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      PARG is recruited to DNA damage sites through poly(ADP-ribose)- and PCNA-dependent mechanisms

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          Abstract

          Post-translational poly(ADP-ribosyl)ation has diverse essential functions in the cellular response to DNA damage as it contributes to avid DNA damage detection and assembly of the cellular repair machinery but extensive modification eventually also induces cell death. While there are 17 human poly(ADP-ribose) polymerase (PARP) genes, there is only one poly(ADP-ribose) glycohydrolase (PARG) gene encoding several PARG isoforms located in different subcellular compartments. To investigate the recruitment of PARG isoforms to DNA repair sites we locally introduced DNA damage by laser microirradiation. All PARG isoforms were recruited to DNA damage sites except for a mitochondrial localized PARG fragment. Using PARP knock out cells and PARP inhibitors, we showed that PARG recruitment was only partially dependent on PARP-1 and PAR synthesis, indicating a second, PAR-independent recruitment mechanism. We found that PARG interacts with PCNA, mapped a PCNA binding site and showed that binding to PCNA contributes to PARG recruitment to DNA damage sites. This dual recruitment mode of the only nuclear PARG via the versatile loading platform PCNA and by a PAR dependent mechanism likely contributes to the dynamic regulation of this posttranslational modification and ensures the tight control of the switch between efficient DNA repair and cell death.

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          Most cited references30

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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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            A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage.

            The molecular role of poly (ADP-ribose) polymerase-1 in DNA repair is unclear. Here, we show that the single-strand break repair protein XRCC1 is rapidly assembled into discrete nuclear foci after oxidative DNA damage at sites of poly (ADP-ribose) synthesis. Poly (ADP-ribose) synthesis peaks during a 10 min treatment with H2O2 and the appearance of XRCC1 foci peaks shortly afterwards. Both sites of poly (ADP-ribose) and XRCC1 foci decrease to background levels during subsequent incubation in drug-free medium, consistent with the rapidity of the single-strand break repair process. The formation of XRCC1 foci at sites of poly (ADP-ribose) was greatly reduced by mutation of the XRCC1 BRCT I domain that physically interacts with PARP-1. Moreover, we failed to detect XRCC1 foci in Adprt1-/- MEFs after treatment with H2O2. These data demonstrate that PARP-1 is required for the assembly or stability of XRCC1 nuclear foci after oxidative DNA damage and suggest that the formation of these foci is mediated via interaction with poly (ADP-ribose). These results support a model in which the rapid activation of PARP-1 at sites of DNA strand breakage facilitates DNA repair by recruiting the molecular scaffold protein, XRCC1.
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              Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death.

              Apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase, is released into the cytoplasm to induce cell death in response to poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation. How PARP-1 activation leads to AIF release is not known. Here we identify PAR polymer as a cell death signal that induces release of AIF. PAR polymer induces mitochondrial AIF release and translocation to the nucleus. PAR glycohydrolase, which degrades PAR polymer, prevents PARP-1-dependent AIF release. Cells with reduced levels of AIF are resistant to PARP-1-dependent cell death and PAR polymer cytotoxicity. These results reveal PAR polymer as an AIF-releasing factor that plays important roles in PARP-1-dependent cell death.
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                Author and article information

                Journal
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                July 2011
                July 2011
                11 March 2011
                11 March 2011
                : 39
                : 12
                : 5045-5056
                Affiliations
                1Ludwig Maximilians University Munich, Department of Biology, Center for Integrated Protein Science Munich, 82152 Planegg-Martinsried, Germany and 2Biotechnologie et Signalisation Cellulaire, UMR7242 CNRS, Université de Strasbourg, ESBS, Bd Sebastien Brant, BP 10413, 67412 Illkirch, France
                Author notes
                *To whom correspondence should be addressed. Tel: +33 3 68 85 47 04; Fax: +33 3 68 85 46 86; Email: valerie.schreiber@ 123456unistra.fr
                Correspondence may also be addressed to Heinrich Leonhardt. Email: h.leonhardt@ 123456lmu.de

                Present address: Oliver Mortusewicz, Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

                Article
                gkr099
                10.1093/nar/gkr099
                3130271
                21398629
                0491ca5c-7910-49bb-8a4d-03887deeb8c8
                © The Author(s) 2011. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 1 September 2010
                : 4 February 2011
                : 7 February 2011
                Page count
                Pages: 12
                Categories
                Genome Integrity, Repair and Replication

                Genetics
                Genetics

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