Analysis of the DNA polymerase active site mutants in yeast

Sakamoto, Ayako; Pavlov, Y. I.*; Kunkel, T. A.*

DNA polymerase (Pol ), a member of family B polymerases, is present in almost all eukaryotic genomes examined to date and is thought to be involved in the replication of damaged DNA in an error-prone manner. For example, in yeast, disruption of Pol reduces both spontaneous and UV-induced mutation rates. However, the mechanisms with which Pol bypasses lesions and induces mutations remain unclear. To elucidate the function of Pol in spontaneous and damage-induced mutagenesis, we have generated a series of amino acid substitutions in the active site of yeast Pol . A conserved leucine residue in the palm subdomain of B-type polymerases has previously been shown to be important for the fidelity of DNA Pol (Niimi et al., 2004). We substituted the corresponding leucine of yeast Pol (Leu979) with other amino acid residues and examined the effect of these substitutions on mutation frequency in vivo. The rev3-L979M and rev3-L979F mutants had wild-type sensitivity to UV-induced cytotoxicity but showed higher UV-induced mutation frequencies compared to the wild-type strain. These results suggest that the substitution of Leu979 with methionine or phenylalanine reduces the fidelity of DNA polymerase without affecting the enzyme activity like the Pol mutants. The mutator phenotype of the rev3-L979F/M alleles should be a useful marker to analyze the functions of DNA polymerase in maintaining the stability of eukaryotic genomes.