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The MRE11-RAD50-NBS1 complex both starts and extends DNA end resection in mouse meiosis [ChIP-seq]

ABSTRACT: Nucleolytic resection of DNA ends is critical for homologous recombination, but its mechanism is not fully understood, particularly in mammalian meiosis. Here we examine roles of the conserved MRN complex (MRE11, RAD50, and NBS1) through genome-wide analysis of meiotic resection in mice with various MRN mutations, including several that cause chromosomal instability in humans. Meiotic DSBs form at elevated levels but remain unresected if Mre11 is conditionally deleted, thus MRN is required for both resection initiation and regulation of DSB numbers. Resection lengths are reduced to varying degrees in MRN hypomorphs or if MRE11 nuclease activity is attenuated in a conditional nuclease-dead Mre11 model. These findings unexpectedly establish that MRN is needed for longer-range extension of resection, not just resection initiation. Finally, resection defects are additively worsened by combining MRN and Exo1 mutations, and mice that are unable to initiate resection or have greatly curtailed resection lengths experience catastrophic spermatogenic failure. Our results elucidate multiple functions of MRN in meiotic recombination, uncover unanticipated relationships between short- and long-range resection, and establish the importance of resection for mammalian meiosis.

ORGANISM(S): Mus musculus

PROVIDER: GSE266151 | GEO | 2024/05/06

REPOSITORIES: GEO

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The MRE11-RAD50-NBS1 complex both starts and extends DNA end resection in mouse meiosis [SSDS]

Project description:Nucleolytic resection of DNA ends is critical for homologous recombination, but its mechanism is not fully understood, particularly in mammalian meiosis. Here we examine roles of the conserved MRN complex (MRE11, RAD50, and NBS1) through genome-wide analysis of meiotic resection in mice with various MRN mutations, including several that cause chromosomal instability in humans. Meiotic DSBs form at elevated levels but remain unresected if Mre11 is conditionally deleted, thus MRN is required for both resection initiation and regulation of DSB numbers. Resection lengths are reduced to varying degrees in MRN hypomorphs or if MRE11 nuclease activity is attenuated in a conditional nuclease-dead Mre11 model. These findings unexpectedly establish that MRN is needed for longer-range extension of resection, not just resection initiation. Finally, resection defects are additively worsened by combining MRN and Exo1 mutations, and mice that are unable to initiate resection or have greatly curtailed resection lengths experience catastrophic spermatogenic failure. Our results elucidate multiple functions of MRN in meiotic recombination, uncover unanticipated relationships between short- and long-range resection, and establish the importance of resection for mammalian meiosis.

2024-05-06 | GSE266271 | GEO

The MRE11-RAD50-NBS1 complex both starts and extends DNA end resection in mouse meiosis [S1_Exo7T_ExoT]

2024-05-06 | GSE266258 | GEO

A Rad50-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells

Project description:Programed DNA double-strand breaks (DSBs) catalyzed by the topoisomerase II-like enzymes, SPO11 and TOPVIBL, initiate meiotic recombination. Following DSB formation, the MRE11-RAD50-NBS1/Xrs2 (MRN/X) complex, along with EXO1 and DNA2, cleave the SPO11-DNA to generate 3′ single-stranded DNA (ssDNA) ends, which are prerequisite for meiotic DSB repair. In both yeast and mammals, MRE11 exhibits endonucleolytic cleavage of the 5′ terminated DNA strand in the vicinity of the DSBs and exonucleolytic resection from 3′ to 5′ towards the DSB ends. RAD50 is a structure maintenance of chromosome (SMC) related protein that contains one ATPase domain at its N- and C- terminal ends, respectively, Zn hook, and anti-parallel coiled coils. RAD50 plays a crucial role in facilitating the MRE11 nuclease activity on DSBs by ATP binding and hydrolysis. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic DSB ends at the molecular level remains elusive. Here, we performed END-seq with synchronized zygotene spermatocytes from control and germ cell specific Rad50 mutant (Rad50-sKO) mice. We find that the number of formed DSB in the mutant spermatocytes was reduced compared to control spermatocytes (6 636 DSBs in Rad50-sKO spermatocytes vs 8 168 in control spermatocytes) and abnormal DSB end resection occurred in mutant spermatocytes (DSB end resection length: 1 279 nts in Rad50-sKO spermatocytes vs 1 923 nts in control spermatocytes). Thus, RAD50 is essential for DSB formation and end resection during mammalian meiosis.

2024-04-02 | GSE262706 | GEO

Molecular structures and mechanisms of DNA break processing in mouse meiosis

Project description:Exonucleolytic resection is a critical step in repair of DNA double-strand breaks (DSBs) via homologous recombination, but resection mechanisms are not well understood, particularly in mammalian meiosis. Here, we use a genome-wide, nucleotide-resolution analysis to define the molecular structure of resected DSBs in mouse spermatocytes. Resection tracts averaged 1100 nucleotides on each side of a DSB, but with a broad distribution and substantial fine-scale heterogeneity at individual hotspots. Surprisingly, eliminating the nuclease activity of EXO1 only modestly decreased resection lengths, thus EXO1 is not the major 5′→3′ exonuclease in mouse meiosis. In contrast, the DSB-responsive kinase ATM proved to be a key regulator of both the initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3′ ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.

2020-07-10 | GSE141850 | GEO

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

Project description:Meiotic recombination is initiated by genome-wide SPO11-induced double-strand breaks (DSBs) that are processed by MRE11-mediated release of SPO11-bound oligonucleotides (SPO11-oligos). The DSB is then resected and loaded with DMC1/RAD51 filaments that invade homologous chromosome templates. In most mammals, DSB locations (“hotspots”) are determined by the DNA sequence specificity of the PRDM9 DNA binding zinc finger array. Here, we demonstrate the first direct detection of meiotic DSBs in vertebrates by performing END-seq on mouse spermatocytes using low sample input. We find that DMC1 limits both the minimum and maximum length of ssDNA produced at all hotspots, whereas 53BP1, BRCA1 and EXO1 play a surprisingly minimal role in meiotic resection. Through enzymatic modifications to the END-seq protocol that mimic the in vivo processing of SPO11, we identify a novel meiotic recombination intermediate (RI) with SPO11 still bound to the 3’ end via a small stretch of dsDNA (“SPO11-RI”) that is dependent on the presence of PRDM9. We propose that SPO11-RI is generated because chromatin-bound PRDM9 blocks MRE11 from releasing SPO11 via 3’-5’ resection. SPO11-RI is present at all hotspots and correlates with the localization and frequency of crossovers and noncrossovers. In Atm–/– spermatocytes, multiple DSBs at the same hotspot reduces SPO11-RI formation, while unresected DNA-bound SPO11 accumulates because of defective MRE11 initiation. Thus in addition to their global roles in governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.

2020-02-20 | GSE138915 | GEO

Optimized methods for mapping DNA double-strand-break ends and resection tracts and application to meiotic recombination in mouse spermatocytes

Project description:DNA double-strand breaks (DSBs) made by SPO11 protein initiate homologous recombination during meiosis. Subsequent to DNA strand breakage, endo- and exo-nucleases process the DNA ends to resect the strands whose 5' termini are at the DSB, generating long 3'-terminal single-stranded tails that serve as substrates for strand exchange proteins. DSB resection is essential for meiotic recombination, but a detailed understanding of its molecular mechanism is currently lacking. Genomic approaches to mapping DSBs and resection endpoints, e.g., S1-sequencing (S1-seq) and similar methods, play a critical role in studies of meiotic DSB processing. In these methods, nuclease S1 or other enzymes that specifically degrade ssDNA are used to trim resected DSB ends, allowing capture and sequencing of the ends of resection tracts. Here, we present optimization of S1-seq that improves its signal:noise ratio and allows its application to analysis of spermatocyte meiosis in adult mice. Furthermore, quantitative features of meiotic resection are evaluated for reproducibility, and we suggest best practices for analysis and interpretation of S1-seq data. We also compare S1-seq to variants that use exonuclease T and or exonuclease VII from Escherichia coli instead of nuclease S1. Detailed step-by-step protocols and suggestions for troubleshooting are provided.

