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      Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break


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          In budding yeast, a single double-strand break (DSB) triggers extensive ATM (Tel1) and ATR (Mec1)-dependent phosphorylation of histone H2A (γ-H2AX) around the DSB. We describe Mec1- and Tel1-dependent phosphorylation of γ-H2B at H2B-T129. γ-H2B formation is impaired by γ-H2AX and its binding partner, Rad9. High-density microarray analyses show similar γ-H2AX and γ-H2B distributions, but γ-H2B is absent near telomeres. Both γ-H2AX and γ-H2B are strongly diminished over highly transcribed regions. When transcription of GAL genes are turned off, γ-H2AX is restored within 5 min, in a Mec1 dependent manner; when these genes are again induced, γ-H2AX is rapidly lost. Moreover, when a DSB is induced near CEN2, γ-H2AX spreads to all other centromeric regions, again depending on Mec1. Taken together, our data provide new insights on the function and establishment of the phosphorylation events occurring onto chromatin after DSB induction.

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          Most cited references36

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          A high-resolution map of transcription in the yeast genome.

          There is abundant transcription from eukaryotic genomes unaccounted for by protein coding genes. A high-resolution genome-wide survey of transcription in a well annotated genome will help relate transcriptional complexity to function. By quantifying RNA expression on both strands of the complete genome of Saccharomyces cerevisiae using a high-density oligonucleotide tiling array, this study identifies the boundary, structure, and level of coding and noncoding transcripts. A total of 85% of the genome is expressed in rich media. Apart from expected transcripts, we found operon-like transcripts, transcripts from neighboring genes not separated by intergenic regions, and genes with complex transcriptional architecture where different parts of the same gene are expressed at different levels. We mapped the positions of 3' and 5' UTRs of coding genes and identified hundreds of RNA transcripts distinct from annotated genes. These nonannotated transcripts, on average, have lower sequence conservation and lower rates of deletion phenotype than protein coding genes. Many other transcripts overlap known genes in antisense orientation, and for these pairs global correlations were discovered: UTR lengths correlated with gene function, localization, and requirements for regulation; antisense transcripts overlapped 3' UTRs more than 5' UTRs; UTRs with overlapping antisense tended to be longer; and the presence of antisense associated with gene function. These findings may suggest a regulatory role of antisense transcription in S. cerevisiae. Moreover, the data show that even this well studied genome has transcriptional complexity far beyond current annotation.
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            DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1.

            A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.
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              Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage.

              Saccharomyces cells suffering a single unrepairable double-strand break (DSB) exhibit a long, but transient arrest at G2/M. hdf1 cells, lacking Ku70p, fail to escape from this RAD9/RAD17-dependent checkpoint. The effect of hdf1 results from its accelerated 5' to 3' degradation of the broken chromosome. Permanent arrest in hdf1 cells is suppressed by rad50 or mre11 deletions that retard this degradation. Wild-type HDF1 cells also become permanently arrested when they experience two unrepairable DSBs. Both DSB-induced arrest conditions are suppressed by a mutation in the single-strand binding protein, RPA. We suggest that escape from the DNA damage-induced G2/M checkpoint depends on the extent of ssDNA created at broken chromosome ends. RPA appears to play a key intermediate step in this adaptation.

                Author and article information

                Nat Struct Mol Biol
                Nat. Struct. Mol. Biol.
                Nature structural & molecular biology
                18 December 2013
                15 December 2013
                January 2014
                01 July 2014
                : 21
                : 1
                : 103-109
                [1 ]Department of Biology and Rosenstiel Center, Brandeis University, Waltham MA, USA
                [2 ]Université de Toulouse, Université Paul Sabatier, Laboratoire de biologie cellulaire et moléculaire du contrôle de la proliferation (LBCMCP), 31062, Toulouse, France
                [3 ]Centre national de la recherche scientifique (CNRS), LBCMCP, F-31062 Toulouse, France
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                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R37 GM020056 || GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM076020 || GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM061766 || GM

                Molecular biology
                Molecular biology


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