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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): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Benjamin Neuditschko

LAB HEAD: Franz Herzog

PROVIDER: PXD042221 | Pride | 2024-02-23

REPOSITORIES: Pride

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Project description:DNA double-strand breaks (DSBs) initiate meiotic recombination. Past DSB-mapping studies have used rad50S or sae2? mutants, which are defective in break processing, to accumulate DSBs, and report large (= 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2? mutants. We therefore developed novel methods that detect DSBs using ssDNA enrichment and microarray hybridization, and that use background-based normalization to allow cross-comparison between array datasets, to map genome-wide the DSBs that accumulate in processing-capable, repair-defective dmc1î and dmc1î rad51î mutants. DSBs were observed at known hotspots, but also in most previously-identified “DSB-cold” regions, including near centromeres and telomeres. While about 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1? shows that most of these regions have significant DSB activity. Thus, DSBs are distributed much more uniformly than was previously believed. Southern-blot assays of DSBs in selected regions in dmc1?, rad50S and wild-type cells confirm these findings. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as the primary strand transfer activity genome-wide, and Spo11-induced lesions as initiating all meiotic recombination. Keywords: DSB mapping, ChIP-chip, single strand DNA , BND cellulose We use two different strategies to map the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) based approach that targets the Spo11p protein, which remains covalently attached to DSB ends in the rad50S mutant background. The second approach involves BND cellulose enrichment of the single strand DNA (ssDNA) recombination intermediate formed by end-resection at DSB sites following Spo11p removal. We use dmc1 and dmc1 rad51 mutants that accumulates meiotic single strand DNA intermediates

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

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