Modeling cancer-associated mutations reveals an integrated role for the Pol epsilon catalytic core in replisome assembly and DNA synthesis

DNA polymerase epsilon (Pol epsilon) plays multiple roles in genome duplication and its catalytic subunit POLE suppresses tumorigenesis in humans. Despite its importance, we have limited understanding of Pol epsilon functional mechanisms and how its loss-of-function affects the evolution of cancer genomes. To address this issue, we used the budding yeast ortholog Pol2 to model cancer-associated POLE mutations that affect an uncharacterized yet highly conserved Pol2/POLE family-specific region. Analysis of pol2 mutants revealed an unexpected role for this region in the incorporation of Pol epsilon into the pre-loading complex during replisome assembly. Cellular and reconstitution experiments further identified a separate role for this region in promoting Pol epsilon polymerase activity. Unlike previously reported POLE mutations leading to hypermutation in cancer cells, the examined pol2 variants increase gross chromosomal rearrangements but not mutation rates. Our findings thus suggest that the Pol epsilon catalytic core integrates replisome assembly and DNA polymerization functions and that POLE tumor suppressive roles likely extend beyond limiting replication errors. Two sets of samples at are included: 1) set 1 contains 3 reference samples, collected at G1, and 30 min, 40 min post G1 release; 2) set 2 contains 3 mutant samples, collected at the same time point as the reference. Whole genome sequencing (Illumina HiSeq) was used to access the copy number change and origin firing in these samples.