Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 “tag-mutate-enrich” mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.

A single mutagen can generate multiple different types of DNA lesions. How different repair pathways cooperate in complex DNA lesions, however, remains largely unclear. Here we measured, clustered, and modeled the kinetics of recruitment and dissociation of 70 DNA repair proteins to laser-induced DNA damage sites in HeLa cells. The precise timescale of protein recruitment reveals that error-prone translesion polymerases are considerably delayed compared to error-free polymerases. We show that this is ensured by the delayed recruitment of RAD18 to double-strand break sites. The time benefit of error-free polymerases disappears when PARP inhibition significantly delays PCNA recruitment. Moreover, removal of PCNA from complex DNA damage sites correlates with RPA loading during 5'-DNA end resection. Our systematic study of the dynamics of DNA repair proteins in complex DNA lesions reveals the multifaceted coordination between the repair pathways and provides a kinetics-based resource to study genomic instability and anticancer drug impact.

PARP inhibitors (PARPi) target homologous recombination defective tumour cells via synthetic lethality. Genome-wide and high-density CRISPR-Cas9 "tag, mutate and enrich" mutagenesis screens identified single amino acid mutations in PARP1 that cause profound PARPi-resistance. These included PARP1 mutations outside of the DNA interacting regions of the protein, such as mutations in solvent exposed regions of the catalytic domain and clusters of mutations around points of contact between ZnF, WGR and HD domains. These mutations altered PARP1 trapping, as did a mutation found in a clinical case of PARPi resistance. These genetic studies reinforce the importance of trapped PARP1 as a key cytotoxic DNA lesion and suggest that interactions between non-DNA binding domains of PARP1 influence cytotoxicity. Finally, different mechanisms of PARPi resistance (BRCA1 reversion, PARP1, 53BP1, REV7 mutation) had differing effects on chemotherapy sensitivity, suggesting that the underlying mechanism of PARPi resistance likely influences the success of subsequent therapies.