Doctor of Philosophy (PhD)
Genetics, Cell Biology & Anatomy
Gargi Ghosal, PhD
DNA repair pathways that recognize and remove damaged DNA are vital for maintenance of genomic stability and prevention of tumorigenesis. Conversely, these pathways may be robust in tumor cells, thus diminishing the anti-cancer potential of available therapies. DNA-protein crosslinks (DPCs) are particularly deleterious DNA adducts that occur when proteins become irreversibly covalently bound to the DNA. DPCs represent a diverse group of lesions, as any protein can be crosslinked to the DNA duplex by non-specific crosslinking agents like reactive aldehydes and radiation. Additionally, functional DNA-binding proteins such as topoisomerases may become permanently crosslinked to DNA ends by abortive enzymatic processes or targeted chemotherapies. DPCs are steric blockades to all DNA metabolic processes, and unrepaired DPCs cause replication fork collapse, error-prone gene transcription, and inefficient DNA repair of non-DPC lesions. However, the process by which DPCs are removed is not well characterized.
Replication forks stall at DPCs and they must be proteolytically degraded before being bypassed by the replisome. Replication-dependent DPC degradation occurs via the proteasome or the DNA-dependent metalloprotease SPRTN. SPRTN cleaves substrates in a sequence-independent manner, and its function must be tightly regulated to prevent aberrant protein cleavage. Mechanisms of SPRTN regulation include DNA-binding, post-translational modification, and auto-proteolysis in trans. Previous work has shown that monoubiquitinated SPRTN is deubiquitinated upon DPC induction; thus, deubiquitinating enzymes are critical regulators of SPRTN function.
USP11 and USP7 have been previously implicated in DNA repair, but their role in promoting DPC removal is not known. We show that USP11 and USP7 interact with SPRTN and deubiquitinate SPRTN in cells and in vitro. Depletion of USP11 or USP7 causes hypersensitivity to DPC-inducing agents, accumulation of unrepaired DPCs, and impaired SPRTN deubiquitination upon formaldehyde treatment. In contrast to previous reports, we find that SPRTN monoubiquitination does not prevent chromatin association but instead enhances SPRTN auto-proteolysis. We predict that disruption of SPRTN deubiquitination depletes the pool of unmodified SPRTN available for DPC cleavage.
Our work identifies USP11 and USP7 as critical regulators of SPRTN protease activity, highlights the role of deubiquitinases in DPC repair and genome maintenance, and implicates new mechanistic targets for chemotherapy development.
Perry, Megan C., "USP11 and USP7 Deubiquitinases Regulate SPRTN Auto-proteolysis and SPRTN-mediated DNA-protein Crosslink Repair" (2021). Theses & Dissertations. 591.
Available for download on Friday, December 01, 2023