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Numerical and Spatial Patterning of Yeast Meiotic DNA Breaks by Tel1

ABSTRACT: The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to examine the contribution of the DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM) to this regulation in Saccharomyces cerevisiae. A tel1Î mutant had globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tel1 mutation also increased Spo11-oligonucleotide levels, but mutating known Tel1 phosphotargets on Hop1 and Rec114 did not. Deep sequencing of Spo11 oligonucleotides from tel1Î mutants demonstrated that Tel1 shapes the nonrandom DSB distribution in ways that are distinct but partially overlapping with previously described contributions of the recombination regulator Zip3. Finally, we uncover a context-dependent role for Tel1 in hotspot competition, in which an artificial DSB hotspot inhibits nearby hotspots. Evidence for Tel1-dependent competition involving strong natural hotspots is also provided. Sixteen samples total: The first 12 are two biological replicate Spo11-oligo maps from each of the following: wild type and tel1 each collected at 4, 5, and 6 hours after meiotic induction; the next 4 are one biological replicate Spo11-oligo map from each of the following: wild type and tel1 bearing an artificial hotspot insertion either on Chr III or on Chr IX.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Neeman Mohibullah

PROVIDER: E-GEOD-84696 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Numerical and Spatial Patterning of Yeast Meiotic DNA Breaks by Tel1

Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to examine the contribution of the DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM) to this regulation in Saccharomyces cerevisiae. A tel1Δ mutant had globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tel1 mutation also increased Spo11-oligonucleotide levels, but mutating known Tel1 phosphotargets on Hop1 and Rec114 did not. Deep sequencing of Spo11 oligonucleotides from tel1Δ mutants demonstrated that Tel1 shapes the nonrandom DSB distribution in ways that are distinct but partially overlapping with previously described contributions of the recombination regulator Zip3. Finally, we uncover a context-dependent role for Tel1 in hotspot competition, in which an artificial DSB hotspot inhibits nearby hotspots. Evidence for Tel1-dependent competition involving strong natural hotspots is also provided.

2016-07-22 | GSE84696 | GEO

Meiotic recombination initiation hotspots in Arabidopsis genes and transposons (SPO11-1-oligonucleotides-seq)

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2017-06-05 | E-MTAB-5041 | biostudies-arrayexpress

Spo11-oligo mapping in zip3 mutants

Project description:Meiotic recombination promotes genetic diversification as well as pairing and segregation of homologous chromosomes, but the double-strand breaks (DSBs) that initiate recombination are dangerous lesions that can cause mutation or meiotic failure. How cells control DSBs to balance between beneficial and deleterious outcomes is not well understood. This study tests the hypothesis that DSB control involves a network of intersecting regulatory circuits. We show that DSBs form in greater numbers in Saccharomyces cerevisiae cells lacking ZMM proteins, a suite of recombination-promoting factors traditionally regarded as acting strictly downstream of DSB formation. This counterintuitive result suggests that homologous chromosomes that have successfully engaged one another stop making DSBs, and provides new insight into phenotypes of zmm and other recombination-defective mutants. A genetically distinct pathway ties DSB formation to meiotic progression through the Ndt80 transcription factor. High-resolution genome-wide DSB maps generated by sequencing short oligonucleotides covalently bound to Spo11 (Spo11 oligos) demonstrate that feedback tied to ZMM function contributes in unexpected ways to spatial patterning of the recombination landscape. Four samples total: two wild type and two zip3 mutant (each an independent culture)

