Rpd3L Contributes to the DNA Damage Sensitivity of Saccharomyces cerevisiae Checkpoint Mutants
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE113869 | GEO | 2019/02/27
REPOSITORIES: GEO
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Project description:Rpd3L Contributes to the DNA Damage Sensitivity of Saccharomyces cerevisiae Checkpoint Mutants
| PRJNA454307 | ENA
Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis
2007-08-01 | GSE8305 | GEO
Project description:DNA damage results in the activation of checkpoint kinases, which phosphorylate downstream effectors that inhibit the cell cycle, activate DNA repair, and cause widespread changes in transcription. However, the specific connections between the checkpoint kinases and downstream transcription factors (TFs) are not well understood. Here, we introduce a strategy for mapping regulatory networks between kinases and TFs involving integration of kinase mutant expression profiles, transcriptional regulatory interactions, and phosphoproteomics. We use this approach to investigate the role of the Saccharomyces cerevisiae checkpoint kinases (Mec1, Tel1, Chk1, Rad53, and Dun1) in the transcriptional response to DNA damage caused by methyl methanesulfonate (MMS). The result is a global kinase-TF regulatory network in which Mec1 and Tel1 signal through Rad53 to synergistically regulate the expression of more than 600 genes. This network implicates at least nine TFs, including Msn4, Gcn4, SBF (Swi4/Swi6), MBF (Swi6/Mbp1), and Fkh2/Ndd1/Mcm1, nearly all of which have sites of Rad53-dependent phosphorylation, as downstream regulators of checkpoint kinase-dependent genes. We also identify a major DNA damage-induced transcriptional network acting independently of Rad53 and other checkpoint kinases to regulate expression of genes involved in general and oxidative stress responses. Expression was profiled with and without MMS treatment in several genetic backgrounds (gene deletion strains).
2013-03-31 | E-GEOD-40351 | biostudies-arrayexpress
Project description:DNA damage results in the activation of checkpoint kinases, which phosphorylate downstream effectors that inhibit the cell cycle, activate DNA repair, and cause widespread changes in transcription. However, the specific connections between the checkpoint kinases and downstream transcription factors (TFs) are not well understood. Here, we introduce a strategy for mapping regulatory networks between kinases and TFs involving integration of kinase mutant expression profiles, transcriptional regulatory interactions, and phosphoproteomics. We use this approach to investigate the role of the Saccharomyces cerevisiae checkpoint kinases (Mec1, Tel1, Chk1, Rad53, and Dun1) in the transcriptional response to DNA damage caused by methyl methanesulfonate (MMS). The result is a global kinase-TF regulatory network in which Mec1 and Tel1 signal through Rad53 to synergistically regulate the expression of more than 600 genes. This network implicates at least nine TFs, including Msn4, Gcn4, SBF (Swi4/Swi6), MBF (Swi6/Mbp1), and Fkh2/Ndd1/Mcm1, nearly all of which have sites of Rad53-dependent phosphorylation, as downstream regulators of checkpoint kinase-dependent genes. We also identify a major DNA damage-induced transcriptional network acting independently of Rad53 and other checkpoint kinases to regulate expression of genes involved in general and oxidative stress responses.
2013-03-31 | GSE40351 | GEO
Project description:The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal – and therefore the cells’ DNA damage load – is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection at a site-specific DNA double-strand break (DSB) in budding yeast to generate quantitatively different DNA damage (ssDNA) signals. Interestingly, two major targets of the Mec1-Ddc2 kinase – Rad53 and γH2A – differ in their response to the ssDNA signal, indicating distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is non-quantitative and unresponsive to increased amounts of damage-associated Mec1-Ddc2 kinase. In contrast, the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. We find that not only Mec1-Ddc2 association, but also loading of the 9-1-1 co-sensor complex is enhanced during ongoing resection. Moreover, we can uncouple global checkpoint activation from the amount of Mec1-Ddc2 kinase at the lesion by using mutant conditions that hyper-activate the 9-1-1 signalling axis and at the same time show reduced amounts of damage-associated Mec1-Ddc2 kinase. We therefore propose that a key function of the 9-1-1 complex and the downstream checkpoint mediators is to generate a checkpoint response, which is quantitative and proportional to the cellular DNA damage load. The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal – and therefore the cells’ DNA damage load – is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection at a site-specific DNA double-strand break (DSB) in budding yeast to generate quantitatively different DNA damage (ssDNA) signals. Interestingly, two major targets of the Mec1-Ddc2 kinase – Rad53 and γH2A – differ in their response to the ssDNA signal, indicating distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is non-quantitative and unresponsive to increased amounts of damage-associated Mec1-Ddc2 kinase. In contrast, the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. We find that not only Mec1-Ddc2 association, but also loading of the 9-1-1 co-sensor complex is enhanced during ongoing resection. Moreover, we can uncouple global checkpoint activation from the amount of Mec1-Ddc2 kinase at the lesion by using mutant conditions that hyper-activate the 9-1-1 signalling axis and at the same time show reduced amounts of damage-associated Mec1-Ddc2 kinase. We therefore propose that a key function of the 9-1-1 complex and the downstream checkpoint mediators is to generate a checkpoint response, which is quantitative and proportional to the cellular DNA damage load.
