Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts

The (6-4) pyrimidine-pyrimidone photoproduct [(6-4)PP] is a major DNA lesion induced by ultraviolet radiation. (6-4)PP induces complex mutations opposite its downstream bases, in addition to opposite 3' or 5' base, as has been observed through a site-specific translesion DNA synthesis (TLS) assay. The mechanism by which these mutations occur is not well understood. To elucidate the mechanisms underlying mutagenesis induced by (6-4)PP, we performed an intracellular TLS assay using a replicative vector with site-specific T(thymidine)-T (6-4)PP. Rev3^-/-p53^-/- mouse embryonic fibroblast (MEF) cells (defective in Polζ) were almost completely defective in bypassing T-T (6-4)PP, whereas both Rev1^-/- and Polh^-/-Poli^-/-Polk^-/- MEF cells (defective in Polη, Polι, and Polκ) presented bypassing activity comparable to that of wild-type cells, indicating that Y-family TLS polymerases are dispensable for bypassing activity, whereas Polζ plays an essential role, probably at the extension step. Among all cells tested, misincorporation occurred most frequently just beyond the lesion (position +1), indicating that the Polζ-dependent extension step is crucial for (6-4)PP-induced mutagenesis. We then examined the effects of sequence context on T-T (6-4)PP bypass using a series of T-T (6-4)PP templates with different sequences at position +1 or -1 to the lesion, and found that the dependency of T-T (6-4)PP bypass on Polζ is not sequence specific. However, the misincorporation frequency at position +1 differed significantly among these templates. The misincorporation of A at position +1 occurred frequently when a purine base was located at position -1. These results indicate that Polζ-dependent extension plays a major role in inducing base substitutions in (6-4)PP-induced mutagenesis, and its fidelity is affected by sequence context surrounding a lesion.