Homologue engagement controls meiotic DNA break number and distribution

Drew, Thacker; Mohibullah, Neeman; Zhu, Xuan; Keeney, Scott

doi:10.1038/nature13120

Cited by 194 publications

(359 citation statements)

References 52 publications

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“…The SC also appears to be sufficient for establishing and maintaining pairing in planarians even in the absence of DSBs. The observation of a more than twofold increase in the number of DSBs in pachytene nuclei that lack SC is consistent with the recent finding in yeast that SC formation and the stabilizing of homolog interactions acts to suppress DSB formation (35,65). We cannot exclude the possibility that the observed increase in RAD51 foci reflects a failure to mature and repair DSBs in the absence of SC.…”

Section: Discussionsupporting

confidence: 77%

Synaptonemal complex extension from clustered telomeres mediates full-length chromosome pairing in Schmidtea mediterranea

Xiang

Miller

Ross

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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In the 1920s, József Gelei proposed that chromosome pairing in flatworms resulted from the formation of a telomere bouquet followed by the extension of synapsis from telomeres at the base of the bouquet, thus facilitating homolog pairing in a processive manner. A modern interpretation of Gelei's model postulates that the synaptonemal complex (SC) is nucleated close to the telomeres and then extends progressively along the full length of chromosome arms. We used the easily visible meiotic chromosomes, a well-characterized genome, and RNAi in the sexual biotype of the planarian Schmidtea mediterranea to test that hypothesis. By identifying and characterizing S. mediterranea homologs of genes encoding synaptonemal complex protein 1 (SYCP1), the topoisomerase-like protein SPO11, and RAD51, a key player in homologous recombination, we confirmed that SC formation begins near the telomeres and progresses along chromosome arms during zygotene. Although distal regions pair at the time of bouquet formation, pairing of a unique interstitial locus is not observed until the formation of full-length SC at pachytene. Moreover, neither full extension of the SC nor homologous pairing is dependent on the formation of double-strand breaks. These findings validate Gelei's speculation that full-length pairing of homologous chromosomes is mediated by the extension of the SC formed near the telomeres. S. mediterranea thus becomes the first organism described (to our knowledge) that forms a canonical telomere bouquet but does not require double-strand breaks for synapsis between homologous chromosomes. However, the initiation of SC formation at the base of the telomere bouquet, which then is followed by full-length homologous pairing in planarian spermatocytes, is not observed in other species and may not be conserved.

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Section: Discussionsupporting

confidence: 77%

Synaptonemal complex extension from clustered telomeres mediates full-length chromosome pairing in Schmidtea mediterranea

Xiang

Miller

Ross

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Wildtype cells displayed the typical kinetics of appearance and disappearance of Spo11-oligo complexes (Fig. 1A,B), previously shown to coincide with DSB formation (Neale et al 2005;Thacker et al 2014). In the tel1Δ mutant, Spo11-oligo complexes first appeared with similar timing and levels as wild type but continued to accumulate, reaching a maximum at 5 h that was 2.2-fold higher on average than in wild type (Fig.…”

Section: Resultsmentioning

confidence: 87%

Numerical and spatial patterning of yeast meiotic DNA breaks by Tel1

2016

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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. Mechanisms of this regulation remain poorly understood. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to reveal aspects of the contribution of the Saccharomyces cerevisiae DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM). A tel1Δ mutant has 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 similarly increases Spo11-oligonucleotide levels but mutating known Tel1 phosphotargets on Hop1 and Rec114 does not, implicating Tel1 kinase activity and clarifying roles of Tel1 phosphorylation substrates. Deep sequencing of Spo11 oligonucleotides demonstrates that Tel1 shapes the genome-wide DSB landscape in unexpected ways. Early in meiosis, Tel1 absence causes widespread changes in DSB distributions across large chromosomal domains. Many of these changes are erased as meiosis proceeds, however, illustrating homeostatic behavior of DSB regulatory systems. We further find that effects of Tel1 are distinct but partially overlapping with previously described contributions of the recombination regulator Cst9 (also known as Zip3). Finally, we provide evidence indicating that Tel1-dependent DSB interference influences the population-average DSB landscape but also demonstrate that locally inhibitory effects of an artificial hotspot insertion can be both Tel1-independent and chromosomal context-dependent. Our findings delineate Tel1 roles in regulating number and location of DSBs and illuminate the complex interplay between Tel1 and other pathways for DSB control.

