Regulation of monoubiquitinated PCNA by DUB autocleavage

Abstract

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...

Deubiquitinases in cancer: new functions and therapeutic options

“…Reactive oxygen or nitrogen species can also post-translationally modify DUBs as illustrated by the hydrogen peroxide-mediated modification of Cezanne (Enesa et al, 2008a). Furthermore, some DUBs such as USP1 and ATXN3 are inactivated by autoproteolytic cleavage, whereas CYLD and A20 are inactivated through the action of other proteases (Huang et al, 2006;Mauri et al, 2006;Coornaert et al, 2008;Staal et al, 2011).…”

“…In this respect, the deubiquitylation of Fanconi's anemia protein (FANCD2) by USP1 and the subsequent stabilization of CHK1 are critical in DNA damage repair (Nijman et al, 2005;Guervilly et al, 2011). Moreover, USP1 modulates proliferating cell nuclear antigen ubiquitylation, a safeguard factor against errorprone DNA translesion synthesis that is ubiquitylated in response to genotoxic stress (Huang et al, 2006). As discussed above, USP1 forms a complex with U2 small nuclear ribonucleoparticle auxilliary factor 1 and promotes double-strand break repair through homologous recombination (Murai et al, 2011).…”

Protein monoubiquitylation: targets and diverse functions

“…In the absence of DNA damage, monoubiquitylation of PCNA is counteracted by USP1, the same DUB involved in ICL repair, and the switch from replicative to TLS polymerases is inhibited (Huang et al . 2006). …”

Translesion synthesis in mammalian cells

“…Poly-ubiquitination of PCNA has been very hard to detect. A further interesting feature of the regulation of PCNA ubiquitination following exposure to DNA damaging treatment is its association with the degradation of the de-ubiquitinating enzyme (DUB) USP1 [27], which is able to remove mono-ubiquitin from PCNA. Thus DNA damaging treatments result in both the activation of proteins that ubiquitinate PCNA (Rad6 and Rad18) and the degradation of the protein that de-ubiquitinates it (USP1).…”