Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro.

DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, Pol delta D316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Pol delta. Human cell lines overexpressing Pol delta D316A;E318A display a mild mutator phenotype, while nuclear extracts of these cells exhibit reduced MMR activity in vitro, and these defects are complemented by overexpression or addition of exogenous human Exonuclease 1 (EXO1). By contrast, another proofreading-deficient mutant, Pol delta D515V, which has a weaker strand displacement activity, does not decrease the MMR activity as significantly as Pol delta D316A;E318A. In addition, Pol delta D515V does not increase the mutation frequency in MMR-proficient cells. Based on our findings, we propose that the proofreading activity restricts the strand displacement activity of Pol delta in MMR. This contributes to maintain the nicks required for EXO1 entry, and in this manner ensures the dominance of the EXO1-dependent MMR pathway.