The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands

Abstract

Abstract Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo−/−, CTF18−/−, PARP1−/−, CTF18−/−/POLE1exo−/−, PARP1−/−/POLE1exo−/−, and CTF18−/−/PARP1−/− cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo−/− and HDR-deficient BRCA1−/− cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo−/− with BRCA1−/− mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.