PARP1 auto-modification promotes faithful Okazaki fragment processing and limits replication fork speed

Abstract

Poly(ADP-ribose) polymerase (PARP) inhibitors have proven their efficacy for treating tumors defective in homologous recombination via synthetic lethality. In response to DNA breaks, PARP1 is the primary ADP-ribosylation writer, modifying itself (auto-modification) and other proteins to facilitate repair. However, enzymatic inhibition blocks both processes, making it difficult to dissect their distinct functional roles. Using proteomics and site-directed mutagenesis, we identified a PARP1 mutant deficient in auto-modification, yet it retains catalytic activity. This separation-of-function mutant revealed that PARP1 auto-modification slows DNA replication fork progression but is dispensable for repair factor recruitment. Instead, auto-modification promotes the timely release of PARP1 at DNA break sites and prevents the formation of replication stress. Simultaneous inhibition of FEN1 and loss of PARP1 auto-modification gives rise to synthetic lethality, implicating auto-modification in Okazaki fragment processing. Our results demonstrate that trapping of PARP at DNA breaks impedes repair factor accessibility, constituting an important dimension of PARP-inhibitor-driven cytotoxicity.