Role of the Nuclease Activity of Saccharomyces cerevisiae Mre11 in Repair of DNA Double-Strand Breaks in Mitotic Cells

AbstractThe Rad50:Mre11:Xrs2 (RMX) complex functions in repair of DNA double-strand breaks (DSBs) by recombination and nonhomologous end-joining (NHEJ) and is also required for telomere stability. The Mre11 subunit exhibits nuclease activities in vitro, but the role of these activities in repair in mitotic cells has not been established. In this study we have performed a comparative study of three mutants (mre11-D16A, -D56N, and -H125N) previously shown to have reduced nuclease activities in vitro. In ends-in and ends-out chromosome recombination assays using defined plasmid and oligonucleotide DNA substrates, mre11-D16A cells were as deficient as mre11 null strains, but defects were small in mre11-D56N and -H125N mutants. mre11-D16A cells, but not the other mutants, also displayed strong sensitivity to ionizing radiation, with residual resistance largely dependent on the presence of the partially redundant nuclease Exo1. mre11-D16A mutants were also most sensitive to the S-phase-dependent clastogens hydroxyurea and methyl methanesulfonate but, as previously observed for D56N and H125N mutants, were not defective in NHEJ. Importantly, the affinity of purified Mre11-D16A protein for Rad50 and Xrs2 was indistinguishable from wild type and the mutant protein formed complexes with equivalent stoichiometry. Although the role of the nuclease activity has been questioned in previous studies, the comparative data presented here suggest that the nuclease function of Mre11 is required for RMX-mediated recombinational repair and telomere stabilization in mitotic cells.

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Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.00156-17 ◽

2017 ◽

Vol 37 (24) ◽

Cited By ~ 8

Author(s):

Sucheta Arora ◽

Rajashree A. Deshpande ◽

Martin Budd ◽

Judy Campbell ◽

America Revere ◽

...

Keyword(s):

Nuclease Activity ◽

Double Strand Breaks ◽

Endonuclease Activity ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Content Type ◽

Strand Breaks ◽

Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

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Coupling crossover and synaptonemal complex in meiosis

Genes & Development ◽

10.1101/gad.349286.121 ◽

2022 ◽

Vol 36 (1-2) ◽

pp. 4-6

Author(s):

Corinne Grey ◽

Bernard de Massy

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Synaptonemal Complex ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Homologous Chromosomes

During meiosis, a molecular program induces DNA double-strand breaks (DSBs) and their repair by homologous recombination. DSBs can be repaired with or without crossovers. ZMM proteins promote the repair toward crossover. The sites of DSB repair are also sites where the axes of homologous chromosomes are juxtaposed and stabilized, and where a structure called the synaptonemal complex initiates, providing further regulation of both DSB formation and repair. How crossover formation and synapsis initiation are linked has remained unknown. The study by Pyatnitskaya and colleagues (pp. 53–69) in this issue of Genes & Development highlights the central role of the Saccharomyces cerevisiae ZMM protein Zip4 in this process.

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Mapping Meiotic Single-Strand DNA Reveals a New Landscape of DNA Double-Strand Breaks in Saccharomyces cerevisiae

PLoS Biology ◽

10.1371/journal.pbio.0050324 ◽

2007 ◽

Vol 5 (12) ◽

pp. e324 ◽

Cited By ~ 151

Author(s):

Cyril Buhler ◽

Valérie Borde ◽

Michael Lichten

Keyword(s):

Saccharomyces Cerevisiae ◽

Double Strand Breaks ◽

Single Strand ◽

Dna Double Strand Breaks ◽

Strand Breaks

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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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CTCF cooperates with CtIP to drive homologous recombination repair of double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkz639 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9160-9179 ◽

Cited By ~ 6

Author(s):

Soon Young Hwang ◽

Mi Ae Kang ◽

Chul Joon Baik ◽

Yejin Lee ◽

Ngo Thanh Hang ◽

...

Keyword(s):

Homologous Recombination ◽

Critical Role ◽

Control Point ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

C Terminus ◽

Dna End Resection ◽

End Resection

Abstract The pleiotropic CCCTC-binding factor (CTCF) plays a role in homologous recombination (HR) repair of DNA double-strand breaks (DSBs). However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, we show that CTCF engages in DNA end resection, which is the initial, crucial step in HR, through its interactions with MRE11 and CtIP. Depletion of CTCF profoundly impairs HR and attenuates CtIP recruitment at DSBs. CTCF physically interacts with MRE11 and CtIP and promotes CtIP recruitment to sites of DNA damage. Subsequently, CTCF facilitates DNA end resection to allow HR, in conjunction with MRE11–CtIP. Notably, the zinc finger domain of CTCF binds to both MRE11 and CtIP and enables proficient CtIP recruitment, DNA end resection and HR. The N-terminus of CTCF is able to bind to only MRE11 and its C-terminus is incapable of binding to MRE11 and CtIP, thereby resulting in compromised CtIP recruitment, DSB resection and HR. Overall, this suggests an important function of CTCF in DNA end resection through the recruitment of CtIP at DSBs. Collectively, our findings identify a critical role of CTCF at the first control point in selecting the HR repair pathway.

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