2014-04-23 | E-GEOD-48299 | biostudies-arrayexpress

Spo11-oligo mapping in bas1 and ino4 mutants

Project description:Meiotic recombination is initiated by developmentally programmed DNA double-strand breaks (DSBs). In S. cerevisiae, the vast majority of DSBs occur in the nucleosome-depleted regions at gene promoters, where transcription factors (TFs) bind. It has been proposed that TF binding can stimulate DSB formation nearby by modulating local chromatin structure. However, a prior study in TF bas1 mutant suggested that the role of TF binding in determining break formation is complex. Here, we examined fine-scale DSB distributions in TF mutant (bas1Δ and ino4Δ) strains. In bas1Δ mutants, 239 out of the 2468 hotspots showed reduced DSB activity, whereas 87 hotspots showed increased DSB activity. Similarly, in ino4Δ mutant, 415 out of the 2468 hotspots showed reduced DSB activity, whereas 322 hotspots showed increased DSB activity. We also mapped Bas1 and Ino4 binding sites in meiosis and found that only a small portion of the affected hotspots contained TF binding sites. This indicates that TF can influence DSB distribution both directly and indirectly. Surprisingly, these DSB changes in TF mutants did not correlate with change in chromatin structure and histone H3K4me3 modification, suggesting that the role of TF on DSB distribution cannot be simply explained by affecting local chromatin status. Nine samples total: two wild type, four bas1 mutant and three ino4 mutant (each an independent culture)

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Meiotic prophase length modulates Tel1-dependent DNA double-strand break interference

Project description:During meiosis, genetic recombination is initiated by the formation of many DNA double-strand breaks (DSBs) catalysed by the evolutionarily conserved topoisomerase-like enzyme, Spo11, in preferred genomic sites known as hotspots. DSB formation activates the Tel1/ATM DNA damage responsive (DDR) kinase, locally inhibiting Spo11 activity in adjacent hotspots via a process known as DSB interference. Intriguingly, in S. cerevisiae, over short genomic distances (<15 kb), Spo11 activity displays characteristics of concerted activity or clustering, wherein the frequency of DSB formation in adjacent hotspots is greater than expected by chance. We have proposed that clustering is caused by a limited number of sub-chromosomal domains becoming primed for DSB formation. Here, we provide evidence that DSB clustering is abolished when meiotic prophase timing is extended via deletion of the NDT80 transcription factor. We propose that extension of meiotic prophase enables most cells, and therefore most chromosomal domains within them, to reach an equilibrium state of similar Spo11-DSB potential, reducing the impact that priming has on estimates of coincident DSB formation. Consistent with this view, when Tel1 is absent but Ndt80 is present and thus cells are able to rapidly exit meiotic prophase, genome-wide maps of Spo11-DSB formation are skewed towards pericentromeric regions and regions that load pro-DSB factors early—revealing regions of preferential priming—but this effect is abolished when NDT80 is deleted. Our work highlights how the stochastic nature of Spo11-DSB formation in individual cells within the limited temporal window of meiotic prophase can cause localised DSB clustering—a phenomenon that is exacerbated in tel1∆ cells due to the dual roles that Tel1 has in DSB interference and meiotic prophase checkpoint control.

2023-10-18 | GSE245327 | GEO

Genome-wide mapping of meiotic double-strand breaks by sequencing Spo11 oligos.

Project description:The nonrandom distribution of meiotic recombination shapes patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in accumulation of Spo11 protein covalently bound to small DNA fragments. We show here that sequencing these fragments provides a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore the influence of large-scale chromosome structures, chromatin, transcription factors, and local sequence composition on DSB distributions. Our analysis supports the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. Mechanistic aspects of DSB formation and early processing steps are also uncovered. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We discuss implications for evolutionary dynamics of recombination hotspots. We deep sequenced 4 samples of Spo11 oligos on Roche 454 platform. Three samples are technical replicates of Spo11 oligo products prepared from one meiotic culture and the fourth sample was prepared from an independent culture.

2011-03-07 | E-GEOD-26449 | biostudies-arrayexpress

Budding yeast ATM/ATR contribute to meiotic double-strand-break (DSB) homeostasis by down-regulating Rec114, an essential component of the DSB-machinery

