Roles for DNA polymerase zeta in translesion synthesis in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, DNA polymerase ζ (Pol ζ) is required in a major lesion bypass pathway. To help understand the role of Pol ζ in lesion bypass, in vitro biochemical analyses of this polymerase were performed in response to several DNA lesions. Purified yeast Pol ζ performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3′ T, and predominantly A opposite the 5′ T. Purified yeast Pol ζ predominantly incorporated a G opposite an acetylaminofluorene (AAF)-adducted guanine. The lesion, however, significantly inhibited subsequent extension. However, purified yeast Rev1p enhanced the incorporation and extension ability of Pol ζ opposite the AAF-adducted guanine. The synergistic result of Pol ζ and Rev1 was independent of the Rev1's dCMP transferase activity. Furthermore, yeast Pol ζ catalyzed extension DNA synthesis from primers annealed opposite the AAF-guanine and the 3′ T of the TT (6-4) photoproduct with varying efficiencies. Extension synthesis was more efficient when A or C was opposite the AAF-guanine, and when G was opposite the 3′ T of the TT (6-4) photoproduct. In contrast, the 3′ T of a cis-syn TT dimer completely blocked purified yeast Pol ζ, whereas the 5′ T was readily bypassed. An in vivo mutagenesis assay was used to examine potential roles of Pol ζ, Rev1, and Pol η in AAF-induced mutagenesis in the yeast system. Results from this assay showed that Pol ζ, Rev1 and Pol η all played significant roles in the AAF-induced mutagenesis. Pol η was responsible for the deletion mutations induced by AAF adducts. Pol ζ and Rev1 were required for the translesion synthesis of AAF adducts. Large fragment insertion and deletion mutations were detected in rev3 mutant and rev1 mutant strains. These results support the following dual-function model of Pol ζ. First, Pol ζ catalyzes nucleotide incorporation opposite AAF-guanine and TT (6-4) photoproduct with a limited efficiency. Second, more efficient bypass of these lesions may require nucleotide incorporation by other DNA polymerases followed by extension DNA synthesis by Pol ζ.