Histone H3 lysine 56 acetylation is required for formation of normal levels of meiotic DNA breaks in S. cerevisiae
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE37487 | GEO | 2019/12/25
REPOSITORIES: GEO
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Project description:During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strand can displace the parental strand to generate single-strand DNA (ssDNA) flaps. Many programmed DSBs and thus many ssDNA flaps occur during meiosis. However, how these flaps are removed remains enigmatic. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) in Saccharomyces cerevisiae causes an abundant accumulation of RPA-ssDNA flaps and an expansion of RPA from DSBs to broader regions. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, inducing Dna2 expression at pachytene is highly effective in removing accumulated RPA and restoring spore viability. Moreover, the depletion of Pif1, an activator of polymerase δ required for meiotic recombination-associated DNA synthesis, and Pif1 inhibitor Mlh2 decreased and increased RPA accumulation in dna2-md, respectively. Together, our findings show that meiotic DSB repair requires Dna2 to remove RPA-ssDNA flaps generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.
2023-08-30 | GSE217628 | GEO
Project description:Meiotic recombination is initiated by Spo11-catalyzed DNA double-strand breaks (DSBs) that are promoted by histone modifications and histone modifying enzymes. Herein we investigated the role of histone H3 lysine 56 acetylation (H3K56ac) located near the entry/exit points of the DNA in the globular H3 domain. We generated a series of mutant cells (asf1Δ, rtt109Δ, hst3/4Δ, and H3K56A) in which the endogenous level of H3K56ac was manipulated and tracked during meiotic growth. We show that complete loss or increased abundance of H3K56ac in these mutants allows timely entry into meiosis and sporulation and does not impair S phase progression, first and second meiotic cell divisions, and spore viability. In the asf1Δ, rtt109Δ, hst3/4Δ mutants, DSBs and crossovers form normal levels with a short (60-min) delay at the HIS4-LEU2 artificial recombination hotspot, however, DSB formation shows a ∼threefold decrease in the H3K56A mutant at the natural BUD23-ARE1 hotspot. The latter DSB phenotype, showing significant DSB reduction in the H3K56A mutant, was also observed at DSB sites using genome-wide mapping of Rfa1-coated single-stranded DNA flanking DSBs (RPA ChIP). Parallel mapping of H3K56-acetylated histones in wild type cells revealed strong depletion of the H3K56ac ChIP signal over Spo11-oligo DSBs, albeit most H3K56-acetylated histones were enriched adjacent to the identified RPA ChIP binding sites. Taken together, these associations demonstrate a prominent role of H3 lysine 56 acetylation in the formation of DNA breaks within recombination hotspot regions.
| S-EPMC6970188 | biostudies-literature
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:DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 (53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether it does so directly is not known. Here we identify Rapl-interacting factor 1 (Rif1) as an Ataxia-Telangiectasia Mutated (ATM) phosphorylation-dependent interactor of 53BP1, and show that absence of Rif1 results in 5M-bM-^@M-^Y-3M-bM-^@M-^Y DNA end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G1 and S phases of the cell cycle, interferes with class switch recombination (CSR) in B lymphocytes, and leads to accumulation of chromosome DSBs. Study of Rif1 DNA-end protection activity against resection via analysis of single-stranded DNA binding protein RPA and Rad51 accumulation at sites of AID-induced DNA damage by ChIP-seq. All samples shown in Fig. 4 are included (controls and test samples, 7 samples in total).
2013-03-01 | E-GEOD-42298 | biostudies-arrayexpress
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:Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ~5-34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Polδ-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ) or rarely by alternative end joining (Alt-EJ). This process is exacerbated in mutants with low levels or mutated RPA (rtt105Δ; rfa1-t33) or extensive resection mutant (sgs1Δ exo1Δ). Templated insertions from various distant genomic locations also increase in these mutants as well as in rad27 and originate from fragile regions of the genome. Among complex insertions, common events are insertions of two sequences, originating from the same locus and with inverted orientation. We propose that these inversions are also formed by microhomology-mediated template switching. Taken together, we propose that a shortage of RPA typical in cancer cells is one possible factor stimulating the formation of templated insertions.
2024-03-07 | GSE260753 | GEO
Project description:AID promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in G1. RPA is a ssDNA-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR) such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, RAG, or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in S-G2/M is extensive, ATM-independent, and associated with Rad51 accumulation. RPA in S-G2/M increases in NHEJ-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during CSR represents salvage of un-repaired breaks by homology-based pathways during the S-G2/M phases of the cell cycle. Chip-Seq of RPA from mouse activated B cells (n = 40), mouse thymocytes (n = 6), and MEFs (n = 1). Different genotypes and/or inhibitors were used.
2013-01-02 | E-GEOD-42451 | biostudies-arrayexpress
Project description:Meiotic crossovers result from homology-directed repair of double strand breaks (DSBs). Unlike yeast and plants, where DSBs are generated near gene promoters, in many vertebrates, DSBs are enriched at hotspots determined by the DNA binding activity of the rapidly evolving zinc finger array of PRDM9 (PR domain zinc finger protein 9), which subsequently catalyzes trimethylation of lysine 4 and lysine 36 of Histone H3 in nearby nucleosomes. Here, we identify the dual histone methylation reader ZCWPW1, which is tightly co-expressed during spermatogenesis with Prdm9 and co-evolved with Prdm9 in vertebrates, as an essential meiotic recombination factor required for efficient synapsis and repair of PRDM9-dependent DSBs. In sum, our results indicate that the evolution of dual histone methylation reader/writer system involving Prdm9 and Zcwpw1 facilitated a shift in genetic recombination away from a static pattern near genes towards a flexible pattern controlled by the rapidly evolving DNA binding activity of PRDM9
2020-05-12 | GSE139289 | GEO
Project description:The nonrandom distribution of meiotic recombination shapes patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in accumulation of Spo11 protein covalently bound to small DNA fragments. We show here that sequencing these fragments provides a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore the influence of large-scale chromosome structures, chromatin, transcription factors, and local sequence composition on DSB distributions. Our analysis supports the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. Mechanistic aspects of DSB formation and early processing steps are also uncovered. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We discuss implications for evolutionary dynamics of recombination hotspots. We deep sequenced 4 samples of Spo11 oligos on Roche 454 platform. Three samples are technical replicates of Spo11 oligo products prepared from one meiotic culture and the fourth sample was prepared from an independent culture.
2011-03-07 | E-GEOD-26449 | biostudies-arrayexpress
Project description:The BRCA2 tumor suppressor is a DNA double-strand break repair factor essential to maintain genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2 interacting partner. We show that DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid helicase activity of DDX5. Impaired BRCA2-DDX5 interaction, as observed in cells bearing the breast cancer variant BRCA2-T207A, reduces DDX5 association with DSBs, decreases the number of RPA foci upon irradiation and impairs RAD51 repair foci formation. Our findings are consistent with DNA-RNA hybrids being an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.
2021-02-26 | GSE150163 | GEO