and Polη-HX:dCTP* complexes, Arg61 is involved in a cation-π stacking interaction with the incoming dCTP* and thereby supporting its optimal position for base pairing. The guanidino group of this residue swivel over the nucleotide and either points toward the phosphate groups of dCTP* (in Polη-HT:dCTP*) or toward the template DNA thereby making a close contact to O4 of dT(N + 1) base (in Polη-XT:dCTP*). Another significant conformational difference resides in the template DNA, in particular the region upstream of the insertion site. While in Polη-XT:dCTP* complex the dT(N + 1) and dA(N + 2) bases are displaced toward the active site interior, in Polη-HX:dCTP*, the dT(N+1) base flips toward the exterior. It looks quite plausible that the interplay between the flexibility of the Arg61 side-chain and the conformational dynamics in the DNA substrate may have an impact on the polymerase fidelity and catalytic efficiency, and can contribute to the kinetic differences reported in this study [5].