br Introduction The ubiquitination status of
Introduction The ubiquitination status of a target protein is achieved via a delicate balance between two opposing forces: ubiquitin E3 ligases and DUBs. It has been postulated that the majority of 3ma in a cell are regulated and modified by ubiquitin at some point (Hershko and Ciechanover, 1998); however, it has proved difficult to demonstrate the ubiquitination status of these proteins, as many of the modifications only exist transiently in vivo, owing largely to the multitude of DUBs present in cells. There are nearly 100 DUBs encoded in the human genome, which comprise approximately one-fifth of all proteases (Rawlings et al., 2010). DUBs are divided into five subfamilies based on catalytic mechanism and the fold of the active site domain (Reyes-Turcu et al., 2009). The largest family are the ubiquitin-specific proteases (USPs), followed by the ovarian tumor DUBs (OTUs), the ubiquitin C-terminal hydrolases (UCHs), the Josephin DUBs, and, last, the JAMM/MPN+ family member DUBs. Except for the JAMM/MPN+ family of DUBs, which are zinc-dependent metalloproteases, all other characterized DUBs are cysteine proteases. As the ubiquitin-modified status of a protein can fundamentally alter its properties and change its biological role, it is critical to capture the ubiquitinated state of a protein in order to investigate its biological function both in vitro and in vivo. The ubiquitin system has recently been exploited with the design of unique ubiquitin mutants that inhibit specific DUBs (Ernst et al., 2013, Zhang et al., 2013). Although the specificity of such mutants is remarkable, generating a novel mutant for each DUB has to begin de novo and is quite laborious, especially when the physiological substrates of many DUBs remain unknown. In this study, we designed and generated a DUB-resistant ubiquitin to capture and identify transiently ubiquitinated DUB substrates. Building on previous work in the SUMO conjugation and deconjugation pathway (Békés et al., 2011), we have generated a ubiquitin mutant (UbL73P) that is pleiotropically resistant to cleavage by multiple DUB families. This uncleavable ubiquitin mutant is conjugated to protein substrates in mammalian cells and leads to ubiquitin-conjugate stabilization. Ectopic expression of the DUB-resistant ubiquitin mutant stabilized monoubiquitinated PCNA, leading to the aberrant recruitment of translesion synthesis (TLS) polymerases in the absence of DNA damage, mimicking the effect of USP1 loss. Additional studies with DUB-resistant ubiquitin revealed a ubiquitin switch in the clearance of the DNA damage response (DDR) at shelterin-deficient chromosomal ends and captured ubiquitin-stabilized substrates by mass spectrometry. Our work provides a framework to study deubiquitination-dependent events both in vitro and in mammalian cells through the generation and use of the DUB-resistant ubiquitin tool.