Mus musculus strain:C57BL/6 (Mm), SK1 (Sc)
Ontology highlight
ABSTRACT:
PROVIDER: PRJDB15814 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| DRR464107_1.fastq.gz | Fastqsanger.gz | |||
| DRR464107_2.fastq.gz | Fastqsanger.gz | |||
| DRR464108_1.fastq.gz | Fastqsanger.gz | |||
| DRR464108_2.fastq.gz | Fastqsanger.gz | |||
| DRR464109_1.fastq.gz | Fastqsanger.gz |
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Project description:Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) and proceeds via binding of RPA, RAD51 and DMC1 to single-stranded DNA (ssDNA) substrates created after the formation of DSBs. Here, we report high-resolution in vivo maps of RPA and RAD51 binding in meiosis, mapping their binding locations and lifespans in a B6 and a genetically modified B6xCAST F1 mouse. We ascribe signals separately to the individual homologous chromosomes in the hybrid mouse, thereby separating the signal of binding to the chromosome where DSBs occurred and the chromosome that was used as template for repair. Together with super-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: whereas DMC1 binds near the break-site, RAD51 binds away from it. We characterize the D-loop, a critical intermediate bound by RPA, in vivo. These data show that DMC1, not RAD51, performs strand exchange in mammalian meiosis. We find that the localisation of D-loop intermediates is similar in crossover and non-crossover pathways, with a longer lifespan for crossover-destined intermediates. These findings answer long-standing questions about the molecular intermediates of recombination.
2020-06-30 | GSE143582 | GEO
Project description:Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks (DSBs). This process requires choreographed binding of RPA, DMC1 and RAD51 to single-stranded DNA (ssDNA) substrates and in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F1-hybrid mice, these maps can distinguish the broken chromosome from the homologous chromosome used as template for repair, which reveals further mechanistic detail and enables the structure of the recombination intermediates to be inferred. By applying CRISPR/Cas9 mutagenesis directly on F1-hybrid embryos, we have extended this powerful analysis technique to explore the molecular detail of recombination when a key component is knocked-out. As a proof-of-concept, we have generated biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in these knockout hybrid mice. Dmc1 mutants undergo meiotic arrest and comparison of these maps with those from wild-type mice is informative about the structure and timing of recombination intermediates in both genotypes. We confirm a complete abrogation of strand exchange in Dmc1 mutants, and observe a redistribution of RAD51 binding across both the distal and proximal ends of the resected DNA. We observe unexpected RPA and DMC1 binding in the wild-type, which suggests multiple rounds of strand invasion with template-switching in mouse. The methodology used involves direct phenotyping of hybrid “founder” mice following CRISPR mutagenesis and provides a high-throughput approach for the analysis of gene function during meiotic recombination, at low animal cost.
2023-10-16 | GSE239997 | GEO
Project description:We report the application of ChIP-seq targeted at the meiosis-specific protein DMC1 to reveal the genome-wide distribution of initiation of meiotic recombination. The mouse model here employed is Hop2-/- because it is unable to repair the DNA double-stranded breaks and therefore the DMC1 signal is more persistent. We also provide the resulting dataset of ChIP-seq targeted at RAD51 which is not meiosis specific but is also targeted at initiation of recombination loci in meiotic tissue. In addition, we report DMC1 ChIP-seq on wild type mouse pup testis. Finally, we present ChIP-seq targeted at H3K4me3 in testis and liver tissues.
2011-04-04 | GSE24438 | GEO
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
2007-12-30 | E-GEOD-8981 | biostudies-arrayexpress
Project description:The formation of RAD51/DMC1 filaments on single-stranded (ss)DNAs, which is essential for homology search and strand exchange in DNA double-strand break (DSB) repair and for the protection of stalled DNA replication forks, is tightly regulated in time and space. FIGNL1 AAA+++ ATPase plays positive and negative roles in the RAD51-mediated recombination in human cells. However, the role of FIGNL1 in gametogenesis remains unsolved. Here, we characterized a germ-line-specific conditional knockout (cKO) mouse of FIGNL1. The Fignl1 cKO male mice showed defective chromosome synapsis and impaired meiotic DSB repair with the accumulation of RAD51/DMC1 on chromosomes in mid-meiotic prophase I, supporting a role of FIGNL1 in a post-assembly stage of RAD51/DMC1 filaments. Fignl1 cKO spermatocytes accumulate RAD51 and DMC1 ensembles on chromosomes not only in early meiotic prophase I but also in meiotic S-phase. These RAD51/DMC1 assemblies are independent of meiotic DSB formation. Finally, we showed that purified FIGNL1 dismantles RAD51 filament on double-stranded (ds)DNA as well as ssDNA. These results suggest a critical role of FIGNL1 to limit the uncontrolled assembly of RAD51 and DMC1 on native dsDNAs during the meiotic S-phase and meiotic prophase I.
2023-03-31 | GSE227944 | GEO
Project description:RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity.
2017-02-18 | GSE95050 | GEO
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
2007-12-31 | GSE8981 | GEO
Project description:Recombinational repair of meiotic DNA double-strand breaks (DSBs) uses the homologous chromosome as a template, although the sister chromatid offers itself as a spatially more convenient substrate. In many organisms, this choice of the homolog rather than the sister is reinforced by the recombination protein Dmc1. We found that, in Tetrahymena, in the absence of two novel proteins, Mcmd1 and Pamd1, Dmc1 is misregulated. Hence, to find out potential interactions, we did LC-MS/MS analysis for Mcmd1 and Pamd1 immunoprecipitation samples.
2019-11-19 | PXD015986 | Pride
Project description:Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, AK125393, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51.
2015-02-19 | GSE66062 | GEO
Project description:The proper maintenance of genetic material is essential for the survival of living organisms. One of the main safeguards of genome stability is homologous recombination involved in the faithful repair of DNA double-strand breaks, the restoration of collapsed replication forks, and the bypass of replication barriers. Homologous recombination relies on the formation of Rad51 nucleofilaments which are responsible for the homology-based interactions between DNA strands. Here we demonstrate that without the regulation of these filaments by Srs2 and Rad54, which are known to remove Rad51 from single-stranded and double-stranded DNA respectively, the filaments strongly inhibit damage-associated DNA synthesis during DNA repair. Furthermore, this regulation is essential for cell survival under normal growth conditions as in the srs2Δ rad54Δ mutants unregulated Rad51 nucleofilaments cause activation of the DNA damage checkpoint, formation of mitotic bridges and loss of genetic material. These genome instability features may stem from the problems at stalled replication forks as the lack of Srs2 and Rad54 in the presence of Rad51 nucleofilaments impedes cell recovery from replication stress. This study demonstrates that the timely and efficient disassembly of recombination machinery is essential for genome maintenance and cell survival.
2023-03-16 | GSE227383 | GEO