DSB formation in replication compromised cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE35666 | GEO | 2012/06/19
SECONDARY ACCESSION(S): PRJNA155867
REPOSITORIES: GEO
Similar Datasets
Project description:DSBs were mapped genome-wide by ssDNA enrichment in cdc6-mn replication comprimsied strains. We mapped DSB sites by detecting DSB-associated ssDNA enrichment on microarrays. To test the role of DNA replication in DSB formation, we mapped ssDNA in a cdc6-mn replication depleted strain. ssDNA was isolated from cells after 5 hours in sporulation medium. As a reference, ssDNA isolated from cells at 0 hrs in sporulation medium prior to DSB formation was differentially labeled and co-hybridized to the same array. For each experiment, we have submitted biological replicates that were hybridized to separate arrays. For each experiment a dye swap was performed and shown to have no effect on the data observed, although not all experiments in this series include the dye swap sample (we have included only two representative experiments for each strain).
2012-06-18 | E-GEOD-35666 | biostudies-arrayexpress
Project description:Mcm2-7 ChIP in pre-meiotic and pre-mitotic cells, axis factor ChIP in wild-type and replication compromised strains in meiosis Multiple studies of meiotic chromosomes were undertaken. To study DNA replication, the locations of replicative helicase (Mcm2-7) were mapped in pre-meiotic and pre-mitotic cells, and DNA replication profiles were created for pre-meiotic S (meiS) and pre-mitotic S (mitS) phases. Early origins were mapped in hydroxyurea for wild-type cells in mitS + 200mM HU, and meiS +20mM HU for wild-type, sml1, rec8 and spo11 deletion cells. Rec8, Hop1 and Red1 binding to meiotic chromosomes was evaluated using ChIP-chip in wild-type cells with and without 20 mM HU, and in cdc6-mn and clb5 clb6 delete cells. Finally, meiotic DNA double-strand breaks (DSBs) were mapped in cdc6-mn dmc1 delete cells by measuring the ssDNA that accumulates at DSB hotspots.
2012-06-18 | E-GEOD-35658 | biostudies-arrayexpress
Project description:During gamete formation, crossover recombination must occur on replicated DNA to ensure proper chromosome segregation in the first meiotic division. We identified a Mec1/ATR-dependent replication checkpoint in budding yeast that prevented the earliest stage of recombination, the programmed induction of DNA double-strand breaks (DSBs), when pre-meiotic DNA replication was delayed. The checkpoint suppressed DSBs through three complementary mechanisms: inhibition of Mer2 phosphorylation by Dbf4-dependent Cdc7 kinase, preclusion of chromosomal loading of Rec114 and Mre11, and lowered abundance of the Spo11 nuclease. Without this checkpoint, cells formed DSBs on partially replicated chromosomes. Importantly, such DSBs frequently failed to be repaired and impeded further DNA synthesis, leading to a rapid loss in cell viability. We conclude that a checkpoint-dependent constraint of DSB formation to duplicated DNA is critical not only for meiotic chromosome assortment, but also to protect genome integrity during gametogenesis. DSB factor association was measured in wild-type and checkpoint mutants strains under non-inducing or replication checkpoint inducing conditions. Additionally, DNA replication and helicase loading were measured in a replication and checkpoint deficient strain (cdc6-mn).
