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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.<\/jats:p>","DOI":"10.64898\/2026.02.25.708068","type":"posted-content","created":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T20:20:13Z","timestamp":1772223613000},"source":"Crossref","is-referenced-by-count":0,"title":["USP1 inhibition promotes RAD18-dependent PCNA degradation and BRCA1 synthetic lethality"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9708-6319","authenticated-orcid":false,"given":"Nicholas W.","family":"Ashton","sequence":"first","affiliation":[{"name":"Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 4 Blackfan Street, Boston, MA 02215, 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