Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

Abstract

We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

We thank Tazuko Tomita for DNA sequencing and Yasutomo Ito for model construction. We are also grateful to Akio Sugino, Yasuo Kawasaki, Akihiko Kikuchi, and Katsunori Sugimoto for critical advice and consistent encouragement.

This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Hori Foundation, and in part by the U.S. National Institutes of Health (grant EB002059 to E.T.K.).