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  • Discussion In summary we have

    2020-07-31

    Discussion In summary, we have introduced and characterized a ubiquitin point mutant capable of conjugating to cognate substrates and incorporating into ubiquitin chains, yet remains refractory to cleavage by DUBs. This provides a unique tool to enable the generation, identification, and study of substrates with stabilized ubiquitination states. Previous work by Békés et al. (2011) on the related ubiquitin-like molecule SUMO2 showed that mutation in the P4 position of the SUMO cleavage site for deSUMOylating enzymes (SENPs) from glutamine to proline (Figure S1A, Q90P, which is also found naturally in the pseudogene, SUMO4 [Bohren et al., 2007]), results in resistance to cleavage by deSUMOylating enzymes, allowing the generation of stabilized SUMO2-conjugates in cells. We then asked whether mutation of the corresponding residue (Leu73) on ubiquitin could affect ubiquitin cleavage reactions by DUBs, and we indeed show that it does. Interestingly, in an alanine-scanning mutagenesis study of yeast ubiquitin more than a decade ago, it was shown that UbL73A allows for ubiquitin conjugation; however, the mutant is partially defective in an S. cerevisiae WAY 208466 dihydrochloride assay (Sloper-Mould et al., 2001). A recent study has also identified bulky Leu73 mutations that differentially affect conjugation and deconjugation activities (Zhao et al., 2012). Furthermore, yeast provided with the UbL73A mutant as the only source of ubiquitin cannot support vegetative growth (Sloper-Mould et al., 2001). It is possible that the Leu73 mutant phenotype of ubiquitin in yeast is a composite of defects in conjugation as well as deconjugation. Leu73 contributes to part of the hydrophobic patch in ubiquitin centered around Ile36, which is utilized by some E3 ligases to synthesize polyubiquitin chains (Plechanovová et al., 2012). Therefore, in accordance with our in vitro E1/E2/E3 conjugation data, it is possible that certain substrates, whose E3 ligases solely rely on ubiquitin Ile36 hydrophobic interactions, would not be efficiently conjugated by UbL73P. This selectivity at the E3 level, together with the selectivity of the E1 enzymes, Ube1 and Uba6, in differentially charging E2 enzymes, suggests that the conjugation of UbL73P in vivo would be skewed toward conjugation pathways that can tolerate it. Nevertheless, those UbL73P conjugates would remain stable and resistant to DUBs in cells, as is the case for monoubiquitinated PCNA, the identified Ubc13, unknown factor(s) in the telomeric DDR pathway, and others. 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 cells 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.