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In vitro reconstitution of meiotic DNA double-strand break formation

ABSTRACT: The Spo11 complex catalyzes the formation of DNA double-strand breaks (DSBs), initiating meiotic recombination-a process essential for fertility and genetic diversity. Although Spo11’s function has been known for 27 years, previous efforts to reconstitute DSB formation in vitro have been unsuccessful. Here, we biochemically characterize mouse SPO11 and TOP6BL protein complex and demonstrate that this complex cleaves DNA and covalently attaches to the 5' terminus of DNA breaks in vitro. Using a point-mutation strategy, we reveal that Mg2+ is essential for DNA cleavage activity of this complex in vitro, as confirmed by knock-in mice carrying a point mutation in SPO11 that disrupts its binding to Mg2+, thereby abolishing DSB formation. However, the activity of the SPO11 complex is ATP-independent. We also present evidence that the mouse SPO11 complex is biochemically distinct from the ancestral topoisomerase VI. Our findings establish a mechanistic framework for understanding the initial steps of meiotic recombination.

ORGANISM(S): Mus musculus

PROVIDER: GSE281557 | GEO | 2025/01/20

REPOSITORIES: GEO

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Physical interaction with Spo11 mediates the localisation of Mre11 to chromatin in meiosis and promotes its nuclease activity

Project description:Meiotic recombination is of central importance for the proper segregation of homologous chromosomes and is initiated by DNA double-strand breaks (DSB) that depend on Spo11 and Mre11. Calibrated chromatin immunoprecipitation (ChIP) for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A C-terminal deletion mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death.

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Meiotic recombination cold spots in chromosomal cohesion sites [ChIP-chip]

Project description:Meiotic chromosome architecture called M-bM-^@M-^\axis-loop structuresM-bM-^@M-^] and histone modifications have been demonstrated to regulate the Spo11-dependent formation of DNA double-strand breaks (DSBs) that trigger meiotic recombination. Using genome-wide chromatin immunoprecipitation (ChIP) analyses followed by deep sequencing, we compared the genome-wide distribution of the axis protein Rec8 (the kleisin subunit of meiotic cohesin) with that of oligomeric DNA covalently bound to Spo11, indicative of DSB sites. The frequency of DSB sites is overall constant between Rec8 binding sites. However, DSB cold spots are observed in regions spanning M-BM-10.8 kb around Rec8 binding sites. The axis-associated cold spots are not due to exclusion of Spo11 localization from the axis, since ChIP experiments revealed that substantial Spo11 persists at Rec8 binding sites during DSB formation. Spo11 fused with Gal4 DNA binding domain (Gal4BD-Spo11) tethered in close proximity (M-bM-^IM-$0.8 kb) to Rec8 binding sites hardly forms meiotic DSBs, in contrast with other regions. In addition, H3K4 tri-methylation (H3K4me3) remarkably decreases at Rec8 binding sites. These results suggest that reduced histone H3K4me3 in combination with inactivation of Spo11 activity on the axis discourages DSB hot spot formation. ChIP-chip analysis of Rec8 on fission yeast meiotic chromosomes

2014-03-31 | E-GEOD-52858 | biostudies-arrayexpress

Meiotic recombination cold spots in chromosomal cohesion sites [ChIP-Seq]

