Human Pol ε-dependent replication errors and the influence of mismatch repair on their correction

Mutations in human DNA polymerase (Pol) epsilon one of three eukaryotic Pols required for DNA replication, have recently been found associated with an ultramutator phenotype in tumors from somatic colorectal and endometrial cancers and in a familial colorectal cancer. Possibly, Pol epsilon mutations reduce the accuracy of DNA synthesis, thereby increasing the mutational burden and contributing to tumor development. To test this possibility in vivo, we characterized an active site mutant allele of human Pol epsilon that exhibits a strong mutator phenotype in vitro when the proofreading exonuclease activity of the enzyme is inactive. This mutant has a strong bias toward mispairs opposite template pyrimidine bases, particularly T.dTTP mispairs. Expression of mutant Pol epsilon in human cells lacking functional mismatch repair caused an increase in mutation rate primarily due to T.dTTP mispairs. Functional mismatch repair eliminated the increased mutagenesis. The results indicate that the mutant Pol epsilon causes replication errors in vivo, and is at least partially dominant over the endogenous, wild type Pol epsilon. Since tumors from familial and somatic colorectal patients arise with Pol epsilon mutations in a single allele, are microsatellite stable and have a large increase in base pair substitutions, our data are consistent with a Pol epsilon mutation requiring additional factors to promote tumor development. (C) 2013 Elsevier B.V. All rights reserved.