2024-04-30 | GSE265863 | GEO

Chromatin remodeler Fun30 promotes efficient DSB end resection and recombination during yeast meiosis

Project description:Resection, nucleolytic processing of DSB ends is necessary to generate 3' single-stranded DNA tails (ssDNA) for homologous recombination (HR). Meiotic recombination initiated by Spo11 induced double-strand breaks (DSBs) is essential for the accurate segregation of homologous chromosomes. After cleavage, Spo11 stays covalently linked to the DSB ends, which requires MRX/Sae2 incision on broken molecules to allow following Exo1-mediated resection. Exonuclease I activity is inhibited by nucleosome bound DNA in vitro. To ensure resection proceeds, resection machineries must overcome chromatin barrier. Here we show that in the absence of Fun30, an SNF2-like ATPase, resection tract lengths shortened, albeit less severe than exo1-nd (with more longer resection tracts than that in exo1nd), across all DSB hotspots, suggesting that Fun30 is required for meiotic resection. Additive resection defect in fun30Δ exo1-nd (nuclease dead) compared to exo1-nd mutant indicates that Fun30 regulates MRX/Sae2 nicking positions. We also observed extremely short resection tracts in the double mutants are mostly confined to NDR, suggesting initial nicking step is blocked by nucleosomes.

2023-05-01 | GSE221377 | GEO

MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

Project description:Rad50 is a component of the conserved MRE11-RAD50-NBS1 (MRN) complex, which functions in genome stability and the cell’s ability to deal with stalled DNA replication forks. We identified Rad50 as a factor important for R-loop tolerance and thus mapped DNA:RNA hybrids in Rad50KO cells and compare them to previously reported wild-type and Sgs1KO profiles.

2019-10-31 | E-MTAB-7885 | biostudies-arrayexpress

Transcription profiling of S. cerevisiae mre11, rad50 and spo11 mutants in premeiosis and meiotic prophase

Project description:Mre11, together with Rad50 and Xrs2/NBS, plays pivotal roles in homologous recombination, repair of DNA double strand breaks (DSBs), activation of damage-induced checkpoint, and telomere maintenance. Using DNA microarray assays to analyze yeast mutants (mre11delta, rad50delta, and spo11Y135F) defective for meiotic DSB formation, we demonstrate that the absence of Mre11 in yeast causes specific effects on regulation of a class of meiotic genes for spore development. The transcriptional deficiency was not observed in other DSB mutants such as rad50delta and spo11Y135F, suggesting the transcriptional defect in mre11delta is due to neither lack of meiotic DSB formation, nor disintegrity of Mre11-Rad50-Xrs2 complex.These defects were confirmed by northern and lacZ reporter gene assays. Experiment Overall Design: Gene expression data from wild type, mre11delta, rad50delta, and spo11Y135F cells in premeiosis (meiosis 0 hr) and prophase (meiosis 4 hr). Affymetrix GeneChip YG-S98 was used. All strains used were SK1 background diploid cells.

2007-10-23 | E-GEOD-6620 | biostudies-arrayexpress

OsMre11 is required for mitosis during rice growth and development

Project description:Meiotic recombination 11 (Mre11) is a relatively conserved nuclease in various species. Mre11 plays important roles in meiosis and DNA damage repair in yeast, humans and Arabidopsis, but little research has been done on mitotic DNA replication and repair in rice. Here, it was found that Mre11 was an extensively expressed gene among the various tissues and organs of rice, and loss-of-function of Mre11 resulted in severe defects of vegetative and reproductive growth, including dwarf plants, abnormally developed male and female gametes, and completely abortive seeds. The decreased number of cells in the apical meristem and the appearance of chromosomal fragments and bridges during the mitotic cell cycle in rice mre11 mutant roots revealed an essential role of OsMre11. Further research showed that DNA replication was suppressed, and a large number of DNA strand breaks occurred during the mitotic cell cycle of rice mre11 mutants. The expression of OsMre11 was up-regulated with the treatment of hydroxyurea and methyl methanesulfonate. Moreover, OsMre11 could form a complex with OsRad50 and OsNbs1, and they might function together in non-homologous end joining and homologous recombination repair pathways. These results indicated that OsMre11 plays vital roles in DNA replication and damage repair of the mitotic cell cycle, which ensure the development and fertility of rice by maintaining genome stability.

2021-01-13 | GSE163616 | GEO

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