Genome-wide mapping of DNA double-strand breaks from eukaryotic cell cultures using Break-seq

AbstractMaintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

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TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

10.1101/2021.11.30.470517 ◽

2021 ◽

Author(s):

Alexandre Nore ◽

Ariadna B Juarez-Martinez ◽

Julie AJ Clement ◽

Christine Brun ◽

Bouboub Diagouraga ◽

...

Keyword(s):

Dna Cleavage ◽

Point Mutations ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Catalytic Complex ◽

Strand Breaks ◽

Genome Wide ◽

Dna Cleavage Activity ◽

Meiotic Dsbs

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. In male and female meiotic cells, DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, but only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity by integrating specific chromosomal features.

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DNA Double-Strand Breaks Trigger Genome-Wide Sister-Chromatid Cohesion Through Eco1 (Ctf7)

Science ◽

10.1126/science.1140637 ◽

2007 ◽

Vol 317 (5835) ◽

pp. 245-248 ◽

Cited By ~ 219

Author(s):

E. Unal ◽

J. M. Heidinger-Pauli ◽

D. Koshland

Keyword(s):

Sister Chromatid ◽

Sister Chromatid Cohesion ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Genome Wide ◽

Chromatid Cohesion

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A global view of meiotic double-strand break end resection

Science ◽

10.1126/science.aak9704 ◽

2017 ◽

Vol 355 (6320) ◽

pp. 40-45 ◽

Cited By ~ 79

Author(s):

Eleni P. Mimitou ◽

Shintaro Yamada ◽

Scott Keeney

Keyword(s):

Meiotic Recombination ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Naked Dna ◽

Global View ◽

Genome Wide ◽

End Resection

DNA double-strand breaks that initiate meiotic recombination are exonucleolytically processed. This 5′→3′ resection is a central, conserved feature of recombination but remains poorly understood. To address this lack, we mapped resection endpoints genome-wide at high resolution inSaccharomyces cerevisiae. Full-length resection requires Exo1 exonuclease and the DSB-responsive kinase Tel1, but not Sgs1 helicase. Tel1 also promotes efficient and timely resection initiation. Resection endpoints display pronounced heterogeneity between genomic loci that reflects a tendency for nucleosomes to block Exo1, yet Exo1 also appears to digest chromatin with high processivity and at rates similar to naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a defining feature of the meiotic resection landscape.

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END-seq: An Unbiased, High-Resolution, and Genome-Wide Approach to Map DNA Double-Strand Breaks and Resection in Human Cells

Homologous Recombination - Methods in Molecular Biology ◽

10.1007/978-1-0716-0644-5_2 ◽

2020 ◽

pp. 9-31

Author(s):

Nancy Wong ◽

Sam John ◽

André Nussenzweig ◽

Andrés Canela

Keyword(s):

High Resolution ◽

Human Cells ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Genome Wide

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Genome-Wide Redistribution of Meiotic Double-Strand Breaks in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.02063-06 ◽

2006 ◽

Vol 27 (5) ◽

pp. 1868-1880 ◽

Cited By ~ 63

Author(s):

Nicolas Robine ◽

Norio Uematsu ◽

Franck Amiot ◽

Xavier Gidrol ◽

Emmanuel Barillot ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Recombination ◽

Binding Sites ◽

Chromosomal Region ◽

Double Strand Breaks ◽

Dna Binding Domain ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Genome Wide ◽

Local Stimulation

ABSTRACT Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. DSBs are not randomly distributed along chromosomes. To better understand factors that control the distribution of DSBs in budding yeast, we have examined the genome-wide binding and cleavage properties of the Gal4 DNA binding domain (Gal4BD)-Spo11 fusion protein. We found that Gal4BD-Spo11 cleaves only a subset of its binding sites, indicating that the association of Spo11 with chromatin is not sufficient for DSB formation. In centromere-associated regions, the centromere itself prevents DSB cleavage by tethered Gal4BD-Spo11 since its displacement restores targeted DSB formation. In addition, we observed that new DSBs introduced by Gal4BD-Spo11 inhibit surrounding DSB formation over long distances (up to 60 kb), keeping constant the number of DSBs per chromosomal region. Together, these results demonstrate that the targeting of Spo11 to new chromosomal locations leads to both local stimulation and genome-wide redistribution of recombination initiation and that some chromosomal regions are inherently cold regardless of the presence of Spo11.

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Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e08-12-1223 ◽

2009 ◽

Vol 20 (13) ◽

pp. 3064-3076 ◽

Cited By ~ 69

Author(s):

Kazuto Kugou ◽

Tomoyuki Fukuda ◽

Shintaro Yamada ◽

Masaru Ito ◽

Hiroyuki Sasanuma ◽

...

Keyword(s):

Chromatin Immunoprecipitation ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Spatial Regulation ◽

Tiling Arrays ◽

Meiotic Chromosomes ◽

Genome Wide ◽

Hydroxyurea Treatment ◽

Temporal And Spatial

Spo11-mediated DNA double-strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To elucidate this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thus, we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation.

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Regional Gene Repression by DNA Double-Strand Breaks in G1 Phase Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00181-19 ◽

2019 ◽

Vol 39 (24) ◽

Cited By ~ 4

Author(s):

Caitlin E. Purman ◽

Patrick L. Collins ◽

Sofia I. Porter ◽

Ankita Saini ◽

Harshath Gupta ◽

...

Keyword(s):

Transcriptional Repression ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Transcriptional Responses ◽

Strand Breaks ◽

Extensive Region ◽

Genome Wide ◽

Cellular Transcription ◽

Regional Gene

ABSTRACT DNA damage responses (DDR) to double-strand breaks (DSBs) alter cellular transcription programs at the genome-wide level. Through processes that are less well understood, DSBs also alter transcriptional responses locally, which may be important for efficient DSB repair. Here, we developed an approach to elucidate the cis-acting responses to DSBs in G1 phase cells. We found that DSBs within a gene body silence its expression, as well as the transcription of local undamaged genes at a distance defined by the spread of γ-H2AX from the DSB. Importantly, DSBs not only repress ongoing transcription but also block the inducible expression of regional genes. DSB-mediated transcriptional repression depends on DDR signaling but does not require the generation of inaccessible chromatin. Our findings demonstrate that in G1 phase cells, DDR signaling establishes a robust and extensive region of transcriptional repression spreading from DSB sites and introduce an approach to study the mechanistic impact of targeted DNA breaks in nearly any chromatin environment.

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