Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[ a ]pyrene dG adduct

Abstract

DNA polymerase κ (Polκ) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N 2 -deoxyguanine adducts efficiently and accurately. The unique features of Polκ, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Polκ variants, which have reduced gap size on both sides [Polκ Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Polκ variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N 2 -dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Polκ-deficient cells stably complemented with PGM1 GFP-Polκ remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Polκ restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Polκ (mPolκ 52-516 ) leads to reduced polymerization activity, and the mutant PGM2 52-516 but not PGM1 52-516 can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolκ 52-516 retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Polκ during translesion synthesis.translesion DNA synthesis | polycyclic aromatic hydrocarbons B enzo[a]pyrene (BP) is one of the best characterized polycyclic aromatic hydrocarbons generated during incomplete fuel combustion, in tobacco smoke, and in cooked food. The most mutagenic and tumorigenic metabolite of BP in vivo is the (+)-7R,8S,9S,10R-BP dihydrodiol epoxide [(+)-anti-BPDE] (1), which reacts readily with the exocyclic amino groups of guanine residues in DNA to form the major 10S (+)-trans-anti-BPDE-N 2 -dG adduct (2) (abbreviated as BPDE-dG hereafter). This adduct typically impedes normal DNA replication. Translesion DNA synthesis (TLS), which provides one mode of DNA damage tolerance, uses specialized bypass polymerases to synthesize DNA opposite and beyond a variety of replication-blocking lesions, thus avoiding replication fork collapse (3). The Y-family DNA polymerases are specialized for TLS. To date, four of them, namely DNA polymerase (Pol) η, Polι, Polκ, and REV1, have been identified in mammals (4-6). Polκ is the only one with homologs in bacteria (DinB; also known as Pol IV in Escherichia coli), archaea [DNA polymerase IV (Dpo4) in Sulfolobus solfataricus and DinB homologue (Dbh) in Sulfolobus acidocaldarius] and all eukaryotes. The mouse and human POLK gene are expressed ubiquitously, with highest levels in testis (7).Human Polκ can accurately incorporate dCMP opposite the BPDE-dG adduct ...

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism

“…E. coli Pol IV is found to efficiently bypass N2-furfuryl-dG adducts (100). The structural gap in Pol κ is particularly large, and perhaps as a result Pol κ is most efficient and accurate in bypassing minor-groove adducts (76, 95, 96, 101, 102). The structural gap, however, is implicated in template slippage / misalignment, which leads to deletional mutations (73, 75, 103).…”

“…4b). The flexible region N-terminal to the N-clasp, which is not yet characterized structurally, also enhances lesion bypass capability (76). …”

Regulation of Rad6/Rad18 Activity During DNA Damage Tolerance

“…At one end of the spectrum are lesions induced by chemicals such as BDPE. Pol κ bypasses BPDE-induced lesions in an error-free manner in vitro and is responsible for their faithful replication in vivo (3, 70, 91, 116, 141). Polη, however, replicates past such lesions in an error-prone manner in vitro and, perhaps owing to the horse-and-carriage model, contributes to the mutagenic bypass of BPDE adducts in vivo (20, 59, 102, 141).…”

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage

“…Mouse Pol κ could bypass N 2 -B[ a ]P G efficiently and accurately, but a mutant with reduced gap size (Pol κ gap mutant, PGM) was strongly blocked by this lesion, suggesting that the presence of this gap is essential for the DNA adduct bypass. From the crystal structure, the gap physically accommodates the bulky aromatic adduct and keeps the active site ordered, explaining crucial functions of the gap in Pol κ in maintenance of the active site for translesion DNA synthesis [65]. …”