Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.Monoubiquitination is a highly regulated process that is conserved in all eukaryotes 1,2 and controls a broad range of cellular functions, including DNA repair. Protein monoubiquitination is a reversible post-translational event that can be influenced by the opposing activities of a ubiquitin E3 ligase and a deubiquitinating enzyme (DUB), similar to the regulation of protein phosphorylation by kinases and phosphatases 3,4 . Protein monoubiquitination regulates the rescue of stalled DNA replication forks -an important cellular process required for cell survival 5 . The E2 ubiquitin conjugating enzyme RAD6 and the E3 ligase RAD18 are conserved in both yeast and human and they coordinate and activate the monoubiquitination of PCNA in response to UV damage or stalled replication forks [6][7][8] . Recent studies have shown that polη, a specialized TLS polymerase, is recruited to the replication fork through a specific interaction with monoubiquitinated PCNA 7 . The deployment of TLS polymerases during replication ensures timely bypass of the diverse DNA lesions encountered by the replication fork. Although many TLS polymerases are intrinsically mutagenic, polη allows replication past UV-damaged bases with high fidelity 9-11 . Thus, a model has emerged in which polη binds to monoubiquitinated PCNA and ensures accurate (error-free) replicative bypass of UV lesions. However, other (error-prone) TLS polymerases (such as polι and Rev1) have recently been shown to rely on monoubiquitinated PCNA for their function 12,13 . How cells limit PCNA monoubiquitination and the unwanted deployment of polη and/or other error-prone TLS polymerases in the absence or presence of extrinsic DNA damage during the synthesis of DNA in S phase is not known.In humans, protein deubiquitination is controlled by a family of approximately 95 distinct DUB enzymes 14,15 but the function of most of these proteins is unknown. DUBs are cysteine proteases that cleave ubiquitin fro...

Proteasome inhibitors, such as the dipeptide boronic acid bortezomib, are emerging as important tools in the treatment of the fatal hematologic malignancy multiple myeloma. Despite the recent US Food and Drug Administration approval of bortezomib (PS341, Velcade) for the treatment of refractory multiple myeloma, many of the basic pharmacologic parameters of bortezomib and its mode of action on myeloma cells remain to be determined. We describe the synthesis and use of a cell-permeant active site-directed probe, which allows profiling of proteasomal activities in living cells. When we compared proteasome activity patterns in cultured cells and crude cell extracts with this probe, we observed substantial differences, stressing the importance for bioassays compatible with live cells to ensure accuracy of such measurements. Using this probe, we investigated the in vivo subunit specificities of bortezomib and another inhibitor, MG132.