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      Hyperactivation of PARP Triggers Nonhomologous End-Joining in Repair-Deficient Mouse Fibroblasts

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          Abstract

          Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.

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          Most cited references48

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          DNA double-strand breaks: signaling, repair and the cancer connection.

          To ensure the high-fidelity transmission of genetic information, cells have evolved mechanisms to monitor genome integrity. Cells respond to DNA damage by activating a complex DNA-damage-response pathway that includes cell-cycle arrest, the transcriptional and post-transcriptional activation of a subset of genes including those associated with DNA repair, and, under some circumstances, the triggering of programmed cell death. An inability to respond properly to, or to repair, DNA damage leads to genetic instability, which in turn may enhance the rate of cancer development. Indeed, it is becoming increasingly clear that deficiencies in DNA-damage signaling and repair pathways are fundamental to the etiology of most, if not all, human cancers. Here we describe recent progress in our understanding of how cells detect and signal the presence and repair of one particularly important form of DNA damage induced by ionizing radiation-the DNA double-strand break (DSB). Moreover, we discuss how tumor suppressor proteins such as p53, ATM, Brca1 and Brca2 have been linked to such pathways, and how accumulating evidence is connecting deficiencies in cellular responses to DNA DSBs with tumorigenesis.
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            Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

            Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism.
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              The PARP side of the nucleus: molecular actions, physiological outcomes, and clinical targets.

              The abundant nuclear enzyme PARP-1, a multifunctional regulator of chromatin structure, transcription, and genomic integrity, plays key roles in a wide variety of processes in the nucleus. Recent studies have begun to connect the molecular functions of PARP-1 to specific physiological and pathological outcomes, many of which can be altered by an expanding array of chemical inhibitors of PARP enzymatic activity. 2010 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                7 November 2012
                : 7
                : 11
                : e49301
                Affiliations
                [1]Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America
                King Faisal Specialist Hospital & Research Center, Saudi Arabia
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: NRG SHW. Performed the experiments: NRG DFS PSK. Analyzed the data: NRG DFS JKH. Wrote the paper: NRG SHW.

                Article
                PONE-D-12-26931
                10.1371/journal.pone.0049301
                3492265
                23145148
                c7fb63bf-e1bb-4e4e-b5bf-aef8a5c01705
                Copyright @ 2012

                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 6 September 2012
                : 8 October 2012
                Page count
                Pages: 10
                Funding
                This research was supported by Research Project Numbers Z01-ES050158 and Z01-ES050159 in the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Nucleic Acids
                DNA
                DNA repair
                Molecular Cell Biology
                Signal Transduction
                Signaling Cascades
                Stress Signaling Cascade
                Signaling in Cellular Processes
                Cell Death
                Cellular Stress Responses

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                Uncategorized

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