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      Rad59 regulates association of Rad52 with DNA double-strand breaks

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          Abstract

          Homologous recombination among repetitive sequences is an important mode of DNA repair in eukaryotes following acute radiation exposure. We have developed an assay in Saccharomyces cerevisiae that models how multiple DNA double-strand breaks form chromosomal translocations by a nonconservative homologous recombination mechanism, single-strand annealing, and identified the Rad52 paralog, Rad59, as an important factor. We show through genetic and molecular analyses that Rad59 possesses distinct Rad52-dependent and -independent functions, and that Rad59 plays a critical role in the localization of Rad52 to double-strand breaks. Our analysis further suggests that Rad52 and Rad59 act in multiple, sequential processes that determine genome structure following acute exposure to DNA damaging agents.

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          Molecular Cloning : A Laboratory Manual

          Joseph Sambrook, David William Russell (2001)
          <p>The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.<br>In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.<br>Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques.<br>The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.<br>These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.<br>The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions.<br>The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.<br>As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. </p>
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            A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli.

            C Hoffman, F Winston (1987)
            A procedure for the rapid isolation of DNA from the yeast Saccharomyces cerevisiae is described. To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform. Centrifugation of this mixture separates the DNA from cellular debris. E. coli can be efficiently transformed with plasmid present in the aqueous layer without further purification of the plasmid DNA. This procedure also releases chromosomal DNA. Following two ethanol precipitations, the chromosomal DNA can be digested by restriction endonucleases and analysed by Southern blot analysis.
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              Genetic steps of mammalian homologous repair with distinct mutagenic consequences.

              Jeremy M. Stark, Andrew J Pierce, Jin Joo Oh … (2004)
              Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52(-)(/)(-) mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Microbiologyopen
                Journal ID (iso-abbrev): Microbiologyopen
                Journal ID (publisher-id): mbo3
                Title: MicrobiologyOpen
                Publisher: Blackwell Publishing Ltd
                ISSN (Print): 2045-8827
                ISSN (Electronic): 2045-8827
                Publication date (Print): September 2012
                Publication date (Electronic): 03 August 2012
                Volume: 1
                Issue: 3
                Pages: 285-297
                Affiliations
                [1 ]Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope Duarte, California, 91010, USA
                [2 ]The Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope Duarte, California, 91010, USA
                [3 ]College of Biological Sciences, University of California Davis, California, 95616, USA
                Author notes
                Adam M. Bailis, Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA. Tel: (626) 359-8111, ext. 64031; Fax: (626) 930-5463; E-mail: abailis@ 123456coh.org

                Funding Information This work was supported by funds from the National Institutes of Health (GM057484 to A. M. B.), the Eugene and Ruth Roberts Summer Academy, and the Beckman Research Institute of the City of Hope.

                [*]

                Current address: Departments of Pathology and Biochemistry & Molecular Biology, USC Norris Comprehensive Cancer Center, Los Angeles, CA, 90089, USA

                Article
                DOI: 10.1002/mbo3.31
                PMC ID: 3496973
                PubMed ID: 23170228
                SO-VID: 9aef743c-70ab-421b-872b-c1256048e575
                Copyright © © 2012 Published by Blackwell Publishing Ltd.
                License:

                Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

                History
                Date received : 17 April 2012
                Date revision received : 06 June 2012
                Date accepted : 02 July 2012
                Categories
                Subject: Original Research

                ScienceOpen disciplines: Microbiology & Virology
                Keywords: saccharomyces cerevisiae,homologous recombination,dna double-strand breaks,single-strand annealing,chromosomal translocations
                Data availability:
                ScienceOpen disciplines: Microbiology & Virology
                Keywords: saccharomyces cerevisiae, homologous recombination, dna double-strand breaks, single-strand annealing, chromosomal translocations

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