A DNA Polymerase ε Mutant That Specifically Causes +1 Frameshift Mutations Within Homonucleotide Runs in Yeast

Abstract

The DNA polymerases δ and ε 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 Polε other than the proofreading motifs to replication accuracy. An allele, pol2-C1089Y, was identified in a screen of Polε 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 Polε.

Fidelity of DNA Polymerase δ Holoenzyme from Saccharomyces cerevisiae: The Sliding Clamp Proliferating Cell Nuclear Antigen Decreases Its Fidelity

“…Another pol ε mutant ( pol2-C1089Y ) was isolated as a +1 frame-shift mutator within homonucleotide runs. In vitro studies show that this mutant has an ≈1000-fold increase in mutation frequency and the mutant enzyme pol ε-C1089Y has a lower intrinsic fidelity, independent of its 3‘−5‘ exonuclease activity ( , ). These results are consistent with the notion that pol ε plays a direct role in chromosomal DNA replication in vivo.…”

A Subset of Microsatellite Unstable Cancer Genomes Prone to Short Insertions over Deletions Is Associated with Elevated Anticancer Immunity

“…For example, it is unclear whether MMRd genomes are largely exclusive of other types of genomic instability, e.g., deficiencies in polymerase proofreading functions (PPd) with loss-of-function mutations in POLD1 and POLE . However, PPd genomes are often accompanied by MMRd [ 18 , 19 ]. With intact MMR functions, POLD1 and POLE mutations contribute to elevated mutation rates, but cancer genomes with combined MMRd and PPd tend to show a higher ID burden than those with only MMRd [ 1 ].…”