2019-01-12 | GSE124948 | GEO
Project description:Transcriptional memory is critical for the faster reactivation of necessary genes upon environmental changes and requires that the genes were previously in an active state. However, whether transcriptional repression also displays “memory” of the prior transcriptionally inactive state remains unknown. In this study, we show that transcriptional repression of approximately 540 genes in yeast occurs much more rapidly if the genes have been previously repressed during carbon source shifts. This novel transcriptional response has been termed transcriptional repression memory(TREM). Interestingly, Rpd3L histone deacetylase (HDAC), targeted to active promoters induces TREM. Mutants for Rpd3L exhibit increased acetylation at active promoters and delay TREM and RNA PolII dissociation significantly. Surprisingly, the interaction between H3K4me3 and Rpd3L via the Pho23 PHD finger is sufficient to induce histone deacetylation and TREM by Rpd3L. Therefore, we propose that an active mark, H3K4me3 enriched at promoters, instructs Rpd3L HDAC to induce histone deacetylation and TREM.
2018-10-23 | E-MTAB-6551 | biostudies-arrayexpress
Project description:Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed. Keywords: ChIP-chip
2009-05-26 | GSE14761 | GEO
Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis S. cerevisiae chromosomes IIIâV, and chromosome VI highdensity oligonucleotide microarrays were provided by Affymetrix Custom Express Service (SC3456a520015F, P/N 520015; rikDACF, P/N 510636, respectively). Sequence and position of oligonucleotides on the microarrays are available from Affymetrix. ChIP was carried out as previously described (Katou et al. 2003; Katou et al. 2006): we disrupted 1.5 x 108 cells byMulti-beads shocker (MB400U, Yasui Kikai) using glass beads. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc.) and anti-Flag monoclonal antibody M2 (Sigma-Aldrich) were used for chromatin immunoprecipitation. ChIPed DNA was purified and amplified by random priming as described (Katou et al. 2003): a total of 10 μg of amplified DNA was digested with DNaseI to a mean size of 100 bp, purified, and the fragments were end-labelled with biotin-N6-ddATP23. Hybridization, washing, staining and scanning were performed according to the manufacturerâs instruction (Affymetrix). Primary data analyses were carried out using the Affymetrix microarray Suite version 5.0 software to obtain hybridization intensity, fold change value, change P-value and detection P-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIPed fraction with supernatant fraction by using three criteria. First, the reliability of strength of signal was judged by detection P-value of each locus (P â¥0.025). Second, reliability of binding ratio was judged by change P-value (P ⥠0.025). Third, clusters consisting of at least three contiguous loci that filled the above Bermejo et al. 26 two criteria were selected, because it was known that a single site of proteinâDNA interaction will result in immunoprecipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbours. For the analyses of BrdU incorporation, cells were fixed by ice-cold buffer containing 0.1% azide, and then total DNA from 3 x108 cells was purified. DNA was sheared to 300 bp by sonication, denatured, and mixed with 2μg anti-BrdU monoclonal antibody (2B1D5F5H4E2; MBL). Antibody-bound and unbound fractions were subsequently purified, amplified, labelled and hybridized to the DNA chip.
2010-06-05 | E-GEOD-8305 | biostudies-arrayexpress
Project description:Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed. Keywords: ChIP-chip ⢠The goal of the experiment Genome-wide localization of Rad52 binding sites in Saccharomyces cerevisiae ⢠Experimental factor Distribution of Rad52 on chromosome III, IV, and V and the right arm of chromosome VI Strain: wild type, rad53 mutant, and rad53 siz2 mutant (W303 background, expressing myc tagged protein from its native promoter) Cell condition 1: G1 arrest with alpha-factor Cell condition 2: HU treatment ⢠Experimental design ChIP analyses: ChIP using anti-c-Myc antibody. ChIP-chip analyses: In all cases, hybridization data of ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F) were used. ⢠Quality control steps taken Confirmation of several loci by quantitative real time PCR. Wild type cells expressing non-tagged Rad52 were used as a negative control of DNA amplification.
2010-05-18 | E-GEOD-14761 | biostudies-arrayexpress
Project description:Yeast cell cycle transcript dynamics in three S. cerevisiae strains grown at 30 degrees Celsius: cdc20 GALL-CDC20 (persistent mitotic CDK activity; CDK on), cdc8-ts (DNA replication checkpoint), GAL-cse4-353 (spindle assembly checkpoint), cdc8-ts cdc20 (DNA replication checkpoint, CDK on), and cdc8-ts cdc20, rad53-1 (DNA replication checkpoint without Rad53 activity, CDK on) in a BF264-15DU background. We compared transcript levels of genes previously shown to be periodically expressed in wild-type cells and in cells lacking all mitotic cyclins (clb1,2,3,4,5,6; CDK off). Two replicate time courses for each of the strains studied are included
2014-07-27 | E-GEOD-49650 | biostudies-arrayexpress