Temporally distinct translesion synthesis pathways for ultraviolet light-induced photoproducts in the mammalian genome

a b s t r a c t Replicative polymerases (Pols) arrest at damaged DNA nucleotides, which induces ubiquitination of the DNA sliding clamp PCNA (PCNA-Ub) and DNA damage signaling. PCNA-Ub is associated with the recruit- ment or activation of translesion synthesis (TLS) DNA polymerases of the Y family that can bypass the lesions, thereby rescuing replication and preventing replication fork collapse and consequent formation of double-strand DNA breaks. Here, we have used gene-targeted mouse embryonic fibroblasts to per- form a comprehensive study of the in vivo roles of PCNA-Ub and of the Y family TLS Pols , , , Rev1 and the B family TLS Pol in TLS and in the suppression of DNA damage signaling and genome instabil- ity after exposure to UV light. Our data indicate that TLS Pols and and the N-terminal BRCT domain of Rev1, that previously was implicated in the regulation of TLS, play minor roles in TLS of DNA pho- toproducts. PCNA-Ub is critical for an early TLS pathway that replicates both strongly helix-distorting (6-4) pyrimidine-pyrimidone ((6-4)PP) and mildly distorting cyclobutane pyrimidine dimer (CPD) pho- toproducts. The role of Pol is mainly restricted to early TLS of CPD photoproducts, whereas Rev1 and, in particular, Pol are essential for the bypass of (6-4)PP photoproducts, both early and late after exposure. Thus, structurally distinct photoproducts at the mammalian genome are bypassed by different TLS Pols in temporally different, PCNA-Ub-dependent and independent fashions.