A thumb-domain insertion balances processivity and fidelity in DNA polymerase ε.

Recent cryo-EM structures of human DNA polymerase ε (Pol ε) bound to PCNA position a Pol ε-specific thumb insertion (polymerase thumb insertion; PTI) adjacent to a PCNA protomer, suggesting a regulatory role in DNA synthesis. To define the functional contribution of this region, we generated alanine-substitution variants in the yeast Pol ε thumb domain, targeting the PTI (SLED1131-1134→AAAA; polε-SLED) and an adjacent conserved loop (PVTE1101-1104→AAAA; polε-PVTE and KPFN1096-1099→AAAA; polε-KPFN). polε-SLED displayed increased intrinsic processivity, efficient bypass of DNA secondary structures, and enhanced synthesis on long templates, consistent with reduced pausing. In contrast, a previous study extended this substitution to six amino acids, SLEDLD1131-1136→AAAAAA, and found a reversed effect, a reduced processivity, indicating that subtle perturbations in this insertion can have opposing functional consequences. polε-PVTE shifted polymerase activity toward exonuclease proofreading and was not fully rescued by PCNA on long templates, whereas polε-KPFN retained near-wild-type activity but showed increased sensitivity to secondary structures that was alleviated by PCNA. In vivo, the corresponding pol2-SLED allele caused a modest mutator phenotype, while pol2-PVTE and pol2-KPFN showed little or no increase. Together, these results indicate that the PTI fine-tunes intrinsic processivity and proofreading to maintain replication fidelity during leading-strand synthesis.