title: Mutator phenotypes caused by substitution at a conserved motif A residue in eukaryotic DNA polymerase delta.
abstract: Eukaryotic DNA polymerase (Pol) delta replicates chromosomal DNA and is also involved in DNA repair and genetic recombination. Motif A in Pol delta, 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 Leu(612) in Motif A of Saccharomyces cerevisiae Pol delta. We show that substitutions at Leu(612) 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 Leu(612) in replication fidelity.
authors.affiliation: Department of Pathology, University of Washington, Seattle, 98195-7705, USA.
authors.forename: Ranga N
authors.initials: RN
authors.lastname: Venkatesan
authors.forename: Jessica J
authors.initials: JJ
authors.lastname: Hsu
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Lawrence
authors.forename: Bradley D
authors.initials: BD
authors.lastname: Preston
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002453:Cell Cycle; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics*; D004261:DNA Replication; D006918:Hydroxyurea / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D008741:Methyl Methanesulfonate; D004258:DNA Polymerase III; D006918:Hydroxyurea