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      Histone H3K56 Acetylation, Rad52, and Non-DNA Repair Factors Control Double-Strand Break Repair Choice with the Sister Chromatid

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          Abstract

          DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation.

          Author Summary

          Double-strand breaks (DSBs) are among the most dangerous DNA lesions and can lead to genomic instability, a process associated with cancer and hereditary diseases. An important source of DSBs is replication, Sister Chromatid Recombination (SCR) being the main mechanism for DSB repair in dividing eukaryotic cells. SCR repair is error-free and uses the sister chromatid as template, generating an identical DNA sequence and therefore preventing genomic instability. In this work, we use an inverted-repeat assay with which we can physically detect SCR intermediates generated by the repair of a replication-born DSB. We hypothesized that SCR defects can result in an increase of recombination with the homologous chromosome, so we assayed SCR in 28 mutants previously described to increase homolog recombination. Our results describe 8 new genes involved in SCR, including functions such as histone acetylation/deacetylation, SUMO-Ubiquitin metabolism, and stress response, as well as an allele of RAD52. This demonstrates the importance of the choice of the sister chromatid as template for DSB repair and provides a broad vision of SCR as a tightly regulated process essential for genome integrity.

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          Break-induced replication and telomerase-independent telomere maintenance require Pol32.

          Break-induced replication (BIR) is an efficient homologous recombination process to initiate DNA replication when only one end of a chromosome double-strand break shares homology with a template. BIR is thought to re-establish replication at stalled and broken replication forks and to act at eroding telomeres in cells that lack telomerase in pathways known as 'alternative lengthening of telomeres' (reviewed in refs 2, 6). Here we show that, in haploid budding yeast, Rad51-dependent BIR induced by HO endonuclease requires the lagging strand DNA Polalpha-primase complex as well as Poldelta to initiate new DNA synthesis. Polepsilon is not required for the initial primer extension step of BIR but is required to complete 30 kb of new DNA synthesis. Initiation of BIR also requires the nonessential DNA Poldelta subunit Pol32 primarily through its interaction with another Poldelta subunit, Pol31. HO-induced gene conversion, in which both ends of a double-strand break engage in homologous recombination, does not require Pol32. Pol32 is also required for the recovery of both Rad51-dependent and Rad51-independent survivors in yeast strains lacking telomerase. These results strongly suggest that both types of telomere maintenance pathways occur by recombination-dependent DNA replication. Thus Pol32, dispensable for replication and for gene conversion, is uniquely required for BIR; this finding provides an opening into understanding how DNA replication re-start mechanisms operate in eukaryotes. We also note that Pol32 homologues have been identified both in fission yeast and in metazoans where telomerase-independent survivors with alternative telomere maintenance have also been identified.
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            CBP / p300-mediated acetylation of histone H3 on lysine 56

            Acetylation within the globular core domain of histone H3 on lysine 56 has recently been shown to play a critical role in packaging DNA into chromatin following DNA replication and repair in budding yeast 1, 2. However, the function or occurrence of this specific histone mark has not been studied in multi-cellular eukaryotes, mainly because the Rtt109 enzyme that is known to mediate acetylation of H3 K56 (H3 K56Ac) is fungal-specific 3 4. Here we demonstrate that in flies and humans the histone acetyl transferases CBP / p300 acetylate H3 K56, while Sir2 / hSirT1 / hSirT2 deacetylate H3 K56Ac. The histone chaperone Asf1 in Drosophila, Asf1a in humans, is required for acetylation of H3 K56 in vivo, while the histone chaperone CAF-1 is required for the incorporation of histones bearing this mark into chromatin. We show that in response to DNA damage, histones bearing acetylated K56 are assembled into chromatin in Drosophila and human cells, forming foci that colocalize with sites of DNA repair. Furthermore, acetylation of H3 K56 is elevated in multiple types of cancer, correlating with elevated levels of Asf1a in these tumors. Our identification of multiple proteins regulating the levels of H3 K56 acetylation in higher eukaryotes will allow future studies of this critical and unique histone modification that couples chromatin assembly to DNA synthesis, cell proliferation and cancer.
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              Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks.

              DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.
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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
                January 2013
                January 2013
                24 January 2013
                : 9
                : 1
                : e1003237
                Affiliations
                [1 ]Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla–CSIC, Sevilla, Spain
                [2 ]Department of Genetics and Development, Columbia University Medical Center, New York, New York, United States of America
                NYU Medical Center, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: SM-G SJ RR AA. Performed the experiments: SM-G SJ. Analyzed the data: SM-G SJ RR AA. Wrote the paper: SM-G AA.

                Article
                PGENETICS-D-12-02368
                10.1371/journal.pgen.1003237
                3554610
                23357952
                3af31b9a-44bd-43cb-8283-6150ef08c162
                Copyright @ 2013

                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
                : 18 September 2012
                : 27 November 2012
                Page count
                Pages: 12
                Funding
                Research was funded by grants from the Spanish Ministry of Science and Innovation (BFU2010-16372 and Consolider Ingenio 2010 CSD2007-0015), the Junta de Andalucía (BIO-102 and P09/CVI4567), and the European Union (FEDER) to AA and from the National Institutes of Health (GM50237 and GM67055) to RR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Genetics
                Epigenetics
                Histone Modification
                Gene Expression
                Histone Modification
                Cancer Genetics
                Gene Function
                Genetic Screens
                Molecular Genetics
                Model Organisms
                Yeast and Fungal Models
                Saccharomyces Cerevisiae
                Molecular Cell Biology
                Nucleic Acids
                DNA
                DNA metabolism
                DNA recombination
                DNA repair
                DNA replication
                DNA synthesis
                Cellular Stress Responses

                Genetics
                Genetics

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