An active site mutant allele of human Pol ε is a mutator in vitro and in vivo

Abstract

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