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      Reversal of PCNA Ubiquitylation by Ubp10 in Saccharomyces cerevisiae

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          Abstract

          Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2B K123 ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae. Our results sustain that Ubp10-dependent deubiquitylation of the sliding clamp PCNA normally takes place during S phase, likely in response to the simple presence of ubPCNA. In agreement with this, we show that Ubp10 forms a complex with PCNA in vivo. Interestingly, we also show that deletion of UBP10 alters in different ways the interaction of PCNA with DNA polymerase ζ–associated protein Rev1 and with accessory subunit Rev7. While deletion of UBP10 enhances PCNA–Rev1 interaction, it decreases significantly Rev7 binding to the sliding clamp. Finally, we report that Ubp10 counteracts Rad18 E3-ubiquitin ligase activity on PCNA at lysine 164 in such a manner that deregulation of Ubp10 expression causes tolerance impairment and MMS hypersensitivity.

          Author Summary

          DNA damage is a major source of genome instability and cancer. A universal mechanism of DNA damage tolerance is based on translesion synthesis (TLS) by specialized low-fidelity DNA polymerases capable of replicating over DNA lesions during replication. Translesion synthesis requires the switch between replicative and TLS DNA polymerases, and this switching is controlled through the ubiquitylation of the proliferating-cell nuclear antigen (PCNA), a processivity factor for DNA synthesis. It is thought that DNA polymerase switching is a reversible process that has a favorable outcome for cells in the prevention of irreversible DNA replication forks collapse. However, the low-fidelity nature of TLS polymerases has unfavorable consequences like the increased risk of mutations opposite to DNA lesions. Here we identify Ubp10 as an enzyme controlling PCNA deubiquitylation in the model yeast S. cerevisiae. The identification of Ubp10 is a first step that will allow us to understand its biological significance and its potential role as part of a safeguard mechanism limiting the residence time of TLS DNA polymerases on replicating chromatin in eukaryotes.

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          Most cited references75

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          A genomic and functional inventory of deubiquitinating enzymes.

          Posttranslational modification of proteins by the small molecule ubiquitin is a key regulatory event, and the enzymes catalyzing these modifications have been the focus of many studies. Deubiquitinating enzymes, which mediate the removal and processing of ubiquitin, may be functionally as important but are less well understood. Here, we present an inventory of the deubiquitinating enzymes encoded in the human genome. In addition, we review the literature concerning these enzymes, with particular emphasis on their function, specificity, and the regulation of their activity.
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            RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.

            The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.
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              PCNA, the maestro of the replication fork.

              Inheritance requires genome duplication, reproduction of chromatin and its epigenetic information, mechanisms to ensure genome integrity, and faithful transmission of the information to progeny. Proliferating cell nuclear antigen (PCNA)-a cofactor of DNA polymerases that encircles DNA-orchestrates several of these functions by recruiting crucial players to the replication fork. Remarkably, many factors that are involved in replication-linked processes interact with a particular face of PCNA and through the same interaction domain, indicating that these interactions do not occur simultaneously during replication. Switching of PCNA partners may be triggered by affinity-driven competition, phosphorylation, proteolysis, and modification of PCNA by ubiquitin and SUMO.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                July 2012
                July 2012
                19 July 2012
                : 8
                : 7
                : e1002826
                Affiliations
                [1 ]Instituto de Biología Molecular y Celular del Cáncer, Universidad de Salamanca/CSIC, Salamanca, Spain
                [2 ]Instituto de Biología Funcional y Genómica, Universidad de Salamanca/CSIC, Salamanca, Spain
                [3 ]Departamento de Microbiología y Genética, Universidad de Salamanca/CSIC, Salamanca, Spain
                University of Washington, United States of America
                Author notes

                Conceived and designed the experiments: AG-S PAS-S AB. Performed the experiments: AG-S SA FC PAS-S. Analyzed the data: AG-S PAS-S AB. Wrote the paper: AB. Performed immunoprecipitation experiments: AG-S SA. Performed nuclear spreads and mutagenesis experiments: AG-S FC PAS-S.

                Article
                PGENETICS-D-11-02523
                10.1371/journal.pgen.1002826
                3400564
                22829782
                b899ce13-ce3b-4bdc-a96d-641ac51fede7
                Gallego-Sánchez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 21 November 2011
                : 25 May 2012
                Page count
                Pages: 16
                Categories
                Research Article
                Biology

                Genetics
                Genetics

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