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Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes

ABSTRACT: Recombination-mediated linking of homologous chromosomes is a unique meiotic feature that enables the halving of chromosomal ploidy in sexual life cycles. Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) that are generated by focal multi-protein clusters that condense onto chromosomal axes by poorly understood mechanisms. We discovered in mice that a DBF4-dependent kinase (DDK)–modulated interaction between IHO1 and the chromosomal axis component HORMAD1 effects the formation of axis-bound IHO1 platforms that enhance nucleation of cytologically distinguishable DSB-machinery clusters. This IHO1-HORMAD1-mediated seeding of the DSB-machinery ensures that sufficient DSBs form for efficient pairing of homologous chromosomes. In the absence of IHO1-HORMAD1 interaction, residual DSB activity depends on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, meiotic DSBs are ensured by complementing pathways that differentially effect seeding and growth of DSB-machinery clusters, and that synergistically enable assembly of the DSB machinery on chromosomal axes.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Benjamin Neuditschko

LAB HEAD: Franz Herzog

PROVIDER: PXD042221 | Pride | 2024-02-23

REPOSITORIES: Pride

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			Action	DRS
	IHO1_NH3_FAIMS_CID_1.mzML	Mzml
	IHO1_NH3_FAIMS_CID_1.mzid.gz	Mzid
	IHO1_NH3_FAIMS_CID_1.raw	Raw
	IHO1_NH3_FAIMS_CID_2.mzML	Mzml
	IHO1_NH3_FAIMS_CID_2.mzid.gz	Mzid

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Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes

Project description:Recombination-mediated linking of homologous chromosomes is a unique meiotic feature that enables the halving of chromosomal ploidy in sexual life cycles. Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) that are generated by focal multi-protein clusters that condense onto chromosomal axes by poorly understood mechanisms. We discovered in mice that a DBF4-dependent kinase (DDK)–modulated interaction between IHO1 and the chromosomal axis component HORMAD1 effects the formation of axis-bound IHO1 platforms that enhance nucleation of cytologically distinguishable DSB-machinery clusters. This IHO1-HORMAD1-mediated seeding of the DSB-machinery ensures that sufficient DSBs form for efficient pairing of homologous chromosomes. In the absence of IHO1-HORMAD1 interaction, residual DSB activity depends on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, meiotic DSBs are ensured by complementing pathways that differentially effect seeding and growth of DSB-machinery clusters, and that synergistically enable assembly of the DSB machinery on chromosomal axes.

2024-02-23 | PXD042179 | Pride

Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes.

Project description:Programmed DNA double-strand break (DSB) formation is a crucial feature of meiosis in most organisms. DSBs initiate recombination-mediated linking of homologous chromosomes, which enables correct chromosome segregation in meiosis. DSBs are generated on chromosome axes by heterooligomeric focal clusters of DSB-factors. Whereas DNA-driven protein condensation is thought to assemble the DSB-machinery, its targeting to chromosome axes is poorly understood. We uncover in mice that efficient biogenesis of DSB-machinery clusters requires seeding by axial IHO1 platforms. Both IHO1 phosphorylation and formation of axial IHO1 platforms are diminished by chemical inhibition of DBF4-dependent kinase (DDK), suggesting that DDK contributes to the control of the axial DSB-machinery. Furthermore, we show that axial IHO1 platforms are based on an interaction between IHO1 and the chromosomal axis component HORMAD1. IHO1-HORMAD1-mediated seeding of the DSB-machinery on axes ensures sufficiency of DSBs for efficient pairing of homologous chromosomes. Without IHO1-HORMAD1 interaction, residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, recombination initiation is ensured by complementary pathways that differentially support seeding and growth of DSB-machinery clusters, thereby synergistically enabling DSB-machinery condensation on chromosomal axes.

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Mouse REC114 is essential for meiotic DNA double-strand break formation and forms a complex with MEI4.

Project description:Programmed formation of DNA double-strand breaks (DSBs) initiates the meiotic homologous recombination pathway. This pathway is essential for proper chromosome segregation at the first meiotic division and fertility. Meiotic DSBs are catalyzed by Spo11. Several other proteins are essential for meiotic DSB formation, including three evolutionarily conserved proteins first identified in Saccharomyces cerevisiae (Mer2, Mei4, and Rec114). These three S. cerevisiae proteins and their mouse orthologs (IHO1, MEI4, and REC114) co-localize on the axes of meiotic chromosomes, and mouse IHO1 and MEI4 are essential for meiotic DSB formation. Here, we show that mouse Rec114 is required for meiotic DSB formation. Moreover, MEI4 forms a complex with REC114 and IHO1 in mouse spermatocytes, consistent with cytological observations. We then demonstrated in vitro the formation of a stable complex between REC114 C-terminal domain and MEI4 N-terminal domain. We further determine the structure of the REC114 N-terminal domain that revealed similarity with Pleckstrin homology domains. These analyses provide direct insights into the architecture of these essential components of the meiotic DSB machinery.

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Caenorhabditis elegans DSB-3 reveals conservation and divergence among protein complexes promoting meiotic double-strand breaks.

