<p>Replication stress is a therapeutic vulnerability in cancer, and the deubiquitinase USP1, in complex with its WD40 cofactor UAF1, is a central editor of the ubiquitin signals that govern tolerance and repair at stalled forks. By reversing monoubiquitination on the FANCI–FANCD2 clamp and on PCNA—preferentially when these complexes are DNA-bound—USP1–UAF1 functions as a reset valve that terminates repair-competent states and restores replisome progression. Structural and mechanistic advances now explain how this enzyme is druggable at a distance: cryo-EM of assembled USP1–UAF1 revealed a cryptic, non–active-site pocket whose occupancy subtly misaligns the catalytic center, establishing allosteric inhibition as the defining pharmacology. These insights enabled potent, selective chemical probes (e.g., ML323) and propelled next-generation agents into the clinic, led by KSQ-4279 (RO7623066/RG6614). In cells, USP1 blockade sustains FANCD2-Ub and Ub-PCNA and can produce “USP1-trapping” lesions—stabilized DUB–DNA complexes that amplify replication stress—providing a mechanistic basis for combination strategies with PARP, ATR/CHK1, and DNA-damaging agents. We synthesize the biological functions, complex architecture, and recognition logic of USP1–UAF1; delineate the druggability landscape and structure–mechanism integration that underwrite allostery; and survey the translational trajectory from first tools to clinical programs, including biomarkers, resistance hypotheses, and safety considerations. We also outline interface-level interventions—disrupting USP1–UAF1 assembly, nuclear import, or ATAD5-mediated platform recruitment—as orthogonal modalities. Together, these developments position USP1–UAF1 inhibition as a frontier approach in replication-stress–directed oncology and map the near-term priorities for delivering durable patient benefit.</p>