Roles of yeast DNA polymerases delta and zeta and of Rev1 in the bypass ofabasic sites

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 acti on of yeast DNA polymerases delta and zeta. In this reaction, Pol delta ins erts an A nucleotide opposite the AP site, and Pol zeta subsequently extend s from the inserted nucleotide. Consistent with these observations, sequenc e analyses of mutations in the yeast CAN1(s) gene indicate that A is the nu cleotide inserted most often opposite AP sites. The nucleotides C, G, and T are also incorporated, but much less frequently. Enzymes such as Rev1 and Pol eta may contribute to the insertion of these other nucleotides; the pre dominant role of Rev1 in AP bypass, however, is likely to be structural. St eady-state kinetic analyses show that Pol zeta is highly inefficient in inc orporating nucleotides opposite the AP site, but it efficiently extends fro m nucleotides, particularly an A, inserted opposite this lesion. Thus, in e ukaryotes, bypass of an AP site requires the sequential action of two DNA p olymerases, wherein the extension step depends solely upon Pol zeta, but th e insertion step can be quite varied, involving not only the predominant ac tion of the replicative DNA polymerase, Pol delta, but also the less promin ent role of various translesion synthesis polymerases.