Mutations in human DNA Polymerase (Pol) ε, 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 ε 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 ε that exhibits a strong mutator phenotype in vitro when the proofreading exonuclease activity of the enzyme is inactive. This mutant has a strong bias towards mispairs opposite template pyrimidine bases, particularly T•dTTP mispairs. Expression of mutant Pol ε 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 ε causes replication errors in vivo, and is at least partially dominant over the endogenous, wild type Pol ε. Since tumors from familial and somatic colorectal patients arise with Pol ε mutations in a single allele, are microsatellite stable and have a large increase in base pair substitutions, our data are consistent with a Pol ε mutation requiring additional factors to promote tumor development.

DNA replication in eukaryotes is carried out primarily by three DNA polymerases (Pols): Pols α, δ and ε. While yeast Pol ε has been shown to function on the leading strand during DNA replication, the precise functions of human Pol ε in vivo remain relatively poorly understood.Wild type pol ε from both yeast and humans is a highly accurate enzyme. Active site mutant alleles of yeast Pol ε were previously shown to be mutators with unique error signatures both in vitro and in vivo. We found that human Exo− M630G Pol ε is a 50‐fold mutator over the wild type enzyme in vitro, with a strong preference for T•dT and T•dG errors.We introduced Exo− M630G Pol ε into human mismatch repair‐deficient HCT116 colorectal cancer cells. Mutations in this cell line arise primarily from errors made during replication. Cells transfected with Exo− M630G Pol ε exhibited an increase in HPRT1 mutant frequency over cells transfected with control vector. The results indicate that transfected Exo− M630G Pol ε is involved in replication, is a mutator during replication in vivo, and is at least partially dominant over the endogenous Exo+ Pol ε. Importantly, Exo− M630G Pol ε mutant lacking the C‐terminal tail, which is known to be essential in yeast, showed no increase in HPRT1 mutant frequency.With the human Exo‐ M630G Pol ε mutator allele, we now have the ability to probe Pol ε function during human DNA replication and repair of damaged DNA.NIH ES016780, NIH RR020152