A conserved thumb domain insertion in DNA polymerase epsilon supports processive DNA synthesis

The leading strand DNA polymerase, Pol ϵ, plays a crucial role in DNA replication and maintenance of genome stability. In contrast to other replicative polymerases, Pol ϵ contains unique structural domains that likely underlie its specialized functions. However, the contribution of these structural elements to the functional capabilities of Pol ϵ remain poorly understood. In this study, we identify a conserved thumb domain insertion as a key determinant of the processivity of Pol ϵ in Saccharomyces cerevisiae. Disruption of this insertion leads to genome instability and significant defects in DNA replication. In vitro DNA binding and polymerase assays demonstrate that this insertion is critical for tight DNA binding and efficient processive synthesis. Our results highlight the essential role of this previously uncharacterized thumb domain insertion in supporting the intrinsic processivity of Pol ϵ.