Spo11 generates gaps through concerted cuts at sites of topological stress [Top2]
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ABSTRACT:
PROVIDER: PRJNA717811 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14085600_1.fastq.gz | Fastqsanger.gz | |||
| SRR14085600_2.fastq.gz | Fastqsanger.gz | |||
| SRR14085601_1.fastq.gz | Fastqsanger.gz | |||
| SRR14085601_2.fastq.gz | Fastqsanger.gz | |||
| SRR14085602_1.fastq.gz | Fastqsanger.gz |
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Project description:Spo11 generates gaps through concerted cuts at sites of topological stress
| PRJNA718235 | ENA
Project description:Spo11 generates gaps through concerted cuts at sites of topological stress [dDSB]
| PRJNA718233 | ENA
Project description:Meiotic recombination is essential for proper meiotic chromosome segregation and fertility, and is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homolog of archaeal topoisomerase VIA. Here we report the discovery of hitherto uncharacterized Spo11-induced lesions, small gaps from 34 bp to several hundred bp, which are generated by coordinated pairs of DSBs (double DSBs or dDSBs). Isolation and genome-wide mapping of the resulting fragments with single base pair precision reveals enrichment at DSB hotspots but also a widely dispersed distribution covering the entire genome. We show that Spo11 prefers to cut at a sequence motif which promotes DNA bending, indicating that bendability of DNA contributes to cleavage site choice. Moreover, fragment lengths display a ~ (10.4n+3) bp periodicity, implying that Spo11 favours cleavage on the same face of underwound DNA. Consistently, dDSB signals overlap and correlate with topoisomerase II binding sites, which points to a role for topological stress and DNA crossings in break formation, and suggests a unified model for DSB and dDSB formation, in which Spo11 traps two DNA strands. Furthermore, gaps resulting from dDSBs, an estimated 20% of all initiation events, can account for full gene conversion events that are independent of both Msh2-dependent heteroduplex repair and MutLγ. Since non-homologous gap repair results in deletions, and ectopically re-integrated dDSB fragments result in insertions, dDSB formation represents a potential source of evolutionary diversity and pathogenic germ-line aberrations.
2021-06-09 | GSE169760 | GEO
Project description:Meiotic recombination is essential for proper meiotic chromosome segregation and fertility, and is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homolog of archaeal topoisomerase VIA. Here we report the discovery of hitherto uncharacterized Spo11-induced lesions, small gaps from 34 bp to several hundred bp, which are generated by coordinated pairs of DSBs (double DSBs or dDSBs). Isolation and genome-wide mapping of the resulting fragments with single base pair precision reveals enrichment at DSB hotspots but also a widely dispersed distribution covering the entire genome. We show that Spo11 prefers to cut at a sequence motif which promotes DNA bending, indicating that bendability of DNA contributes to cleavage site choice. Moreover, fragment lengths display a ~ (10.4n+3) bp periodicity, implying that Spo11 favours cleavage on the same face of underwound DNA. Consistently, dDSB signals overlap and correlate with topoisomerase II binding sites, which points to a role for topological stress and DNA crossings in break formation, and suggests a unified model for DSB and dDSB formation, in which Spo11 traps two DNA strands. Furthermore, gaps resulting from dDSBs, an estimated 20% of all initiation events, can account for full gene conversion events that are independent of both Msh2-dependent heteroduplex repair and MutLγ. Since non-homologous gap repair results in deletions, and ectopically re-integrated dDSB fragments result in insertions, dDSB formation represents a potential source of evolutionary diversity and pathogenic germ-line aberrations.
2021-06-09 | GSE171042 | GEO
Project description:DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo. This data set contains maps of Top2 CCs in the S. cerevisiae genome, generated by CC-seq of BY4741 cells -/+ etoposide (VP16).
2019-09-28 | GSE136675 | GEO
Project description:DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.
2019-09-28 | GSE136943 | 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 are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes - and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy enables us to reveal the influence primary DNA sequence has upon Top2 cleavage - distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.
2019-09-28 | GSE137685 | 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: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