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  • Increased levels of PCNA monoubiquitination

    2019-09-18

    Increased levels of PCNA monoubiquitination by UbL73P expression in a damage-independent manner mimics the phenotype observed for USP1 knockdown (Huang et al., 2006, Jones et al., 2012). USP1 is the only DUB to date shown to remove ubiquitin from PCNA in vivo. This finding reveals the highly dynamic nature of PCNA monoubiquitination in undamaged SR1078 mass by the Rad18-USP1 E3-DUB cycle and underscores the crucial regulatory role of USP1 in maintaining PCNA in an unubiquitinated state. This regulation ensures that PCNA monoubiquitination will not serve as a platform to recruit low-fidelity translesion synthesis (TLS) polymerases (Lehmann et al., 2007) in the absence of DNA damage. Failure to maintain appropriate PCNA monoubiquitination levels could result in increased TLS polymerase recruitment, such as that of polymerase kappa (Jones et al., 2012), which increases genomic instability. Despite the important role of ubiquitination-dependent mechanisms in DSB repair (Jackson and Durocher, 2013), UbL73P displayed no global defects in DNA repair mechanisms following bleomycin treatment or ionizing radiation, perhaps because UbL73P was incompletely utilized or because the need for deubiquitination was bypassed. However, the phenotype brought about by UbL73P, in the context of the activation of spontaneous DNA damage response in the absence of the shelterin complex at telomeres, suggests that UbL73P is a functional player and results in the Ub conjugate stabilization of one or more factors that are required for efficient 53BP1 clearance at chromosomal ends. This is in accordance with a recent finding showing that telomeric and genomic DDR pathways are quite different (Cesare et al., 2013): telomeric DDR does not activate checkpoint signaling and could rely on entirely different sets of effector proteins, some of which could be sensitive to stabilized ubiquitination. The telomeric UbL73P phenotype mimics the one observed with the knockdown of BRCC3, which opposes the activity of RNF168 at telomeres (Okamoto et al., 2013). The identity of the ubiquitinated substrate(s) that are responsible for the activation/regulation of telomeric DDR is currently unknown. In the future, it will be important to discern global versus telomeric ubiquitination substrates in DNA damage response pathways. In our exploratory UbL73P substrate identification survey, we validated the approach by identifying known ubiquitination sites on PCNA and others (Povlsen et al., 2012, Zhou et al., 2008). We also identified several ubiquitinated substrates stabilized by UbL73P. We were able to confirm the ubiquitination of Ubc13 on Lys92, a previously indicated site of ISG15 modification (Giannakopoulos et al., 2005). Additionally, we identified the site of ubiquitination on Ube2T, another E2 enzyme that has been previously shown to be ubiquitinated (Machida et al., 2006). Intriguingly, several other E2 Ub-conjugating enzymes were found in our mass spectrometry results. Whether reversible E2 ubiquitination is a regulatory posttranslational modification common to a variety of E2 enzymes remains to be determined. The utility of DUB-resistant ubiquitin tools is highly promising. First, it is possible to ubiquitinate substrates using UbL73P in vitro and assay the stable Ub conjugate in subsequent binding or affinity purification experiments in search of binding partners. Second, the UbL73P-stabilized ubiquitin chains of a defined linkage could also be utilized to identify substrates modified by a particular ubiquitin chain linkage or identify binding proteins unique to a particular ubiquitin chain linkage. This approach will be most beneficial for chains utilizing hitherto uncharacterized linkages, such as Lys29 and Lys33, and will also allow the purification and identification of ubiquitin receptors that recognize these unique chains. Finally, as unanchored oligomeric ubiquitin chains appear to be stabilized by both UbG76A and by UbL73P, such DUB-resistant ubiquitin mutants have the potential to shed light on the nature and the dynamics of de novo unanchored ubiquitin chain formation, both in resting cells and in a signal inducible manner. This has been a relatively unexplored area of research, as only recently have unanchored ubiquitin chains been implicated in kinase activation (Xia et al., 2009) and in antiviral immunity (Zeng et al., 2010). As long as the conjugation competency for a particular pathway is adequate, we envision the use of DUB-resistant ubiquitins and other isopeptidase-resistant ubiquitin-like molecules by researchers who seek to interrogate ubiquitin and ubiquitin-like-mediated biological processes in a wide range of experimental settings.