Mutator phenotypes caused by substitution at a conserved motif A residue in eukaryotic DNA polymerase

Eukaryotic DNA polymerase (Pol) δ replicates chromosomal DNA and is also involved in DNA repair and genetic recombination. Motif A in Pol δ, containing the sequence DXXXLYPSI, includes a catalytically essential aspartic acid as well as other conserved residues of unknown function. Here, we used site-directed mutagenesis to create all 19 amino acid substitutions for the conserved Leu⁶¹² in Motif A of Saccharomyces cerevisiae Pol δ. We show that substitutions at Leu⁶¹² differentially affect viability, sensitivity to genotoxic agents, cell cycle progression, and replication fidelity. The eight viable mutants contained Ile, Val, Thr, Met, Phe, Lys, Asn, or Gly substitutions. Individual substitutions varied greatly in the nature and extent of attendant phenotypic deficiencies, exhibiting mutation rates that ranged from near wild type to a 37-fold increase. The L612M mutant exhibited a 7-fold elevation of mutation rate but essentially no detectable effects on other phenotypes monitored; the L612T mutant showed a nearly wild type mutation rate together with marked hypersensitivity to genotoxic agents; and the L612G and L612N strains exhibited relatively high mutation rates and severe deficits overall. We compare our results with those for homologous substitutions in prokaryotic and eukaryotic DNA polymerases and discuss the implications of our findings for the role of Leu⁶¹² in replication fidelity.