Saccharomyces cerevisiae
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ABSTRACT:
PROVIDER: PRJNA168290 | ENA |
REPOSITORIES: ENA
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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 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: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:Meiotic double-strand break end resection mapping by S1Seq
| PRJNA337955 | ENA
Project description:The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5′ and 3′ ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30-target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5′ end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2 and +3 nucleosomes at the 5′ end of target genes were significantly shifted, while position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity purified single-component Fun30 exhibited nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in regulation of transcription and indicate that Fun30 remodels chromatin at the 5′ end of genes by sliding promoter proximal nucleosomes.
2013-07-08 | GSE48570 | GEO
Project description:The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5′ and 3′ ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30-target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5′ end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2 and +3 nucleosomes at the 5′ end of target genes were significantly shifted, while position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity purified single-component Fun30 exhibited nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in regulation of transcription and indicate that Fun30 remodels chromatin at the 5′ end of genes by sliding promoter proximal nucleosomes.
2013-07-08 | GSE48562 | GEO
Project description:We determined nucleosome positions throughout the genome in diploid S. cerevisiae undergoing early stages of synchronous meiosis. This study sought to assess if systematic reorganization of nucleosomes occurs during meiotic prophase at or near sites of DNA double strand break formation.
2011-03-07 | GSE26452 | GEO
Project description:We used a plamsid-based assay to identify novel S. cerevisiae mutants with abnormal Break Induced Replication (BIR) efficiencies. We pooled the ca. 5000 yeast deletion mutants from the systematic deletion library and compared the relative tranformation efficiencies of a circular versus linear truncated minichromosome using tag arrays. Our main result is the identification of Fun30 as a novel chromatin remodelr facilitating DNA end resection.
2012-06-15 | GSE38715 | GEO