A method to select for mutator DNA polymerase deltas in Saccharomyces cerevisiae.
Proofreading DNA polymerases share common short peptide motifs that bind Mg(2+) in the exonuclease active center; however, hydrolysis rates are not the same for all of the enzymes, which indicates that there are functional and likely structural differences outside of the conserved residues. Since structural information is available for only a few proofreading DNA polymerases, we developed a genetic selection method to identify mutant alleles of the POL3 gene in Saccharomyces cerevisiae, which encode DNA polymerase delta mutants that replicate DNA with reduced fidelity. The selection procedure is based on genetic methods used to identify "mutator" DNA polymerases in bacteriophage T4. New yeast DNA polymerase delta mutants were identified, but some mutants expected from studies of the phage T4 DNA polymerase were not detected. This would indicate that there may be important differences in the proofreading pathways catalyzed by the two DNA polymerases.
Murphy
Kelly
K
Department of Biological Sciences, CW405 BioSciences Building, University of Alberta, Edmonton, AB T6G 2E9, Canada.
Darmawan
Hariyanto
H
Schultz
Amy
A
Fidalgo da Silva
Elizabeth
E
Reha-Krantz
Linda J
LJ
D000595:Amino Acid Sequence; D020137:Base Pair Mismatch / Q000235:genetics; D002104:Cadmium / Q000009:adverse effects; D004252:DNA Mutational Analysis / Q000379:methods*; D004258:DNA Polymerase III / Q000235:genetics*; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D012641:Selection, Genetic; D016415:Sequence Alignment; D014764:Viral Proteins / Q000235:genetics
D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D002104:Cadmium; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase