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Mechanism of in vivo activation of the MutL-Exo1 complex for meiotic crossover formation

ABSTRACT: Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination and their subsequent repair culminates in crossover (CO) formation. COs result from the asymmetric cleavage of double-Holliday junction (dHJ) intermediates, that requires the MutLγ endonuclease and a non-catalytic function of Exo1, an activity essential for fertility but at risk of generating unwanted chromosome rearrangements. Here we show how crossover formation by MutLγ is activated at the right time and at the right place. MutLγ forms a constitutive complex with Exo1, and in meiotic cells transiently contacts the upstream MutSγ (Msh4-Msh5) heterodimer. MutLγ associates with DSB hotspots only once recombination intermediates have been stabilized and engaged in the crossover repair pathway. MutLγ-Exo1 is recruited to DSB hotspots independently of the polo-like Cdc5 kinase, but to activate dHJ resolution, Cdc5 is recruited to the recombination intermediates and interacts individually with both MutLγ and Exo1, suggesting their direct modification. in vivo, MutLγ occupancy is restrained on recombination intermediates, and MutLγ associates with the vast majority of DSB hotspots, but at a lower frequency in centromeres, consistent with a strategy to reduce at-risk crossover events in these regions, and in late replicating regions. Our data highlight the tight temporal and spatial control of the activity of this constitutive, potentially harmful, nuclease.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE133108 | GEO | 2020/01/15

REPOSITORIES: GEO

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Mechanism of in vivo activation of the MutLgamma-Exo1 complex for meiotic crossover formation

Project description:Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination and their subsequent repair culminates in crossover (CO) formation. COs result from the asymmetric cleavage of double-Holliday junction (dHJ) intermediates, that requires the MutLγ complex together with a non-catalytic function of Exo1, an activity essential for fertility but at risk of generating unwanted chromosome rearrangements. Here we show how crossover formation by MutLγ is activated at the right time and at the right place. MutLγ forms a constitutive complex with Exo1, and in meiotic cells transiently contacts the upstream MutSγ (Msh4-Msh5) heterodimer. MutLγ associates with DSB hotspots at a late step in the recombinational repair, once recombination intermediates have been stabilized and engaged in the crossover repair pathway. MutLγ-Exo1 is recruited to DSB hotspots independently of the polo-like Cdc5 kinase, but to activate dHJ resolution, Cdc5 is recruited to the recombination intermediates and interacts individually with both MutLγ and Exo1, suggesting their direct modification. in vivo, MutLγ occupancy is restrained on recombination intermediates, and genome-wide, MutLγ associates with the vast majority of DSB hotspots, but at a lower frequency in centromeres, consistent with a strategy to reduce at-risk crossover events in these regions, and in late replicating regions. Our data highlight the highly temporally and spatially control of the activity of this constitutive, potentially harmful, nuclease

2019-12-01 | GSE132850 | GEO

Molecular structures and mechanisms of DNA break processing in mouse meiosis

Project description:Exonucleolytic resection is a critical step in repair of DNA double-strand breaks (DSBs) via homologous recombination, but resection mechanisms are not well understood, particularly in mammalian meiosis. Here, we use a genome-wide, nucleotide-resolution analysis to define the molecular structure of resected DSBs in mouse spermatocytes. Resection tracts averaged 1100 nucleotides on each side of a DSB, but with a broad distribution and substantial fine-scale heterogeneity at individual hotspots. Surprisingly, eliminating the nuclease activity of EXO1 only modestly decreased resection lengths, thus EXO1 is not the major 5′→3′ exonuclease in mouse meiosis. In contrast, the DSB-responsive kinase ATM proved to be a key regulator of both the initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3′ ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.

2020-07-10 | GSE141850 | GEO

Turning coldspots into hotspots: targeted recruitment of axis protein Hop1 stimulates meiotic recombination in Saccharomyces cerevisiae

Project description:The DNA double strand breaks (DSBs) that initiate meiotic recombination are formed in the context of the meiotic chromosome axis, which in budding yeast contains a meiosis-specific cohesin isoform and the meiosis-specific proteins Hop1 and Red1. Hop1 and Red are important for DSB formation; DSB levels are reduced in their absence and their levels, which vary along the lengths of chromosomes, are positively correlated with DSB levels. How axis protein levels influence DSB formation and recombination remains unclear. To address this question, we developed a novel approach that uses a bacterial ParB-parS partition system to recruit axis proteins at high levels to inserts at recombination coldspots where Hop1 and Red1 levels are normally low. Recruiting Hop1 markedly increased DSBs and homologous recombination at target loci, to levels equivalent to those observed at endogenous recombination hotspots. This local increase in DSBs did not require Red1 or the meiosis-specific cohesin component Rec8, indicating that, of the axis proteins, Hop1 is sufficient to promote DSB formation. However, while most crossovers at endogenous recombination hotspots are formed by the meiosis-specific MutLγ resolvase, only a small fraction of crossovers that formed at an insert locus required MutLγ, regardless of whether or not Hop1 was recruited to that locus. Thus, while local Hop1 levels determine local DSB levels, the recombination pathways that repair these breaks can be determined by other factors, raising the intriguing possibility that different recombination pathways operate in different parts of the genome.

2022-07-12 | GSE201240 | GEO

Fun30-Myc ChIP-seq in Spo11 wild type and spo11-yf mutants

2023-05-01 | GSE221033 | GEO

Recombination initiation maps of individual human genomes

Project description:DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and to generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here we present the first high-resolution map of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors dictate the efficiency of DSB formation. Furthermore, we find that in human males, the frequency of DSB formation is the primary determinant of crossover rate. Patterns of sequence polymorphisms around meiotic DSB hotspots provide evidence for both GC-biased gene conversion and for a mutagenic role of DSB repair and/or recombination. Finally, we provide compelling evidence that the aberrant repair of meiotic DSBs is a driver of human genomic disorders. Detection of meiotic double strand breaks in testis of several human male individuals.

2014-11-17 | E-GEOD-59836 | biostudies-arrayexpress

Network rewiring of homologous recombination during mitotic proliferation and meiosis

Project description:Homologous recombination is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid, or enforce, formation of reciprocal exchanges – crossovers – between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity-purified the 7 DNA-processing enzymes with known roles in channelling HR intermediates into crossovers, or non-crossovers, from vegetative cells, or cells undergoing meiosis. Using mass spectrometry, we provide the first global characterization of their composition. The resulting mitosis- and meiosis-specific interaction maps reveal intricate changes in enzyme architecture and a concerted rewiring of the interaction networks to support flexible control to the recombination outcome. Moreover, functional analyses of 31 novel interactions uncovered 8 meiosis-specific network components that remodel HR to support crossing-over. Chd1, which transiently associates with Exo1 during meiosis, enables the formation of MutLγ-Exo1-dependent crossovers through its conserved ability to bind and displace nucleosomes.

2019-07-31 | PXD012486 | Pride

Chromatin remodeler Fun30 promotes efficient DSB end resection and recombination during yeast meiosis

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

Recombination initiation maps of individual human genomes

2014-11-17 | GSE59836 | GEO

Histone H3 lysine9 acetylation, rather than lysine4 trimethylation, marks meiotic recombination hotspots and promotes recombination initiation in fission yeast

Project description:Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double strand break (DSB) formation, but the role and precise landscape of histone modifications at hotspots remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is strikingly elevated, and H3K4me3 is not significantly enriched. Remarkably, elimination of H3K9ac reduced binding of the DSB-inducing enzyme Rec12 and DSB at hotspots. We also found that the H3K4 metyltransferase Set1 promotes DSB formation at some loci, but it restricts Rec12 binding to hotspots. These results suggest that H3K9ac rather than H3K4me3 is a hotspot-associated mark involved in meiotic DSB formation in fission yeast.

2013-03-07 | GSE31648 | GEO

R-loop landscapes in yeast meiosis

Project description:Meiotic recombination is initiated by the Spo11-dependent programmed DNA double-strand breaks (DSBs) that are preferentially concentrated within genomic regions known as hotspots, but the factor(s) which specify the positions for meiotic DSB hotspots remain unclear. Here, we show that the frequency and distribution of R-loops, a type of functional chromatin structure comprising single-stranded DNA and a DNA:RNA hybrid, change dramatically throughout meiosis. We detected the formation of multiple de novo R-loops in the pachytene stage, and found they co-localize with meiotic DSB hotspots. We further show that transcription-replication head-on collisions could promote R-loop formation during meiosis, and apparently direct the initiation of meiotic DSB formation. Furthermore, the hotspots can be eliminated by reverse the direction of either transcription or replication, and reconstituted by reverse both of their direction. Our study reveals that R-loops may play dual roles in meiotic recombination. In addition to participation in meiotic DSB processing, some meiotic DSB hotspots may be originated from the transcription-replication head-on collisions during meiotic DNA replication.

2023-05-14 | GSE167915 | GEO

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