DNA polymerase ε relies on a unique domain for efficient replisome assembly and strand synthesis.

DNA polymerase epsilon (Pol ε) is required for genome duplication and tumor suppression. It supports both replisome assembly and leading strand synthesis; however, the underlying mechanisms remain to be elucidated. Here we report that a conserved domain within the Pol ε catalytic core influences both of these replication steps in budding yeast. Modeling cancer-associated mutations in this domain reveals its unexpected effect on incorporating Pol ε into the four-member pre-loading complex during replisome assembly. In addition, genetic and biochemical data suggest that the examined domain supports Pol ε catalytic activity and symmetric movement of replication forks. Contrary to previously characterized Pol ε cancer variants, the examined mutants cause genome hyper-rearrangement rather than hyper-mutation. Our work thus suggests a role of the Pol ε catalytic core in replisome formation, a reliance of Pol ε strand synthesis on a unique domain, and a potential tumor-suppressive effect of Pol ε in curbing genome re-arrangements.