A common cancer-associated DNA polymerase ε mutation causes an exceptionally strong mutator phenotype, indicating fidelity defects distinct from loss of proofreading

Abstract

Exonucleolytic proofreading and DNA mismatch repair (MMR) act in series to maintain high-fidelity DNA replication and to avoid mutagenesis. MMR defects elevate the overall mutation rate and are associated with increased cancer incidence. Hypermutable colorectal and endometrial tumors with functional MMR were recently reported to carry amino acid substitutions in the exonuclease domain of DNA polymerase ε (Polε). This created a notion that loss of the proofreading activity of Polε is an initiating cause of some sporadic human cancers. In this study, we identified a somatic P286R substitution in the conserved ExoI motif of Polε in a collection of 52 sporadic colorectal tumor specimens. This change has been repeatedly observed in colorectal and endometrial tumors in previous studies despite many possible ways to inactivate Polε proofreading. To understand the reasons for the recurrent appearance of the P286R variant, we characterized its functional consequences using the yeast model system. An analogous substitution in the yeast Polε produced an unusually strong mutator phenotype exceeding that of proofreading-deficient mutants by two orders of magnitude. This argues that the P286R mutation acts at some level other than loss of exonuclease to elevate cancer risk. Heterozygosity for the variant allele caused a strong mutator effect comparable with that of complete MMR deficiency, providing an explanation for why loss of heterozygosity is not required for the development of Polε-mutant human tumors.

Figure 1

Mutation at codon 286 of POLE in a sporadic colon tumor. (A) DNA sequencing analysis of sample T-04-364 and matching normal mucosa. (B) Conservation of Pro286 in B-family DNA polymerases. Hatched boxes on the POLE schematic show conserved exonuclease and polymerase motifs. Hs, human; Sc, S. cerevisiae; Ec, E. coli. Asterisks mark Pro286 and the catalytic carboxylates affected by the pol2-4 mutation.

Figure 2

Yeast mimic of POLE-P286R is the strongest Polε mutator known. (A) Location of the known mutator mutations on the yeast Pol2 schematic. Hatched boxes, conserved exonuclease and polymerase motifs. (B) Rate of Can^r mutation (relative to wild-type) in haploid pol2 mutants. Data are from Table 2 and Supplementary Table S3.