Chromatin remodeler Fun30 promotes efficient DSB end resection and recombination during yeast meiosis
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ABSTRACT:
PROVIDER: PRJNA914208 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22825939.fastq.gz | Fastqsanger.gz | |||
| SRR22825940.fastq.gz | Fastqsanger.gz | |||
| SRR22825941.fastq.gz | Fastqsanger.gz | |||
| SRR22825942.fastq.gz | Fastqsanger.gz | |||
| SRR22825943.fastq.gz | Fastqsanger.gz |
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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
Project description:Chromosomal double-strand breaks (DSBs) are resected by 5’-nucleases to form 3’ single-strand DNA (ssDNA) substrates for binding by homologous recombination and DNA damage checkpoint proteins. Two redundant pathways of extensive resection were described both in cells and in vitro, one relying on Exo1 exonuclease and the other on Sgs1 helicase and Dna2 nuclease. However, it remains unknown how resection proceeds within the context of chromatin where histones and histone-bound proteins represent barriers for resection enzymes. Here, we have identified the yeast nucleosome remodeling enzyme Fun30 as novel factor promoting DSB end resection. Fun30 is the major nucleosome remodeler promoting extensive Exo1- and Sgs1-dependent resection of DSBs while the RSC and INO80 chromatin remodeling complexes play redundant roles with Fun30 in resection adjacent to DSB ends. ATPase and helicase domains of Fun30, which are needed for nucleosome remodeling, are also required for resection. Fun30 is robustly recruited to DNA breaks and spreads around the DSB coincident with resection. Fun30 becomes less important for resection in the absence of the histone-bound Rad9 checkpoint adaptor protein known to block 5’ strand processing and in the absence of either histone H3 K79 methylation or γ-H2A, which mediate recruitment of the Rad9 . Together these data suggest that Fun30 helps to overcome the inhibitory effect of Rad9 on DNA resection.
2012-10-04 | GSE38601 | GEO
Project description:Chromosomal double-strand breaks (DSBs) are resected by 5M-bM-^@M-^Y-nucleases to form 3M-bM-^@M-^Y single-strand DNA (ssDNA) substrates for binding by homologous recombination and DNA damage checkpoint proteins. Two redundant pathways of extensive resection were described both in cells and in vitro, one relying on Exo1 exonuclease and the other on Sgs1 helicase and Dna2 nuclease. However, it remains unknown how resection proceeds within the context of chromatin where histones and histone-bound proteins represent barriers for resection enzymes. Here, we have identified the yeast nucleosome remodeling enzyme Fun30 as novel factor promoting DSB end resection. Fun30 is the major nucleosome remodeler promoting extensive Exo1- and Sgs1-dependent resection of DSBs while the RSC and INO80 chromatin remodeling complexes play redundant roles with Fun30 in resection adjacent to DSB ends. ATPase and helicase domains of Fun30, which are needed for nucleosome remodeling, are also required for resection. Fun30 is robustly recruited to DNA breaks and spreads around the DSB coincident with resection. Fun30 becomes less important for resection in the absence of the histone-bound Rad9 checkpoint adaptor protein known to block 5M-bM-^@M-^Y strand processing and in the absence of either histone H3 K79 methylation or M-NM-3-H2A, which mediate recruitment of the Rad9 . Together these data suggest that Fun30 helps to overcome the inhibitory effect of Rad9 on DNA resection. Genome-wide expression profiling of Yeast gene expression in two cell type including the wild type and a FUN30 knockout cell, each cell type is tested in two duplicates. Test relative gene integration efficiency in yeast genome-wide homozygous diploid deletion mutants.
2012-10-04 | E-GEOD-38601 | biostudies-arrayexpress
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 DSB dependent enrichment of Fun30 proteins at hotspots and meiotic axes.
2023-05-01 | GSE221033 | GEO
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
Project description:Analysis of the Saccharomyces cerevisiae transcriptome in the absence of the chromatin remodeler Fun30
2012-06-15 | GSE38735 | GEO
Project description:Analysis of the Saccharomyces cerevisiae transcriptome in the absence of the chromatin remodeler Fun30 A wild type and a fun30 null strain were grown in rich medium. Each culture was divided in two before RNA extraction. Two hybridizations were performed, both using independent RNA preparations from WT and the fun30 null mutant.
2012-06-14 | E-GEOD-38735 | biostudies-arrayexpress
Project description:The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. This implies the presence of insulator elements that separate adjacent domains, allowing them to maintain different chromatin structures. We show that the Fun30 chromatin remodeler, Fft3, is essential for maintaining a proper chromatin structure at centromeres and subtelomeres. Fft3 is localized to insulator elements and inhibits euchromatin assembly in silent chromatin domains. In its absence, euchromatic histone modifications and histone variants invade centromeres and subtelomeres, causing a mis-regulation of gene expression and severe chromosome segregation defects. Our data strongly suggest that Fft3 controls the identity of chromatin domains by protecting these regions from euchromatin assembly.
2011-04-02 | E-MEXP-3044 | biostudies-arrayexpress
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 during spermatogenesis 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 beyond that carried out by the orthologous proteins in budding yeast meiosis. 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 MRN roles in meiotic DSB end processing and establish the importance of resection for mammalian meiosis.
2024-05-06 | GSE266271 | GEO
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