Nucleosome position mapping by micrococcal nuclease analysis in S. cerevisiae of Top1-AID/Top2-AID strains with auxin in galactose-rich media
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE196945 | GEO | 2023/04/28
REPOSITORIES: GEO
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Project description:We analyed the nucleosome positions by using 2 concentrations of micrococcal nuclease of yeast strains that were grown in raffinose and galactose containing media (synthetic complete).
2019-05-14 | GSE116337 | GEO
Project description:We analyed the nucleosome positions by using 2 concentrations of micrococcal nuclease on yeast strains that were grown in raffinose or galactose containing media (synthetic complete). Strains contained FRB-tags to Sth1, Swi2, Sth1 & Swi2 or Sth1 and TBP, where TBP was tagged on one of two alleles in a diploid strain. Cells were treated for 60 min either with 7.5 uM rapamycin or the same volume of DMSO.
2023-01-11 | GSE190737 | 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
2007-08-01 | GSE8305 | 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:Physiological and pathogen-induced oscillations in the plant cells' redox environment trigger response mechanisms that involve redox-sensitive cysteines and consequent changes in proteins' biochemical and functional characteristics. Arabidopsis thimet oligopeptidases (TOPs) TOP1 and TOP2 are components of the immune signaling and oxidative stress response. Here, TOP1 and TOP2 were evaluated comparatively to understand their role as redox sensors. We show that TOPs catalytic activity is augmented in plants undergoing an immune response and in mutant lines with altered expression of the chloroplastic NTRC impaired in the chloroplast thiol-disulfide regulatory system; ntrc was unable to mount a systemic immune response. Oxidation promoted TOP1 self-interaction to dimers and oligomers; Cys-to-Ala mutagenesis of multiple Cys residues inhibited TOP1 oxidative self-interaction. In contrast, TOP1 lacking the signal peptide (ΔSPTOP1) and TOP2 were detected solely as monomers. Treatment with oxidized glutathione increased the catalytic activities of TOPs; Ala substitution of the unique Cys405 of TOP2 abolished its oxidative activation. The immune regulator ROC1 was identified as a candidate TOPs substrate. TOPs cleaved a ROC1 peptide (ROC1p) at similar and unique cleavage sites; ROC1p concentration helped determine TOPs cleavage specificity. We propose that TOP1 and TOP2 form a proteolysis couple with divergent redox-sensing mechanisms and specificity.
2021-04-19 | PXD024059 | Pride
Project description:The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anti-cancer agents such as etoposide operate by stabilising TOP2ccs, ultimately generating genotoxic TOP2-DNA protein crosslinks that require processing and repair. Here, we identify RAD54L2 as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with ZNF451 and independent of TDP2. Our work suggests a model wherein RAD54L2 recognises sumoylated-TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer-therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.
2024-01-26 | PXD046611 | Pride
Project description:Topoisomerase II (Top2) is an essential enzyme that decatenates DNA via a transient Top2-DNA covalent intermediate. This intermediate can be stabilised by a class of drugs termed Top2 poisons, resulting in massive DNA damage. Thus, Top2 activity is a double-edged sword that needs to be carefully controlled to maintain genome stability. We show that Uls1, an ATP-dependent chromatin remodelling (Snf2) enzyme, can alter Top2 chromatin binding and prevent Top2 poisoning in yeast. Deletion mutants of ULS1 are hypersensitive to the Top2 poison acriflavine (ACF), activating the DNA damage checkpoint. We map Uls1’s Top2 interaction domain and show that this, together with its ATPase activity, is essential for Uls1 function. By performing ChIP-seq, we show that ACF leads to a general increase in Top2 binding across the genome. We map Uls1 binding sites and identify tRNA genes as key regions where Uls1 associates after ACF treatment. Importantly, the presence of Uls1 at these sites prevents ACF-dependent Top2 accumulation. Our data reveal the effect of Top2 poisons on the global Top2 binding landscape and highlights the role of Uls1 in antagonising Top2 function. Remodelling Top2 binding is thus an important new means by which Snf2 enzymes promote genome stability.
2019-04-17 | GSE123707 | GEO
Project description:Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated Top2 contribution in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes without influencing their transcription. The Top2-bound loci exhibit low nucleosome density and accumulate yH2A when Top2 is defective. These intergenic loci associate with the HMG-protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate a S-phasen architectural pathway controlling replicon dynamics when forks encounter transcriptionto preserve genome integrity. Signal tracks in BED format suitable for visualization on the UCSC genome browser can be found at http://bio.ifom-ieo-campus.it/supplementary/Bermejo_et_al_CELL_2009
2009-09-05 | GSE16258 | GEO
Project description:DNA topoisomerase-2 and high mobility group protein Hmo1 are known to regulate chromatin architecture by regulating gene boundaries. Here we report how these proteins affect global RNA level after inactivation of Top2 and Hmo1. Our data indicate that inactivating Hmo1 has a drastic effect on transcription levels of 20% yeast genes, however, this phenomenon can slightly be rescued by inactivating Top2 functions. Also, we study the Top2 and Top1 role in nucleosome architecture with and without expressing E.coli TopA. In top2-1;top1∆ condition with TopA expressed, it affects the nucleosome occupancy at the global level compared with top2-1;top1∆-Control plasmid. The ChIA-PET (Chromatin interaction analysis by paired-end tag sequencing) method is used to address whether a specific protein is engaged in the chromosomal interactions. Epitope tagged Top2 protein is used as probe in ChIA-PET experiments to map Top2 mediated chromatin-chromatin interactions.
2018-07-02 | GSE114444 | GEO
Project description:We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8-10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4-5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant.
| S-EPMC3314939 | biostudies-literature