Project description:Meiotic chromosome architecture called M-bM-^@M-^\axis-loop structuresM-bM-^@M-^] and histone modifications have been demonstrated to regulate the Spo11-dependent formation of DNA double-strand breaks (DSBs) that trigger meiotic recombination. Using genome-wide chromatin immunoprecipitation (ChIP) analyses followed by deep sequencing, we compared the genome-wide distribution of the axis protein Rec8 (the kleisin subunit of meiotic cohesin) with that of oligomeric DNA covalently bound to Spo11, indicative of DSB sites. The frequency of DSB sites is overall constant between Rec8 binding sites. However, DSB cold spots are observed in regions spanning M-BM-10.8 kb around Rec8 binding sites. The axis-associated cold spots are not due to exclusion of Spo11 localization from the axis, since ChIP experiments revealed that substantial Spo11 persists at Rec8 binding sites during DSB formation. Spo11 fused with Gal4 DNA binding domain (Gal4BD-Spo11) tethered in close proximity (M-bM-^IM-$0.8 kb) to Rec8 binding sites hardly forms meiotic DSBs, in contrast with other regions. In addition, H3K4 tri-methylation (H3K4me3) remarkably decreases at Rec8 binding sites. These results suggest that reduced histone H3K4me3 in combination with inactivation of Spo11 activity on the axis discourages DSB hot spot formation. ChIP-seq analyses of Rec8, Spo11, and Gal4BD-Spo11 on budding yeast meiotic chromosomes M-bM-^@M-" Distribution of Rec8 in wt and Gal4BD-Spo11-expressing cells at 4h after meiotic induction M-bM-^@M-" Distribution of Spo11 at 3h, 4h, and 5h after meiotic induction M-bM-^@M-" Distribution of Gal4BD-Spo11 at 0h after meiotic induction

2014-03-31 | E-GEOD-52862 | biostudies-arrayexpress

Spo11-oligo mapping of double-strand breaks in pch2 and sir2 mutants

Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to map the distribution of meiotic DSBs in pch2 and sir2 mutant strains of Saccharomyces cerevisiae.

2018-11-25 | GSE122882 | GEO

Genome wide map of DMC1 in testis of Stag3-/-, Rec8-/- and Stag3-/-Rec8-/- mice.

Project description:During mouse meiosis, DNA double-strand breaks (DSBs) are initiated by SPO11 at recombination hotspots (HSs), activated by PRDM9. Although activated HSs are marked by H3K4me3 and H3K36me3 histone modifications at open chromatin, most of the DSB-initiating and repair proteins are associated with the chromosome axis. This study addresses the mechanistic importance of the axis-associated cohesin proteins in DSB formation. We demonstrate that interactions between PRDM9 and the meiotic axis proteins STAG3 and REC8 are essential for efficient DSB formation. The absence of STAG3 or REC8 leads to inefficient meiotic DSB formation at HSs. STAG3 is critical for DSB formation, even at PRDM9-independent DSB-sites. Also, STAG3 and REC8 facilitate recruitment of the DSB-promoting proteins HORMAD1, IHO1 and MEI4 required for SPO11 activity. Together, these results support an evolutionarily conserved model in which axis-associated cohesin complexes recruit recombination-initiating proteins to DSB sites to promote meiotic recombination initiation.

2019-03-07 | GSE112110 | GEO

Spo11-oligo mapping of DSBs in an SK1 x S288C F1 hybrid strain

Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to map the distribution of meiotic DSBs in an SK1 x S88C F1 hybrid strain of Saccharomyces cerevisiae.

2017-07-13 | GSE101339 | GEO

Meiotic recombination cold spots in chromosomal cohesion sites [ChIP-Seq]

Project description:Meiotic chromosome architecture called “axis-loop structures” and histone modifications have been demonstrated to regulate the Spo11-dependent formation of DNA double-strand breaks (DSBs) that trigger meiotic recombination. Using genome-wide chromatin immunoprecipitation (ChIP) analyses followed by deep sequencing, we compared the genome-wide distribution of the axis protein Rec8 (the kleisin subunit of meiotic cohesin) with that of oligomeric DNA covalently bound to Spo11, indicative of DSB sites. The frequency of DSB sites is overall constant between Rec8 binding sites. However, DSB cold spots are observed in regions spanning ±0.8 kb around Rec8 binding sites. The axis-associated cold spots are not due to exclusion of Spo11 localization from the axis, since ChIP experiments revealed that substantial Spo11 persists at Rec8 binding sites during DSB formation. Spo11 fused with Gal4 DNA binding domain (Gal4BD-Spo11) tethered in close proximity (≤0.8 kb) to Rec8 binding sites hardly forms meiotic DSBs, in contrast with other regions. In addition, H3K4 tri-methylation (H3K4me3) remarkably decreases at Rec8 binding sites. These results suggest that reduced histone H3K4me3 in combination with inactivation of Spo11 activity on the axis discourages DSB hot spot formation.

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Meiotic recombination cold spots in chromosomal cohesion sites [ChIP-chip]

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

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