Checkpoints Couple Transcription Network Oscillator Dynamics to Cell-Cycle Progression
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae Schizosaccharomyces pombe
PROVIDER: GSE49650 | GEO | 2014/07/27
SECONDARY ACCESSION(S): PRJNA214609
REPOSITORIES: GEO
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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). Two replicate time courses for each of the strains studied are included
2014-07-27 | E-GEOD-49650 | biostudies-arrayexpress
Project description:Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.
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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:Accurate chromosome segregation is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), to inhibit the anaphase-promoting complex or cyclosome (APC/C). Cdc20 is an essential mitotic regulator since it promotes mitotic exit through activating the APC/C and monitors kinetochore-microtubule attachment through activating the SAC. The proper functioning of Cdc20 requires multiple interactions with APC/C and MCC subunits. To functionally assess each of these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNAi. Here we generated Cdc20 RNAi sensitive cell lines by CRISPR/Cas9 which display a penetrant metaphase arrest phenotype by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron was found to be critical for the SAC by promoting theMCC formation and stabilizing the interaction between the MCC and APC/C. These data reveal additionalregulatory components within the SAC and establish a novel method to interrogate Cdc20 function.
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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:The Anaphase Promoting Complex/Cyclosome (APC/C) is a mega-dalton ubiquitin ligase that initiates mitotic exit by targeting substrates for degradation. The APC/C is activated by Cdc20, which acts as a substrate receptor, and is inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated. We previously identified apcin, a small molecule ligand of Cdc20, as an inhibitor of APC/CCdc20. Surprisingly, we found that apcin paradoxically accelerates substrate degradation and promotes mitotic exit in cells with high SAC activity. Biochemical studies indicate that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin’s behavior as an antagonist of Cdc20 can thus result in either net inhibition of APC/C, delaying mitotic exit when SAC activity is slow, or net activation of APC/C, promoting mitotic exit when SAC activity is high. Genetic experiments suggest that the dual behaviors of apcin arise from targeting a common binding site in Cdc20 that is required for both substrate ubiquitination as well as efficient APC/C inhibition by MCC. We therefore establish a new mechanism through which a small molecule, by targeting a single site on a dynamic protein interface, can lead to opposing biological effects depending on the regulatory context.
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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
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