ANKRD31 anchors meiotic double-strand break formation as a direct partner of REC114
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE118913 | GEO | 2019/12/25
REPOSITORIES: GEO
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Project description:DNA double-strand breaks (DSBs) initiate meiotic recombination. ANKRD31 recently emerged as a DSB regulator particularly important in the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including DSB-promoting REC114. To test if this interaction is important, we generated mice with Ankrd31 mutations that either reduced (missense) or eliminated (C-terminal truncation) the ANKRD31–REC114 interaction without diminishing contacts with other partners. Eliminating the interaction mimicked an Ankrd31 null, with delayed DSB formation, defective DSB repair, and altered DSB locations including absence of PAR breaks. When the ANKRD31–REC114 interaction was instead attenuated, DSB formation was again delayed globally, but DSB locations and repair were affected little if at all. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes. Our results show that ANKRD31 function is critically dependent on its interaction with REC114, and that defects in ANKRD31 activity correlate with the severity of interaction disruption.
2023-04-18 | GSE229450 | GEO
Project description:DNA duplication is intimately connected to setting up post-replicative chromosome structures and events, but molecular details of this coordination are not well understood. A striking example occurs during yeast meiosis, where replication locally influences timing of the DNA double-strand breaks (DSBs) that initiate recombination. We show here that replication-DSB coordination is eliminated by overexpressing Dbf4-dependent Cdc7 kinase (DDK) or removing Tof1 or Csm3, components of the replication fork protection complex (FPC). DDK physically associates with Tof1, and Tof1 is dispensable for replication-DSB coordination if DDK is artificially tethered to replisomes. Furthermore, DDK phosphorylation of the DSB-promoting factor Mer2 is locally coordinated with replication, dependent on Tof1. These findings indicate that DDK recruited by FPC to replisomes phosphorylates chromatin-bound Mer2 in the wake of the replication fork, thus synchronizing replication with an early prerequisite for DSB formation. This may be a general mechanism to ensure spatial and temporal coordination of replication with other chromosomal processes. Ninety-six samples total: 12 time points (each time points contains ChIP and input samples) from Rec114-myc ARS+, Rec114-myc arsM-bM-^HM-^F strains, Rec114-myc tof1M-bM-^HM-^FARS+ and Rec114-myc tof1M-bM-^HM-^F arsM-bM-^HM-^F strains
2014-07-21 | E-GEOD-52970 | biostudies-arrayexpress
Project description:In most organisms, meiotic recombination begins with programmed DNA double strand break (DSB) formation by Spo11. Here, we present evidence that Tel1/Mec1, the budding yeast ATM/ATR, regulate DSB formation by phosphorylating Rec114, an essential Spo11-accessory protein. Analyses of a non-phosphorylatable- or phosphomimetic- alleles of rec114 revealed that DSB-dependent phosphorylation of Rec114 limited its association with DSB-hotspots resulting in reduction in DSB formation. Also observed were the impact of Rec114 phosphorylation on its homolog synapsis-associated removal from chromosomes and NDT80-dependent turnover. Specifically, we found that the synapsis- and NDT80-dependent Rec114 downregulation occurred later in the rec114 mutant with a reduced Spo11-catalysis, but earlier in the other with an enhanced catalysis, strongly implicating the existence of a feedback mechanism coupling the extent of Spo11-catalysis to Rec114 activity. Taken together, these observations suggest that three different mechanisms of down regulating Rec114 contribute to meiotic DSB homeostasis, a feedback mechanism to maintain the number of meiotic DSBs at the developmentally programmed level. 6 genome wide ChIPchip sets: 3 for meiotic DSB formation (Spo11-ChIP) and 3 for protein-DNA association (Rec114-ChIP), each for wild type and two mutants during meiosis (corresponding to the main Figure 3, as well as to Figures S3, S4, S5).