Project description:In most organisms, meiotic recombination begins with programmed DNA double strand break (DSB) formation by Spo11. Here, we present evidence that Tel1/Mec1, the budding yeast ATM/ATR, regulate DSB formation by phosphorylating Rec114, an essential Spo11-accessory protein. Analyses of a non-phosphorylatable- or phosphomimetic- alleles of rec114 revealed that DSB-dependent phosphorylation of Rec114 limited its association with DSB-hotspots resulting in reduction in DSB formation. Also observed were the impact of Rec114 phosphorylation on its homolog synapsis-associated removal from chromosomes and NDT80-dependent turnover. Specifically, we found that the synapsis- and NDT80-dependent Rec114 downregulation occurred later in the rec114 mutant with a reduced Spo11-catalysis, but earlier in the other with an enhanced catalysis, strongly implicating the existence of a feedback mechanism coupling the extent of Spo11-catalysis to Rec114 activity. Taken together, these observations suggest that three different mechanisms of down regulating Rec114 contribute to meiotic DSB homeostasis, a feedback mechanism to maintain the number of meiotic DSBs at the developmentally programmed level. 6 genome wide ChIPchip sets: 3 for meiotic DSB formation (Spo11-ChIP) and 3 for protein-DNA association (Rec114-ChIP), each for wild type and two mutants during meiosis (corresponding to the main Figure 3, as well as to Figures S3, S4, S5).

2013-06-01 | E-GEOD-43384 | biostudies-arrayexpress

Mapping of Rec12 oligonucleotides in Schizosaccharomyces pombe

Project description:Meiotic recombination facilitates accurate pairing and faithful segregation of homologous chromosomes by forming physical connections (crossovers) between homologs. Developmentally programmed DNA double-strand breaks (DSBs) generated by Spo11 protein (Rec12 in fission yeast) initiate meiotic recombination. Until recently, attempts to address the basis of the highly non-random distribution of DSBs on a genome-wide scale have been limited to 0.1M-bM-^@M-^S1 kb resolution of DSB position. We have assessed individual DSB events across the Schizosaccharomyces pombe genome at near-nucleotide resolution by deep-sequencing the short oligonucleotides connected to Rec12 following DNA cleavage. The single oligonucleotide size-class generated by Rec12 allowed us to effectively analyze all break events. Our high-resolution DSB map shows that the influence of underlying nucleotide sequence and chromosomal architecture differs in multiple ways from that in budding yeast. Rec12 action is not strongly restricted to nucleosome-depleted regions but is nevertheless spatially biased with respect to chromatin structure. Furthermore, we find strong evidence across the genome for differential DSB repair previously predicted to account for crossover invariance (constant cM/kb in spite of DSB hotspots). Our genome-wide analyses demonstrate evolutionarily fluid factors contributing to crossover initiation and its regulation. Three samples total: one sample sequenced by 454 and two technical replicates (independent adaptor ligations from material purified from one culture) sequenced by SOLiD

2013-08-20 | E-GEOD-49977 | biostudies-arrayexpress

Budding yeast ATM/ATR contribute to meiotic double-strand-break (DSB) homeostasis by down-regulating Rec114, an essential component of the DSB-machinery

2013-06-01 | GSE43384 | GEO

Physical basis for long-distance communication along meiotic chromosomes (ChIP-chip))

Project description:Viable gamete formation requires segregation of homologous chromosomes connected, in most species, by crossovers. DNA double-strand break (DSB) formation and the resulting crossovers are regulated at multiple levels to prevent overabundance along chromosomes. Meiotic cells coordinate these events between distant sites, but the physical basis of long-distance chromosomal communication has been unknown. We show that DSB hotspots up to ~200 kb (~35 cM) apart form clusters via hotspot-binding proteins Rec25 and Rec27 in fission yeast. Clustering coincides with hotspot competition and interference over similar distances. Without Tel1 (ATM tumor-suppressor homolog), DSB and crossover interference become negative, reflecting coordinated action along a chromosome. These results indicate that DSB hotspots within a limited chromosomal region and bound by their protein determinants form a clustered structure that, via Tel1, allows only one DSB per region. Such a “roulette” process among clusters explains the observed pattern of crossover interference in fission yeast. Key structural and regulatory components of clusters are phylogenetically conserved, suggesting conservation of this vital regulation. Based on these observations, we discuss variations on a model in which clustering and competition between DSB sites leads to DSB interference and in turn produces crossover interference.

2018-09-17 | GSE119920 | GEO

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