Structure of Human DNA Polymerase κ Inserting dATP Opposite an 8-OxoG DNA Lesion

et al. (2009) Structure of Human DNA Polymerase k Inserting dATP Opposite an 8-OxoG DNA Lesion. PLoS ONE 4(6): e5766. doi:10.1371/journal.pone.0005766 Structure of Human DNA Polymerase k Inserting dATP Opposite an 8-OxoG DNA Lesion Rodrigo Vasquez-Del Carpio 0 Timothy D. Silverstein 0 Samer Lone 0 Michael K. Swan 0 Jayati R. 0 Choudhury 0 Robert E. Johnson 0 Satya Prakash 0 Louise Prakash 0 Aneel K. Aggarwal 0 Hilal Lashuel, Swiss Federal Institute of Technology Lausanne, Switzerland 0 1 Department of Structural & Chemical Biology, Mount Sinai School of Medicine , New York , New York, United States of America, 2 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch , Galveston, Texas , United States of America Background: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase k (Polk) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polk's preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polk in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. Methodology and Principal Findings: We show that the Polk active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP. Conclusions and Significance: The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polk is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polk is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases. - Funding: This work was supported by USPHS research grant ES016666. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Oxidative damage to DNA has been proposed to have a role in cancer and ageing [1]. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro8-oxoguanine (8-oxoG) is one of the most common adducts formed [2,3]. Although the high-fidelity replicative DNA polymerases (Pols) can insert an A opposite 8-oxoG, they are inhibited very considerably at both the nucleotide insertion and subsequent extension steps. The recently discovered Y-family of DNA polymerases permit the continuity of the replication fork by allowing replication through such lesions that impede the replicative polymerases [4]. Humans have four Y-family polymerases Polg, Poli, Polk, and Rev1 each with a unique DNA damage bypass and fidelity profile. Polg, for example, is unique in its ability to replicate through an ultraviolet (UV)-induced cis-syn thymine-thymine (T-T) dimer by inserting two As opposite the two Ts of the dimer with the same efficiency and accuracy as opposite undamaged Ts [58]. Because of the involvement of Polg in promoting error-free replication through cyclobutane pyrimidine dimers, its inactivation in humans causes the variant form of xeroderma pigmentosum, a genetic disorder characterized by a greatly enhanced predisposition to sun induced skin cancers [9,10]. Poli, on the other hand, is unable to replicate through a cissyn T-T dimer but it can proficiently incorporate nucleotides opposite N2-adducted guanines and opposite adducts such as 1, N6-ethanodeoxyadenosine which impair the ability of the purine to engage in Watson-Crick (W-C) base-pairing [1115]. Rev1 is highly specialized for incorporation of C opposite template G and promotes efficient dCTP incorporation opposite bulky N2-dG adducts via a protein-template directed mechanism of DNA synthesis [1619]. In all, Y-family polymerases in eukaryotes display a large degree of functional divergence, rendering them highly specialized for specific roles in lesion bypass [4]. Polk is the only human Y-family polymerase with homologues in prokaryotes and archaea, including DinB (PolIV) in Escherichia coli and Dbh and Dpo4 in Sufolobus solfataricus [2022]. However, the amino acid (aa) sequence of Polk differs from PolIV and Dpo4 (and other Y-family polymerases) by an extension at the Nterminus of approximately 75 amino acids [23]. This N-terminal extension is indispensable for Polk activity and is conserved only amongst eukaryotic Polk proteins. The crystal structure of Polk in ternary complex with a template-primer DNA and an incoming nucleotide, reveals encirclement of the DNA by this unique Nterminal extension, referred to as the N-clasp [24]. The N-clasp effectively locks the polymerase around the template-primer, perhaps as a means to keep it engaged on a sugar-phosphate backbone distorted by a DNA lesion. Biochemical studies with yeast and human Y family polymerases indicate that Polg and Polk have the most proficient ability to replicate through the 8-oxoG lesion [4]. However, whereas yeast and human Polg replicate through 8-oxoG by predominantly inserting a C [25], human Polk is more efficient at inserting an A opposite the lesion than a C [26]. In this respect, Polk differs even from Dpo4 (its homologue in Sufolobus solfataricus) which prefers to insert a C opposite 8-oxoG [27,28]. To understand the basis of Polks preference for insertion of A opposite 8-oxoG, we have solved the structure of Polk in ternary complex with a templateprimer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. We show that the Polk active site is welladapted to accommodate the 8-oxoG lesion in the syn conformation for base pairing with incoming dATP. Structure determination We crystallized the Polk catalytic core (aa 19-526) in ternary complex with a 13-nt/18-nt primer/template presenting the 8oxoG lesion as the templating base and with dATP as the incoming nucleotide. The cocrystals diffract to 3.2 A resolution with synchrotron radiation (Brookhaven National Laboratory) and there are two ternary complexes (A and B) in the crystallographic asymmetric unit (Table 1). The structure was determined by molecular replacement using the polymerase from the Polk ternary complex with undamaged DNA as a search model [24]. Electron density maps showed clear den (...truncated)