2013-06-01 | E-GEOD-43384 | biostudies-arrayexpress
Project description:To segregate accurately during meiosis, homologous chromosomes in most species must recombine. Very small chromosomes would risk missegregation if recombination were randomly distributed, so the double-strand breaks (DSBs) that initiate recombination are not haphazard. How this nonrandomness is controlled is not understood. Here we demonstrate that Saccharomyces cerevisiae integrates multiple, temporally distinct pathways to regulate chromosomal binding of pro-DSB factors Rec114 and Mer2, thereby controlling duration of a DSB-competent state. Homologous chromosome engagement regulates Rec114/Mer2 dissociation late in prophase, whereas replication timing and proximity to centromeres or telomeres influence timing and amount of Rec114/Mer2 accumulation early. A distinct early mechanism boosts Rec114/Mer2 binding quickly to high levels specifically on the shortest chromosomes, dependent on chromosome axis proteins and subject to selection pressure to maintain hyperrecombinogenic properties of these chromosomes. Thus, an organism’s karyotype and its attendant risk of meiotic missegregation influence the shape and evolution of its recombination landscape.
2018-09-12 | GSE119787 | GEO
Project description:In most organisms, meiotic recombination begins with programmed DNA double strand break (DSB) formation by Spo11. Here, we present evidence that Tel1/Mec1, the budding yeast ATM/ATR, regulate DSB formation by phosphorylating Rec114, an essential Spo11-accessory protein. Analyses of a non-phosphorylatable- or phosphomimetic- alleles of rec114 revealed that DSB-dependent phosphorylation of Rec114 limited its association with DSB-hotspots resulting in reduction in DSB formation. Also observed were the impact of Rec114 phosphorylation on its homolog synapsis-associated removal from chromosomes and NDT80-dependent turnover. Specifically, we found that the synapsis- and NDT80-dependent Rec114 downregulation occurred later in the rec114 mutant with a reduced Spo11-catalysis, but earlier in the other with an enhanced catalysis, strongly implicating the existence of a feedback mechanism coupling the extent of Spo11-catalysis to Rec114 activity. Taken together, these observations suggest that three different mechanisms of down regulating Rec114 contribute to meiotic DSB homeostasis, a feedback mechanism to maintain the number of meiotic DSBs at the developmentally programmed level.
2013-06-01 | GSE43384 | GEO
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:Meiotic recombination promotes genetic diversification as well as pairing and segregation of homologous chromosomes, but the double-strand breaks (DSBs) that initiate recombination are dangerous lesions that can cause mutation or meiotic failure. How cells control DSBs to balance between beneficial and deleterious outcomes is not well understood. This study tests the hypothesis that DSB control involves a network of intersecting regulatory circuits. We show that DSBs form in greater numbers in Saccharomyces cerevisiae cells lacking ZMM proteins, a suite of recombination-promoting factors traditionally regarded as acting strictly downstream of DSB formation. This counterintuitive result suggests that homologous chromosomes that have successfully engaged one another stop making DSBs, and provides new insight into phenotypes of zmm and other recombination-defective mutants. A genetically distinct pathway ties DSB formation to meiotic progression through the Ndt80 transcription factor. High-resolution genome-wide DSB maps generated by sequencing short oligonucleotides covalently bound to Spo11 (Spo11 oligos) demonstrate that feedback tied to ZMM function contributes in unexpected ways to spatial patterning of the recombination landscape. Four samples total: two wild type and two zip3 mutant (each an independent culture)
2014-04-23 | E-GEOD-48299 | biostudies-arrayexpress
Project description:DNA duplication is intimately connected to setting up post-replicative chromosome structures and events, but molecular details of this coordination are not well understood. A striking example occurs during yeast meiosis, where replication locally influences timing of the DNA double-strand breaks (DSBs) that initiate recombination. We show here that replication-DSB coordination is eliminated by overexpressing Dbf4-dependent Cdc7 kinase (DDK) or removing Tof1 or Csm3, components of the replication fork protection complex (FPC). DDK physically associates with Tof1, and Tof1 is dispensable for replication-DSB coordination if DDK is artificially tethered to replisomes. Furthermore, DDK phosphorylation of the DSB-promoting factor Mer2 is locally coordinated with replication, dependent on Tof1. These findings indicate that DDK recruited by FPC to replisomes phosphorylates chromatin-bound Mer2 in the wake of the replication fork, thus synchronizing replication with an early prerequisite for DSB formation. This may be a general mechanism to ensure spatial and temporal coordination of replication with other chromosomal processes.
2014-07-21 | GSE52970 | GEO
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:Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2 and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S-phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in a domain-specific fashion by cohesin. Loss of Rec114, Mer2 and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.
2011-08-28 | GSE29860 | GEO