PROJECT SUMMARY DNA-Protein crosslinks (DPCs) are irreversible covalent crosslinks of proteins to DNA that stall replication forks during DNA replication. Unrepaired stalled forks lead to DNA breaks and fork collapse, leading to genome instability, cell death or senescence. SPRTN is a DNA-dependent replication-coupled metalloprotease that catalyzes proteolysis of DPCs during DPC repair. SPRTN also regulates replication fork progression and translesion DNA synthesis. Ruijs-Aalfs (RJALS) syndrome patients with bi-allelic mutations in SPRTN protease domain are prone to genome instability, segmental progeria and early-onset hepatocellular carcinoma. Regulation of SPRTN protease function is critical for accurate DNA replication fork progression and DPC repair. This proposed study is designed to characterize novel regulators of SPRTN and investigate the molecular mechanism underlying SPRTN-mediated replication-coupled DPC repair. Investigating the regulation of SPRTN and SPRTN-mediated DPC repair pathway will further our understanding of the DPC repair pathway, delineate the mechanism of RJALS syndrome, and help develop novel strategies for sensitizing cancer cells to chemotherapy by targeting SPRTN-mediated DPC repair pathway.