archives@tulane.edu / The bulk of nuclear DNA synthesis during replication of the eukaryotic genome is carried out by three DNA Polymerases (Pols): Pols α, δ and ε. Through its role in leading strand synthesis, Pol ε is responsible for replicating up to half of the genome. As such, DNA synthesis errors made Pol ε during replication or other DNA repair processes pose a considerable source of potential genomic mutagenesis. Pol ε normally displays a high degree of fidelity, which can be attributed to the action of two physically distinct sites of catalysis: the polymerase domain, which is responsible for catalyzing the preferential addition of an incoming deoxynucleoside triphosphate (dNTP) to a nascent DNA strand resulting in correct Watson-Crick base pairing and the 3’ to 5’ exonuclease domain that proofreads the nascent DNA strand through the removal of misincorporated bases. Mutations in either of these domains that adversely affect their function can lead to a decrease in replication fidelity. Indeed, mutations localized to the exonuclease domain of Pol ε have been observed in tumors bearing drastically elevated genomic mutation burdens. We set out to determine the contribution of individual Pol ε mutants to mutagenesis and mutation spectrum through a combination of in vitro biochemistry and cell culture. We show that even in the face of functional mismatch repair (MMR) these mutants can lead to a variable yet substantial level of mutagenesis and recapitulate some but not all aspects of the anticipated mutation spectrum. These results indicate that Pol ε exonuclease domain mutants are capable of making these errors but other factors may be necessary to achieve the entirety of the observed patient tumor mutation profiles. In a mouse model heterozygous for the most recurrent Pol ε mutant we observe a massive reduction in tumor-free survival (100% mortality at 10 months) comprise exclusively of lymphomas. Additionally, the tumor mutation spectrum reveals a significant bias for TCT>TAT, TCG>TTG and TTT>TGT errors. These data suggest that replication errors made by Pol ε are directly contributing to tumorigenesis and may be solely responsible for the Pol ε mutant mutation profile. / 1 / Karl Hodel