Project description:Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: Recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2, and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have coevolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.

| S-EPMC8379965 | biostudies-literature

Four-pronged negative feedback of DSB machinery in meiotic DNA-break control in mice.

Project description:In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.

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Genome-wide redistribution of meiotic double-strand breaks in Saccharomyces cerevisiae.

Project description: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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MEIOK21: a new component of meiotic recombination bridges required for spermatogenesis.

Project description:Repair of DNA double-strand breaks (DSBs) with homologous chromosomes is a hallmark of meiosis that is mediated by recombination 'bridges' between homolog axes. This process requires cooperation of DMC1 and RAD51 to promote homology search and strand exchange. The mechanism(s) regulating DMC1/RAD51-ssDNA nucleoprotein filament and the components of 'bridges' remain to be investigated. Here we show that MEIOK21 is a newly identified component of meiotic recombination bridges and is required for efficient formation of DMC1/RAD51 foci. MEIOK21 dynamically localizes on chromosomes from on-axis foci to 'hanging foci', then to 'bridges', and finally to 'fused foci' between homolog axes. Its chromosome localization depends on DSBs. Knockout of Meiok21 decreases the numbers of HSF2BP and DMC1/RAD51 foci, disrupting DSB repair, synapsis and crossover recombination and finally causing male infertility. Therefore, MEIOK21 is a novel recombination factor and probably mediates DMC1/RAD51 recruitment to ssDNA or their stability on chromosomes through physical interaction with HSF2BP.

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DNA-driven condensation assembles the meiotic DNA break machinery.

Project description:The accurate segregation of chromosomes during meiosis-which is critical for genome stability across sexual cycles-relies on homologous recombination initiated by DNA double-strand breaks (DSBs) made by the Spo11 protein1,2. The formation of DSBs is regulated and tied to the elaboration of large-scale chromosome structures3-5, but the protein assemblies that execute and control DNA breakage are poorly understood. Here we address this through the molecular characterization of Saccharomyces cerevisiae RMM (Rec114, Mei4 and Mer2) proteins-essential, conserved components of the DSB machinery2. Each subcomplex of Rec114-Mei4 (a 2:1 heterotrimer) or Mer2 (a coiled-coil-containing homotetramer) is monodispersed in solution, but they independently condense with DNA into reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive this condensation. Mutations that weaken protein-DNA interactions strongly disrupt both condensate formation and DSBs in vivo, and thus these processes are highly correlated. In vitro, condensates fuse into mixed RMM clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centres of DSB activity. We propose that multilayered control of Spo11 arises from the recruitment of regulatory components and modulation of the biophysical properties of the condensates.

| S-EPMC8016751 | biostudies-literature

Chromosomal synapsis defects can trigger oocyte apoptosis without elevating numbers of persistent DNA breaks above wild-type levels.

Project description:Generation of haploid gametes depends on a modified version of homologous recombination in meiosis. Meiotic recombination is initiated by single-stranded DNA (ssDNA) ends originating from programmed DNA double-stranded breaks (DSBs) that are generated by the topoisomerase-related SPO11 enzyme. Meiotic recombination involves chromosomal synapsis, which enhances recombination-mediated DSB repair, and thus, crucially contributes to genome maintenance in meiocytes. Synapsis defects induce oocyte apoptosis ostensibly due to unrepaired DSBs that persist in asynaptic chromosomes. In mice, SPO11-deficient oocytes feature asynapsis, apoptosis and, surprisingly, numerous foci of the ssDNA-binding recombinase RAD51, indicative of DSBs of unknown origin. Hence, asynapsis is suggested to trigger apoptosis due to inefficient DSB repair even in mutants that lack programmed DSBs. By directly detecting ssDNAs, we discovered that RAD51 is an unreliable marker for DSBs in oocytes. Further, SPO11-deficient oocytes have fewer persistent ssDNAs than wild-type oocytes. These observations suggest that oocyte quality is safeguarded in mammals by a synapsis surveillance mechanism that can operate without persistent ssDNAs.

| S-EPMC9177993 | biostudies-literature

The DNA Damage Checkpoint Eliminates Mouse Oocytes with Chromosome Synapsis Failure.

Project description:Pairing and synapsis of homologous chromosomes during meiosis is crucial for producing genetically normal gametes and is dependent upon repair of SPO11-induced double-strand breaks (DSBs) by homologous recombination. To prevent transmission of genetic defects, diverse organisms have evolved mechanisms to eliminate meiocytes containing unrepaired DSBs or unsynapsed chromosomes. Here we show that the CHK2 (CHEK2)-dependent DNA damage checkpoint culls not only recombination-defective mouse oocytes but also SPO11-deficient oocytes that are severely defective in homolog synapsis. The checkpoint is triggered in oocytes that accumulate a threshold level of spontaneous DSBs (∼10) in late prophase I, the repair of which is inhibited by the presence of HORMAD1/2 on unsynapsed chromosome axes. Furthermore, Hormad2 deletion rescued the fertility of oocytes containing a synapsis-proficient, DSB repair-defective mutation in a gene (Trip13) required for removal of HORMADs from synapsed chromosomes, suggesting that many meiotic DSBs are normally repaired by intersister recombination in mice.

| S-EPMC5621520 | biostudies-literature

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