Structural basis for proficient nucleotide incorporation across a major cisplatin‐DNA lesion by human DNA polymerase kappa (polκ)

Abstract

Cisplatin (cis‐diamminedichloroplatinum) is a widely used chemotherapeutic compound. It reacts with the N7 atoms of adjacent guanines in DNA to form the Pt‐1,2‐d(GpG) intrastrand cross‐link (Pt‐GG) as a major product. This bulky cisplatin adduct blocks DNA replication by replicative polymerases, thereby promoting apoptosis of rapidly dividing cancer cells. However, cancer cells have developed resistance to cisplatin, partly because of the bypass of cisplatin adducts by specialized DNA polymerases (mostly belonging to the Y‐family) through translesion synthesis (TLS). The human Y‐family DNA polymerase eta (polη) is very efficient in replicating opposite the Pt‐GG lesions, but not in extending after this adduct. Recently, it was shown that human Y‐family DNA polymerase kappa (polκ) is indispensable for DNA repair synthesis in dorsal root ganglion neurons exposed to cisplatin. Polκ gene knockdown experiments in human cells have also indicated the involvement of polκ in TLS of the Pt‐GG lesion. In this study, we investigated the bypass potential and nucleotide incorporation preference of polκ opposite DNA containing the Pt‐GG adduct and determined two crystal structures of polκ complexed with such DNA. The ternary complex structures represent two consecutive stages of lesion bypass: nucleotide insertion opposite the 5′G (Pt‐GG2) and primer extension immediately after the lesion (Pt‐GG3). Our in vitro DNA replication assays using polκ and DNA containing the Pt‐GG lesion indicate that polκ inserts the correct dCTP opposite the 5′G of Pt‐GG (Pt‐GG2), when a nucleotide is already inserted opposite the 3′G (Pt‐GG1). Polκ also inserts the correct dATP opposite the T base immediately after the Pt‐GG (Pt‐GG3). However, polκ is not able to insert a nucleotide opposite the 3′G of Pt‐GG (Pt‐GG1). On the other hand, polη is very efficient in replicating the Pt‐GG lesion at two insertion stages (Pt‐GG1 and Pt‐GG2), but the enzyme is blocked at the extension stage (Pt‐GG3). In terms of fidelity, we found that polκ has the fewest mis‐insertions compared to two other human Y‐family polymerases polη and polι. Our steady‐state kinetic analysis showed that polκ incorporates dCTP and dATP opposite the 5′G of Pt‐GG (Pt‐GG2) and T template 5′ to the lesion (Pt‐GG3), respectively, with very similar relative efficiencies of 0.15–0.20, which are only ~5 fold lower than for normal DNA replication (GG2 and GG3). Our biochemical data indicate that polκ is very proficient and accurate in inserting a nucleotide after the first G of the Pt‐GG lesion. The structures revealed that the accuracy is achieved by stably accommodating the bases containing the cisplatin adducts in the enzyme's active site for proper Watson‐Crick base pairing with the incoming nucleotide. Together, our biochemical and structural data provide the molecular basis for the accurate and proficient incorporation by polκ of nucleotides opposite the cisplatin adduct and suggest an important role of polκ as a second polymerase (extender) in the efficient bypass of the Pt‐GG lesion in vivo. This work holds promise for polκ as a potential target for drug design, along with polη, which together could improve the efficacy of cisplatin treatment for cancer therapy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.