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“…Clipping of library adapters and mapping of reads to the sacCer2 genome assembly was performed using a custom pipeline as described (Pan et al 2011;Thacker et al 2014). Sequence read totals and mapping statistics are described in Table S2.…”

Section: Spo11-oligo Mappingmentioning

confidence: 99%

“…The supernatant was collected using a SPIN-X tube (Corning) and ethanol precipitated with 0.3 volume of 9 M ammonium acetate, 10 mg of DNA-free glycogen, and 2.5 volumes of 100% ethanol. Spo11 oligos were then quantified and used for library preparation as described (Thacker et al 2014). Sequencing was performed using Illumina HiSeq in the Memorial Sloan Kettering Cancer Center (MSKCC) Integrated Genomics Operation core facility.…”

Section: Spo11-oligo Mappingmentioning

confidence: 99%

“…Cell lysates were prepared by grinding frozen cell paste in a bead mill, similar to methods described (Thacker et al 2014). The lysate was then diluted with an equal volume of 23 immunoprecipitation (IP) buffer (2% Triton X-100, 30 mM Tris-HCl, pH 8.0, 300 mM NaCl, 2 mM EDTA) and incubated with protein G agarose beads (Roche) for mock IP (4 hr at 4°mixing end over end, 400 ml beads per 50 ml extract).…”

Section: Spo11-oligo Mappingmentioning

confidence: 99%

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High-Resolution Global Analysis of the Influences of Bas1 and Ino4 Transcription Factors on Meiotic DNA Break Distributions in Saccharomyces cerevisiae

Zhu

Keeney

2015

Genetics

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Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by Spo11. In Saccharomyces cerevisiae, many DSBs occur in "hotspots" coinciding with nucleosome-depleted gene promoters. Transcription factors (TFs) stimulate DSB formation in some hotspots, but TF roles are complex and variable between locations. Until now, available data for TF effects on global DSB patterns were of low spatial resolution and confined to a single TF. Here, we examine at high resolution the contributions of two TFs to genome-wide DSB distributions: Bas1, which was known to regulate DSB activity at some loci, and Ino4, for which some binding sites were known to be within strong DSB hotspots. We examined fine-scale DSB distributions in TF mutant strains by deep sequencing oligonucleotides that remain covalently bound to Spo11 as a byproduct of DSB formation, mapped Bas1 and Ino4 binding sites in meiotic cells, evaluated chromatin structure around DSB hotspots, and measured changes in global messenger RNA levels. Our findings show that binding of these TFs has essentially no predictive power for DSB hotspot activity and definitively support the hypothesis that TF control of DSB numbers is context dependent and frequently indirect. TFs often affected the fine-scale distributions of DSBs within hotspots, and when seen, these effects paralleled effects on local chromatin structure. In contrast, changes in DSB frequencies in hotspots did not correlate with quantitative measures of chromatin accessibility, histone H3 lysine 4 trimethylation, or transcript levels. We also ruled out hotspot competition as a major source of indirect TF effects on DSB distributions. Thus, counter to prevailing models, roles of these TFs on DSB hotspot strength cannot be simply explained via chromatin "openness," histone modification, or compensatory interactions between adjacent hotspots. KEYWORDS Spo11; meiotic recombination; double-strand break; transcription factor; chromatin M EIOSIS is a specialized cell division in which one round of DNA replication is followed by two successive rounds of chromosome segregation to produce haploid gametes from diploid cells. In meiotic prophase, most sexually reproducing organisms use homologous recombination to form physical connections between homologous chromosomes that are essential for accurate chromosome segregation (Petronczki et al. 2003). Recombination also disrupts linkage of sequence polymorphisms on the same chromosome and thus promotes genome diversity and evolution (Kauppi et al. 2004).Recombination is initiated by the programmed formation of DNA double-strand breaks (DSBs). Approximately 150-200 DSBs are formed per meiosis in Saccharomyces cerevisiae (Buhler et al. 2007;Chen et al. 2008;Mancera et al. 2008;Pan et al. 2011), generated by a dimer of the topoisomerase-like protein Spo11 (Bergerat et al. 1997;Keeney et al. 1997). After break formation, Spo11 remains covalently bound to the 59 DNA strand termini (de Massy et al. 1995;Keeney and Kleckner 1995;Liu et al. 1995). Single-stranded n...

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Homologue engagement controls meiotic DNA break number and distribution

Cited by 194 publications

References 52 publications

Synaptonemal complex extension from clustered telomeres mediates full-length chromosome pairing in Schmidtea mediterranea

Synaptonemal complex extension from clustered telomeres mediates full-length chromosome pairing in Schmidtea mediterranea

Numerical and spatial patterning of yeast meiotic DNA breaks by Tel1

High-Resolution Global Analysis of the Influences of Bas1 and Ino4 Transcription Factors on Meiotic DNA Break Distributions in Saccharomyces cerevisiae

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