A RAD18 SAP domain PIP motif enables PCNA mono-ubiquitination and USP1-BRCA1 synthetic lethality
The proliferating cell nuclear antigen (PCNA) sliding clamp is mono-ubiquitinated by RAD6-RAD18 in response to DNA damage, initiating the DNA damage tolerance pathway of translesion synthesis. The molecular basis by which RAD18 engages PCNA has, however, remained incompletely defined. Mono-ubiquitinated PCNA is subsequently poly-ubiquitinated with K48-linked chains that target PCNA for degradation. Ubiquitin-specific protease 1 (USP1) reverses PCNA mono- and poly-ubiquitination; accordingly, inhibiting USP1 causes the accumulation of mono-ubiquitinated PCNA at replication forks and a reduction in total PCNA levels. USP1 inhibitors promote the accumulation of ssDNA gaps (ssGAPs) in newly replicated DNA and are synthetic lethality in BRCA1-deficient cells. Here, we combine computational and structural approaches to identify and characterize a PCNA-interacting peptide (PIP) motif in RAD18. This PIP motif is required for RAD18-dependent DNA damage-induced PCNA ubiquitination and PCNA turnover. Mutation of the RAD18-PCNA interface reduces ssGAP accumulation and USP1 inhibitor sensitivity in BRCA1-deficient cells. Furthermore, cells adapted to prolonged USP1 inhibition exhibit reduced RAD18 levels, suggesting that deregulation of RAD18 contributes to a biologically relevant drug resistance mechanism. This resistance could be overcome by inhibiting the Ataxia telangiectasia and Rad3-related (ATR) kinase. Together, these findings define a molecular interface required for RAD18-dependent PCNA mono-ubiquitination and identify it as a key determinant of USP1-BRCA1 synthetic lethality.