Roles of yeast DNA polymerases δ and ζ and of Rev1 in the bypass of abasic sites

Abstract

Abasic (AP) sites are one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA machinery. Here we show efficient bypass of an AP site by the combined action of yeast DNA polymerases δ and ζ. In this reaction, Polδ inserts an A nucleotide opposite the AP site, and Polζ subsequently extends from the inserted nucleotide. Consistent with these observations, sequence analyses of mutations in the yeastCAN1s gene indicate that A is the nucleotide inserted most often opposite AP sites. The nucleotides C, G, and T are also incorporated, but much less frequently. Enzymes such as Rev1 and Polη may contribute to the insertion of these other nucleotides; the predominant role of Rev1 in AP bypass, however, is likely to be structural. Steady-state kinetic analyses show that Polζ is highly inefficient in incorporating nucleotides opposite the AP site, but it efficiently extends from nucleotides, particularly an A, inserted opposite this lesion. Thus, in eukaryotes, bypass of an AP site requires the sequential action of two DNA polymerases, wherein the extension step depends solely upon Polζ, but the insertion step can be quite varied, involving not only the predominant action of the replicative DNA polymerase, Polδ, but also the less prominent role of various translesion synthesis polymerases.