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Characterization of SPRTN, the first mammalian metalloprotease that repairs DNA-protein-crosslinks
(2019)
DNA is constantly exposed to various endogenous and exogenous sources causing different kinds of DNA damage. To overcome this threat, cells have evolved various repair mechanisms. Impairments of these repair mechanisms result in diverse diseases. Ruijs-Aalfs syndrome is a monogenic disease characterized by accelerated ageing and carcinogenesis, typical features of impaired DNA repair and was shown to be caused by germline mutations of SPRTN, a newly identified and only partially understood protein. A role of SPRTN in DNA damage response was previously shown and an involvement in translesion synthesis (TLS) proposed. However, later discoveries revealed an essential function of SPRTN, being indispensible for embryonic development of vertebrates and cellular survival, whereby this function is independent of SPRTN’s proposed function in TLS. The essential function of SPRTN was proposed to be contained in its protease domain but remained unclear.
In this study we identify SPRTN as the first mammalian metalloprotease that repairs DNA-protein-crosslinks (DPCs). DPCs represent a specific type of DNA-lesions with bulky protein adducts covalently linked to DNA thereby being highly toxic as they potentially stall replication forks and lead to double strand breaks and genomic instability. DPC-repair remains only partially understood despite their frequent appearance and toxicity. With this study we discover and characterize a new mechanism of DPC-repair in mammalian cells - a proteolytic cleavage of the protein adduct by the metalloprotease SPRTN. Accordingly, a proteolytic activity of SPRTN is demonstrated and s SPRTN-recruitment to DNA upon DPC-induction displayed. Furthermore, SPRTN exhibits degradation of different proteins covalently bound to DNA in form of DPCs, but not of unbound fractions of the same protein substrates. Consequently, mutations of SPRTN’s proteolytic core as well as a mislocalization or depletion of SPRTN result in impaired DPC-repair. The importance of SPRTN-mediated DPC-removal is confirmed by a severely compromised response to DPC-inducing agents for cells with impaired SPRTN function. Additionally to the discovery of SPRTN’s essential function this study further provides an explanation of the molecular mechanism underlying Ruijs-Aalfs syndrome (RJALS), the segmental progeroid syndrome resulting from SPRTN mutation. The effects of the identified clinical mutations on the DPC-repair function of SPRTN are explained and a DPC-accumulation in cells carrying clinical SPRTN-mutation displayed. The obtained data provides sufficient evidence that an impaired DPC-repair is the pathophysiologic cause of RJALS-syndrome, confirming the importance of SPRTN’s newly identified function. In conclusion, SPRTN is the first identified mammalian metalloprotease with a DPC-repairing function and the impairment of SPRTN-mediated DPC-removal is the underlying mechanism of RJALS syndrome.
Ruijs-Aalfs syndrome is a segmental progeroid syndrome resulting from mutations in the SPRTN gene. Cells derived from patients with SPRTN mutations elicit genomic instability and people afflicted with this syndrome developed hepatocellular carcinoma. Here we describe the molecular mechanism by which SPRTN contributes to genome stability and normal cellular homeostasis. We show that SPRTN is a DNA-dependent mammalian protease required for resolving cytotoxic DNA-protein crosslinks (DPCs)— a function that had only been attributed to the metalloprotease Wss1 in budding yeast. We provide genetic evidence that SPRTN and Wss1 function distinctly in vivo to resolve DPCs. Upon DNA and ubiquitin binding, SPRTN can elicit proteolytic activity; cleaving DPC substrates and itself. SPRTN null cells or cells derived from patients with Ruijs-Aalfs syndrome are impaired in the resolution of covalent DPCs in vivo. Collectively, SPRTN is a mammalian protease required for resolving DNA-protein crosslinks in vivo whose function is compromised in Ruijs-Aalfs syndrome patients.