Faculty Opinions recommendation of DNA double-strand breaks trigger genome-wide sister-chromatid cohesion through Eco1 (Ctf7).

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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Double-strand-break-induced homologous recombination in mammalian cells

Biochemical Society Transactions ◽

10.1042/bst0290196 ◽

2001 ◽

Vol 29 (2) ◽

pp. 196-201 ◽

Cited By ~ 186

Author(s):

R. D. Johnson ◽

M. Jasin

Keyword(s):

Homologous Recombination ◽

Sister Chromatid ◽

Mammalian Cells ◽

Indirect Evidence ◽

Strand Break ◽

Double Strand Breaks ◽

Crossing Over ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

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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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Double-strand breaks are not the main cause of spontaneous sister chromatid exchange in wild-type yeast cells

10.1101/164756 ◽

2017 ◽

Cited By ~ 1

Author(s):

Clémence Claussin ◽

David Porubský ◽

Diana C.J. Spierings ◽

Nancy Halsema ◽

Stefan Rentas ◽

...

Keyword(s):

Sister Chromatid ◽

Sister Chromatid Exchange ◽

Genome Instability ◽

Single Cells ◽

Double Strand Breaks ◽

Yeast Cells ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Template Strand ◽

Sister Chromatids

SummaryHomologous recombination involving sister chromatids is the most accurate, and thus most frequently used, form of recombination-mediated DNA repair. Despite its importance, sister chromatid recombination is not easily studied because it does not result in a change in DNA sequence, making recombination between sister chromatids difficult to detect. We have previously developed a novel DNA template strand sequencing technique, called Strand-seq, that can be used to map sister chromatid exchange (SCE) events genome-wide in single cells. An increase in the rate of SCE is an indicator of elevated recombination activity and of genome instability, which is a hallmark of cancer. In this study, we have adapted Strand-seq to detect SCE in the yeast Saccharomyces cerevisiae. Contrary to what is commonly thought, we find that most spontaneous SCE events are not due to the repair of DNA double-strand breaks.

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Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex

PLoS Genetics ◽

10.1371/journal.pgen.1009663 ◽

2021 ◽

Vol 17 (7) ◽

pp. e1009663

Author(s):

Maria Velkova ◽

Nicola Silva ◽

Maria Rosaria Dello Stritto ◽

Alexander Schleiffer ◽

Pierre Barraud ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Sister Chromatid ◽

Meiotic Recombination ◽

Sister Chromatid Exchange ◽

Double Strand Breaks ◽

Scaffolding Protein ◽

Scaffolding Proteins ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Functional Homolog

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.

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