A DNA polymerase epsilon mutant that specifically causes +1 frameshift mutations within homonucleotide runs in yeast.
The DNA polymerases delta and epsilon are the major replicative polymerases in the yeast Saccharomyces cerevisiae that possess 3' --> 5' exonuclease proofreading activity. Many errors arising during replication are corrected by these exonuclease activities. We have investigated the contributions of regions of Polepsilon other than the proofreading motifs to replication accuracy. An allele, pol2-C1089Y, was identified in a screen of Polepsilon mutants that in combination with an exonuclease I (exo1) mutation could cause a synergistic increase in mutations within homonucleotide runs. In contrast to other polymerase mutators, this allele specifically results in insertion frameshifts. When pol2-C1089Y was combined with deletions of EXO1 or RAD27 (homologue of human FEN1), mutation rates were increased for +1 frameshifts while there was almost no effect on -1 frameshifts. On the basis of genetic analysis, the pol2-C1089Y mutation did not cause a defect in proofreading. In combination with a deletion of the mismatch repair gene MSH2, the +1 frameshift mutation rate for a short homonucleotide run was increased nearly 100-fold whereas the -1 frameshift rate was unchanged. This suggests that the Pol2-C1089Y protein makes +1 frameshift errors during replication of homonucleotide runs and that these errors can be corrected by either mismatch repair (MMR) or proofreading (in short runs). This is the first report of a +1-specific mutator for homonucleotide runs in vivo. The pol2-C1089Y mutation defines a functionally important residue in Polepsilon.
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
J M
JM
Kirchner
H
H
Tran
M A
MA
Resnick
D000483:Alleles; D000595:Amino Acid Sequence; D004257:DNA Polymerase II / Q000737:chemistry / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D045643:DNA Repair Enzymes; D004706:Endodeoxyribonucleases / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000235:genetics; D045585:Flap Endonucleases; D016368:Frameshift Mutation; D017353:Gene Deletion; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D029701:Saccharomyces cerevisiae Proteins; D004257:DNA Polymerase II; C114331:EXO1 protein, human; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; C028450:exodeoxyribonuclease I; C468323:RAD27 protein, S cerevisiae; D045643:DNA Repair Enzymes