2013-09-20 | E-GEOD-46841 | biostudies-arrayexpress
Project description:DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, due to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination 1. Within the budding yeast repetitive ribosomal (r)DNA array, meiotic DSB formation is prevented in part through Sir2-dependent heterochromatin 2,3. Here, we demonstrate that the edges of the rDNA array are exceptionally susceptible to meiotic DSBs, revealing an inherent heterogeneity within the rDNA array. We find that this localised DSB susceptibility necessitates a border-specific protection system consisting of the meiotic ATPase Pch2 and the origin recognition complex subunit Orc1. Upon disruption of these factors, DSB formation and recombination specifically increased in the outermost rDNA repeats, leading to NAHR and rDNA instability. Strikingly, the Sir2-dependent heterochromatin of the rDNA itself was responsible for the induction of DSBs at the rDNA borders in pch2? cells. Thus, while Sir2 activity globally prevents meiotic DSBs within the rDNA, it creates a highly permissive environment for DSB formation at the heterochromatin/euchromatin junctions. Heterochromatinised repetitive DNA arrays are abundantly present in most eukaryotic genomes. Our data define the borders of such chromatin domains as distinct high-risk regions for meiotic NAHR, whose protection may be a universal requirement to prevent meiotic genome rearrangements associated with genomic diseases and birth defects. This SuperSeries is composed of the following subset Series: GSE30071: ssDNA mapping in dmc1 strains GSE30072: ChIP-chip of DSB factors in wild type and pch2 strains Two types of study were undertaken to understand the regulation of meiotic DSB formation close to repetitive DNA elements in yeast. First, DSBs were mapped using ssDNA enrichment in strains isogenic for a dmc1 mutation, and also including pch2 delete, orc1-161, rdna delete and a reciprocal translocation between chromosomes 2 and 12 (trans2to12). Second, the association of the DSB factors Hop1, Rec114, Mer2, and Mre1, as well as total histone H3 and H3K4-trimethylation were measured by ChIP-chip analysis in wild-type and pch2 delete strains.
2011-08-14 | E-GEOD-30073 | biostudies-arrayexpress
Project description:The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication, and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis, and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not directly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes, and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms. Multiple studies of meiotic chromosomes were undertaken. To study DNA replication, the locations of replicative helicase (Mcm2-7) were mapped in pre-meiotic and pre-mitotic cells, and DNA replication profiles were created for pre-meiotic S (meiS) and pre-mitotic S (mitS) phases. Early origins were mapped in hydroxyurea for wild-type cells in mitS + 200mM HU, and meiS +20mM HU for wild-type, sml1, rec8 and spo11 deletion cells. Rec8, Hop1 and Red1 binding to meiotic chromosomes was evaluated using ChIP-chip in wild-type cells with and without 20 mM HU, and in cdc6-mn and clb5 clb6 delete cells. Finally, meiotic DNA double-strand breaks (DSBs) were mapped in cdc6-mn dmc1 delete cells by measuring the ssDNA that accumulates at DSB hotspots. This SuperSeries is composed of the following subset Series: GSE35658: Chromatin IP for Mcm2-7, Rec8, Hop1 and Red1 GSE35662: S phase and HU profiles in wild-type and mutant cells GSE35666: DSB formation in replication compromised cells
2012-06-14 | E-GEOD-35667 | biostudies-arrayexpress
Project description:Comparative genome hybridization (CGH) was used to measure relative DNA replication after 8 hours in meiosis for wild-type and cdc6-mn cells. We measured DNA replication after 8 hours in meiosis for wild-type and cdc6-mn cells on microarrays using CGH.
2013-09-20 | E-GEOD-46840 | biostudies-arrayexpress
Project description:Comparative genome hybridization (CGH) was used to measure relative DNA replication after 8 hours in meiosis for wild-type and cdc6-mn cells.
2013-09-20 | GSE46840 | GEO
Project description:Meiotic DNA double stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. Although DSBs can be directly mapped using ChIP-Seq and antibody against ssDNA-associated proteins, genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method, single-stranded DNA (ssDNA) sequencing (SSDS), that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS consists of a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. When applied to mapping meiotic DSBs, the use of SSDS reduces the non-specific dsDNA background more than ten-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired. Development and validation of the method, SSDS, for the specific detection of ssDNA-derived and dsDNA-derived fragments in sequencing libraries and enrichment of ssDNA-derived fragments. SSDS was used to detect meiotic DSBs in 9R/13R mice.
2012-02-21 | E-GEOD-34592 | biostudies-arrayexpress
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:Meiotic DNA double stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. Although DSBs can be directly mapped using ChIP-Seq and antibody against ssDNA-associated proteins, genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method, single-stranded DNA (ssDNA) sequencing (SSDS), that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS consists of a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. When applied to mapping meiotic DSBs, the use of SSDS reduces the non-specific dsDNA background more than ten-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.
2012-02-21 | GSE34592 | GEO