Saccharomyces cerevisiae
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ABSTRACT:
PROVIDER: PRJNA112255 | ENA |
REPOSITORIES: ENA
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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.
2010-05-18 | E-GEOD-14761 | biostudies-arrayexpress
Project description:In the yeast Saccharomyces cerevisiae, cleavage factor I (CFI) and cleavage and polyadenylation factor (CPF) build the core of the transcription termination machinery. CFI comprises the Rna14, Rna15, Pcf11, and Clp1 proteins, as well as the associated Hrp5 RNA-binding protein. We found that CFI participates in the DNA damage response and that rna14-1 shows synthetic growth defects with mutants of different repair pathways, including homologous recombination, non-homologous end joining, post replicative repair, mismatch repair, and nucleotide excision repair, implicating that impaired RNAPII termination and 3â-end processing decreases the cellular tolerance for DNA damage. Beyond replication progression defects, we found that bypass of the G1/S checkpoint in rna14-1 cells leads to synthetic sickness, accumulation of phosphorylated H2A, as well as increase in Rad52-foci and in recombination. Our data provide evidence that CFI dysfunction impairs RNAPII turnover, leading to replication hindrance and lower tolerance to exogenous DNA damage. These findings underscore the importance of coordination between transcription termination, DNA repair and replication in the maintenance of genomic stability. S. cerevisiae strains were grown in YPAD liquid culture at 30°C, total RNA was isolated and hybridized on Affymetrix microarrays.
2014-03-31 | E-GEOD-50947 | biostudies-arrayexpress
Project description:In the yeast Saccharomyces cerevisiae, cleavage factor I (CFI) and cleavage and polyadenylation factor (CPF) build the core of the transcription termination machinery. CFI comprises the Rna14, Rna15, Pcf11, and Clp1 proteins, as well as the associated Hrp5 RNA-binding protein. We found that CFI participates in the DNA damage response and that rna14-1 shows synthetic growth defects with mutants of different repair pathways, including homologous recombination, non-homologous end joining, post replicative repair, mismatch repair, and nucleotide excision repair, implicating that impaired RNAPII termination and 3’-end processing decreases the cellular tolerance for DNA damage. Beyond replication progression defects, we found that bypass of the G1/S checkpoint in rna14-1 cells leads to synthetic sickness, accumulation of phosphorylated H2A, as well as increase in Rad52-foci and in recombination. Our data provide evidence that CFI dysfunction impairs RNAPII turnover, leading to replication hindrance and lower tolerance to exogenous DNA damage. These findings underscore the importance of coordination between transcription termination, DNA repair and replication in the maintenance of genomic stability.
2014-03-31 | GSE50947 | GEO
Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip
2008-04-10 | GSE9420 | GEO
Project description:The DNA replication checkpoint maintains expression homeostasis during replication stress
| PRJNA415284 | ENA
Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip
2008-04-10 | GSE9233 | GEO
Project description:A network governing DNA integrity was identified in yeast by a global genetic analysis of synthetic fitness or lethality defect (SFL) interactions. Within this network, multiple functional modules or mini-pathways were defined according to their common patterns of global SFL interactions and available protein-protein interaction information. Modules or genes involved in DNA replication, DNA replication checkpoint signaling, and oxidative stress response were identified as the major guardians against lethal spontaneous DNA damage, efficient repair of which requires the functions of the DNA damage checkpoint signaling and multiple DNA repair pathways. This genome-wide genetic interaction network also revealed potential roles of a number of genes and modules in mitotic DNA replication and maintenance of genomic stability. These include DIA2, NPT1, HST3, HST4, and the CSM1/LRS4 module (CSM1m). Likewise, the CTF18 module (CTF18m), previously implicated in sister chromatid cohesion, was found to participate in the DNA replication checkpoint. Keywords: dose response
2006-02-16 | GSE3574 | GEO
Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip • The goal of the experiment Genome-wide localization of Ino80 on chromosome in Saccharomyces cerevisiae • Keywords DNA replication, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI) • Experimental factor Distribution of Ino80 in random culture Distribution of Ino80 in G1 phase Distribution of Ino80 in early S phase • Experimental design ChIP analyses: W303 background cells expressing Myc-tagged Ino80 were used for the ChIP using anti-Myc monoclonal antibody (9E11). ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI) was used. • Quality control steps taken Confirmation of several loci by quantitative real time PCR.
2008-04-10 | E-GEOD-9420 | biostudies-arrayexpress
Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip • The goal of the experiment Genome-wide localization of Ino80 and Arp5 on chromosome in Saccharomyces cerevisiae • Keywords DNA replication, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI) • Experimental factor Distribution of Ino80 and Arp5 in wild type in random culture Distribution of Ino80 in G1 cells Distribution of Ino80 in early S phase cells • Experimental design ChIP analyses: W303 background cells expressing Myc tagged Ino80 were used for the ChIP using anti-Myc monoclonal antibody (9E11). ChIP analyses: W303 background cells expressing Myc tagged Ino80 were used for the ChIP using anti-Arp5 polyclonal antibody. ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI) was used. • Quality control steps taken Confirmation of several loci by quantitative real time PCR.
2008-04-10 | E-GEOD-9233 | biostudies-arrayexpress