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Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation.

Genome Stability Laboratory, Department of Biochemistry and National Centre for Biomedical Engineering Science, National University of Ireland, University Road, Galway, Ireland.
DNA Repair (Impact Factor: 3.36). 07/2006; 5(6):693-703. DOI: 10.1016/j.dnarep.2006.03.005
Source: PubMed

ABSTRACT In budding yeast, the Rad9 protein is an important player in the maintenance of genomic integrity and has a well-characterised role in DNA damage checkpoint activation. Recently, roles for different post-translational histone modifications in the DNA damage response, including H2A serine 129 phosphorylation and H3 lysine 79 methylation, have also been demonstrated. Here, we show that Rad9 recruitment to foci and bulk chromatin occurs specifically after ionising radiation treatment in G2 cells. This stable recruitment correlates with late stages of double strand break (DSB) repair and, surprisingly, it is the hypophosphorylated form of Rad9 that is retained on chromatin rather than the hyperphosphorylated, checkpoint-associated, form. Stable Rad9 accumulation in foci requires the Mec1 kinase and two independently regulated histone modifications, H2A phosphorylation and Dot1-dependent H3 methylation. In addition, Rad9 is selectively recruited to a subset of Rad52 repair foci. These results, together with the observation that rad9Delta cells are defective in repair of IR breaks in G2, strongly indicate a novel post checkpoint activation role for Rad9 in promoting efficient repair of DNA DSBs by homologous recombination.

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    • "In response to IR damage, the Dot1/Rad6/Bre1 pathway mediates DNA damage repair through G1 homologous recombination (HR) (Game et al. 2006). Although Dot1 deletion mutants do not display a G2 arrest phenotype (Game et al. 2006), Dot1 is required for Rad9 recruitment to the damage foci and phosphorylation of Rad53 upon IR (Toh et al. 2006). Dot1-mediated H3K79 methylation is believed to play two distinct roles in Rad9-mediated DNA damage response (Grenon et al. 2007): the activation of the G1/S checkpoint, and the repair of DNA damage at late G2 phase. "
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    ABSTRACT: DOT1 (disruptor of telomeric silencing; also called Kmt4) was initially discovered in budding yeast in a genetic screen for genes whose deletion confers defects in telomeric silencing. Since the discovery ∼10 years ago that Dot1 and its mammalian homolog, DOT1L (DOT1-Like), possess histone methyltransferase activity toward histone H3 Lys 79, great progress has been made in characterizing their enzymatic activities and the role of Dot1/DOT1L-mediated H3K79 methylation in transcriptional regulation, cell cycle regulation, and the DNA damage response. In addition, gene disruption in mice has revealed that mouse DOT1L plays an essential role in embryonic development, hematopoiesis, cardiac function, and the development of leukemia. The involvement of DOT1L enzymatic activity in leukemogenesis driven by a subset of MLL (mixed-lineage leukemia) fusion proteins raises the possibility of targeting DOT1L for therapeutic intervention.
    Genes & development 07/2011; 25(13):1345-58. DOI:10.1101/gad.2057811 · 12.64 Impact Factor
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    • "G2/M checkpoint assays in yeast commonly use nocodazole, a benzimidazole drug that destabilizes tubulin dimers [72]. Nocodazole treatment disrupts mitotic spindle formation and arrests cells prior to anaphase by activating the mitotic spindle checkpoint [73]. Previous analysis of the response to ionizing radiation in nocodazole-treated hta1, 2-S129A, dot1Δ, and rad9-Y798Q strains showed that each strain fully activates Rad53 and exhibits a characteristic large budded arrest [8] [10] [74] [75]. "
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    ABSTRACT: In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition--much like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for the activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5' strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle.
    DNA repair 10/2009; 8(12):1452-61. DOI:10.1016/j.dnarep.2009.09.010 · 3.36 Impact Factor
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    • "Indeed, it has been reported that Rad53 is required for DSB-induced SCR (Fasullo et al. 2005). However, a ''late'' role for Rad9 in DSB repair by HR, distinct from its ''early'' checkpoint function on Rad53 activation, has been proposed because Rad9 chromatin retention and colocalization with a subset of Rad52 foci correlate with checkpoint signaling downregulation and reconstitution of intact chromosomes after IR treatment (Toh et al. 2006). Moreover, the impaired G2/M checkpoint arrest of the rad9 mutant cannot solely explain the defect in SCR, because the dot1 and H2A-S129 mutants are quite proficient at DNA-damageinduced G2/M arrest (Wysocki et al. 2005; Javaheri et al. 2006), but they display similar SCR defects. "
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    ABSTRACT: Genomic integrity is threatened by multiple sources of DNA damage. DNA double-strand breaks (DSBs) are among the most dangerous types of DNA lesions and can be generated by endogenous or exogenous agents, but they can arise also during DNA replication. Sister chromatid recombination (SCR) is a key mechanism for the repair of DSBs generated during replication and it is fundamental for maintaining genomic stability. Proper repair relies on several factors, among which histone modifications play important roles in the response to DSBs. Here, we study the role of the histone H3K79 methyltransferase Dot1 in the repair by SCR of replication-dependent HO-induced DSBs, as a way to assess its function in homologous recombination. We show that Dot1, the Rad9 DNA damage checkpoint adaptor, and phosphorylation of histone H2A (gammaH2A) are required for efficient SCR. Moreover, we show that Dot1 and Rad9 promote DSB-induced loading of cohesin onto chromatin. We propose that recruitment of Rad9 to DSB sites mediated by gammaH2A and H3K79 methylation contributes to DSB repair via SCR by regulating cohesin binding to damage sites. Therefore, our results contribute to an understanding of how different chromatin modifications impinge on DNA repair mechanisms, which are fundamental for maintaining genomic stability.
    Genetics 04/2009; 182(2):437-46. DOI:10.1534/genetics.109.101899 · 4.87 Impact Factor
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Geraldine Wei-Ling Toh