Checkpoints Couple Transcription Network Oscillator Dynamics to Cell-Cycle Progression
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Steven Haase
PROVIDER: E-GEOD-49650 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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).
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Project description:Across eukaryotes, inhibition of the progression of the DNA replication machinery causes an S-phase checkpoint response. This response regulates multiple processes, including inhibition of replication initiation and fork stabilisation. How these events are coordinated remains poorly understood. Here we show that the replicative helicase component Cdc45 targets the checkpoint kinase Rad53 to distinct replication complexes in the budding yeast Saccharomyces cerevisiae. Rad53 binds to FHA-interaction motifs in an unstructured loop region of Cdc45, which is phosphorylated by Rad53 itself, and this interaction is necessary for the inhibition of origin firing through Sld3. In addition to regulating replication initiation, Cdc45 also recruits Rad53 to stalled replication forks, which we demonstrate is important for the response to replication stress. Finally we show that a Cdc45 mutation that is orthologous to an allele found in patients with Meier-Gorlin Syndrome disrupts the functional interaction with Rad53. Together we present a single mechanism by which Rad53 targets replication initiation and elongation complexes, which may be relevant to human disease.
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Project description:MBF and SBF transcription factors regulate a large family of coordinately expressed G1/S genes required for early cell cycle functions including DNA replication and repair. SBF is inactivated upon S phase entry by Clb/CDK whereas MBF targets are repressed by the corepressor, Nrm1. Using genome-wide expression analysis, we show that genotoxic stress during S phase specifically induces MBF-regulated genes via direct phosphorylation of Nrm1 by Rad53, the effector checkpoint kinase, and prevents its binding to MBF target promoters. Interestingly, some previously defined targets of SBF, including Swe1, are activated by replication stress. Swe1 undergoes a transcription factor exchange leading to its repression by Nrm1/MBF, which renders it sensitive to derepression by the S phase checkpoint. We conclude that MBF-regulated genes are distinguished from SBF-regulated genes by their sensitivity to activation by the S phase checkpoint, thereby, providing an effective mechanism for enhancing DNA replication and repair and promoting genome stability. Modified Loop Time-series design, time points at 0, 30, 45, 60 and 75 minutes. Conditions are untreated (UT), methyl methane sulfonate (MMS-0.033%), camptothecin (CPT-50uM), and hydroxyurea (HU-0.2M)
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Project description:We have developed a method to analyze single-stranded DNA (ssDNA) formation on a genomic scale by using microarrays. Using this technique we have assessed the location and the amount of ssDNA in S. cerevisiae during DNA replication. We have observed that when replication is impeded by hydroxyurea, ssDNA formation can be detected in both wild type and the checkpoint-deficient rad53 cells. However, while wild type cells showed ssDNA formation at only a subset of origins, rad53 cells formed ssDNA at virtually all known origins. Moreover, in rad53 cells the ssDNA regions did not expand over time, presumably due to collapsed replication forks. We also applied this method to map origins in S. pombe, taking advantage of the conserved replication checkpoint function by Cds1, the homolog of Rad53 in S. pombe. Keywords: ssDNA, HU, replication, time course
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Project description:In response to DNA replication stress, DNA replication checkpoint is activated to maintain fork stability, a process critical for maintenance of genome stability. However, how DNA replication checkpoint regulates replication forks remain elusive. Here we show that Rad53, a highly conserved replication checkpoint kinase, functions to couple leading and lagging strand DNA synthesis. In wild type cells under HU induced replication stress, synthesis of lagging strand, which contains ssDNA gaps, is comparable to leading strand DNA. In contrast, synthesis of lagging strand is much more than leading strand, and consequently, leading template ssDNA coated with ssDNA binding protein RPA was detected in rad53-1 mutant cells, suggesting that synthesis of leading strand and lagging strand DNA is uncoupled. Mechanistically, we show that replicative helicase MCM and leading strand DNA polymerase Pole move beyond actual DNA synthesis and that an increase in dNTP pools largely suppresses the uncoupled leading and lagging strand DNA synthesis. Our studies reveal an unexpected mechanism whereby Rad53 regulates replication fork stability.
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Project description:MBF and SBF transcription factors regulate a large family of coordinately expressed G1/S genes required for early cell-cycle functions including DNA replication and repair. SBF is inactivated upon S-phase entry by Clb/CDK whereas MBF targets are repressed by the co-repressor, Nrm1. Using genome-wide expression analysis of cells treated with methyl methane sulfonate (MMS), hydroxyurea (HU) or camptothecin (CPT), we show that genotoxic stress during S phase specifically induces MBF-regulated genes. This occurs via direct phosphorylation of Nrm1 by Rad53, the effector checkpoint kinase, which prevents its binding to MBF target promoters. We conclude that MBF-regulated genes are distinguished from SBF-regulated genes by their sensitivity to activation by the S-phase checkpoint, thereby, providing an effective mechanism for enhancing DNA replication and repair and promoting genome stability.
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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: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.
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Project description:The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression. S.cerevisiae oligonucleotide microarrays were provided by Affymetrix (S.cerevisiae Tiling 1.0R, P/N 900645). BrdU and proteins ChIP-chip analyses were carried out as described (Fachinetti et al., M Cell, 2010).
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Project description:Across eukaryotes, checkpoints maintain the order of cell cycle events in the face of DNA damage or incomplete replication. Although a wide array of DNA lesions activates the checkpoint kinases, whether and how this response differs in different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in the budding yeast Saccharomyces cerevisiae is caused by Rad53 kinase-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M phase, preventing inappropriate gene amplification events. In addition we show that inhibition of Sld3 and Dbf4 after DNA damage in G1 phase prevents premature replication initiation at all origins at the G1/S transition. This study redefines the scope and specificity of the ‘S-phase checkpoint’ with implications for understanding the roles of this checkpoint in the majority of cancers that lack proper cell cycle controls.
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