Plasmid-based BIR screen in S. cerevisiae
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE38715 | GEO | 2012/06/15
SECONDARY ACCESSION(S): PRJNA168576
REPOSITORIES: GEO
Similar Datasets
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. A pool of ca. 5000 yeast deletion mutants from the systematic deletion library was transformed with a circular minichromosome on one hand, and with a linear truncated minichromosome on the other hand. Transformants were recovered, DNA was extracted and the systematic tags were PCR amplified with complementary dyes for the two transformations. Amplified tags were mixed and hybridized on a tag array. The overall experiment was done twice.
2012-06-14 | E-GEOD-38715 | biostudies-arrayexpress
Project description:We applied our ribonucleotide mapping technique HydEn-seq to study DNA polymerase usage during break-induced replication (BIR) in the budding yeast Saccharomyces cerevisiae.
2019-07-01 | GSE133558 | GEO
Project description:Fun30 is the prototype of the Fun30-SMARCAD1-ETL sub-family of nucleosome remodelers involved in DNA repair and gene silencing. These proteins appear to act as single subunit nucleosome remodelers, but their molecular mechanisms are, at this point, poorly understood. Using multiple sequence alignment and structure prediction, we identify an evolutionarily conserved domain that is modeled to contain a SAM-like fold with one long, protruding helix, which we term SAM-key. Deletion of the SAM-key within budding yeast Fun30 leads to a defect in DNA repair and gene silencing similar to that of the fun30 mutant. In vitro, Fun30 protein lacking the SAM key is able to bind nucleosomes but is deficient in DNA-stimulated ATPase activity as well as nucleosome sliding and eviction. A structural model based on AlphaFold2 prediction and verified by crosslinking-MS indicates an interaction of the long SAM-key helix with protrusion I, a subdomain located between the two ATPase lobes that is critical for control of enzymatic activity. Mutation of the interaction interface phenocopies the domain deletion with a lack of DNA-stimulated ATPase activation and a nucleosome remodeling defect, thereby confirming a role of the SAM-key helix in regulating ATPase activity. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, thus highlighting the importance of allosteric activation for this class of enzymes.
2023-07-18 | PXD037249 | Pride
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:Although Poly(ADP-ribose)-polymerases (PARPs) are key regulators of genome stability, how site-specific ADP-ribosylation regulates DNA repair is unclear. Here, we describe a novel role for PARP1 and PARP2 in regulating Rad52-dependent replication fork repair to maintain cell viability when HR is dysfunctional, suppress replication-associated DNA damage, and maintain genome stability. Mechanistically, Mre11 is required for induction of PARP activity in response to replication stress that in turn promotes break-induced replication (BIR) through assembly of Rad52 at stalled/damaged replication forks. Further, by mapping ADP-ribosylation sites induced upon replication stress, we identify that PolD3 is a target for PARP1/PARP2 and importantly, that its site-specific ADP-ribosylation is required for BIR activity, replication fork recovery and genome stability. Overall, these data identify a critical role for Mre11-dependent PARP activation and site-specific ADP-ribosylation in regulating BIR to maintain genome integrity during DNA synthesis.
2023-10-24 | PXD035661 | Pride
Project description:We analyzed the effect of SAC3, SUS1 and SRC1 on the transcription rates, mRNA stabilities and mRNA levels by doing GRO experiments in a deletion mutants and the partial C-truncated version of Sac3 comparing with a wild type. Some data for mRNA amounts (sus1 ans src1 mutants) are not included because were already in GEO database: GSE920 and GSE6370 accession numbers.
2015-12-31 | GSE63769 | GEO
Project description:Chromatin remodeling factors utilize ATP hydrolysis to modulate chromatin dynamics, and the Fun30-subfamily members in yeast and animals are a class of well-studied remodelers implicated in diverse biological processes. However, the molecular activity of plant Fun30 orthologues and their functions in plant growth still remain obscure yet. Compared with yeast and animal orthologues, Arabidopsis Fun30 possesses conserved ATP-dependent nucleosome sliding activity but loses the CUE domain functioning as protein adapter in evolution. Fun30 gene shows a tissue-specific expression pattern in vegetative meristems and various vascular tissues, and is functionally implicated in transcriptional repression of a large number of responsive genes under uninduced conditions. We focused on the interconnected phytohormones SA/JA pathways, and proved the Fun30 recruitment and changes of local nucleosome occupancy within chromatin regions of several key signaling and effector genes in each pathway. Besides SUVR2 involved in RdDM pathway, Fun30 also interacts with non-catalytic subunits of histone deacetylase complex, and Fun30-binding proteins exert in vitro TSA-inhibited histone deacetylase activity. In fun30 mutant, we detected increased H3 acetylation level within local chromatin regions of Fun30 target genes in vivo. Intriguingly, Fun30 transcription is severely inhibited by SA treatment, which thus releases its repression on SA-induced gene during the activation of the downstream SA pathway. Meanwhile, basal transcription level and JA-induced activation of key transcription factor MYC2 gene, as well as the consequent susceptibility to necrotrophic pathogen Botrytis cinerea, are abnormally increased in Fun30-deficient plants.
2020-08-25 | GSE156722 | 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 | 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:Heterozygous mutations in the transcription factor GATA3 are identified in 10-15% of all breast cancer cases. Most of these are protein-truncating mutations, concentrated within or downstream of the second GATA-type zinc-finger domain. Here, we investigated the functional consequences of expression of two truncated GATA3 mutants, in vitro in breast cancer cell lines and in vivo in the mouse mammary gland. We found that the truncated GATA3 mutants display altered DNA binding activity caused by preferred tethering through FOXA1. In addition, expression of the truncated GATA3 mutants reduces E-cadherin expression and promotes anchorage-independent growth in vitro. However, we could not identify any effects of truncated GATA3 expression on mammary gland development or mammary tumor formation in mice. Together, our results demonstrate that both truncated GATA3 mutants promote cistromic re-programming of GATA3 in vitro, but these mutants are not sufficient to induce tumor formation in mice.
2019-06-12 | GSE122848 | GEO