scholarly journals A dCas9/CRISPR-based targeting system identifies a central role for Ctf19 in kinetochore-derived suppression of meiotic recombination

2020 ◽  
Author(s):  
Lisa-Marie Kuhl ◽  
Vasso Makrantoni ◽  
Sarah Recknagel ◽  
Animish N. Vaze ◽  
Adele L. Marston ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Genome Stability ◽  
Budding Yeast ◽  
Homologous Chromosomes ◽  
Experimental Platform ◽  
Chromosome Missegregation ◽  
Meiotic Crossover ◽  
Insight Into

AbstractIn meiosis, crossover formation between homologous chromosomes is essential for faithful segregation. However, improperly controlled or placed meiotic recombination can have catastrophic consequences on genome stability. Specifically, within centromeres and surrounding regions (i.e. pericentromeres), crossovers are associated with chromosome missegregation and developmental aneuploidy. In organisms ranging from yeast to humans, crossovers are repressed within (peri)centromeric regions. We previously identified a key role for the multi-subunit, kinetochore-associated Ctf19 complex (Ctf19c; the budding yeast equivalent of the human CCAN) in regulating pericentromeric crossover formation. Here, we develop a dCas9/CRISPR-based system that allows ectopic targeting of Ctf19c-subunits to a non-centromeric locus during meiosis. Using this approach, we query sufficiency in meiotic crossover suppression, and identify Ctf19 (the budding yeast homologue of vertebrate CENP-P) as a central mediator of kinetochore-associated crossover control. We show that the effect of Ctf19 is encoded in its NH2-terminal tail, and depends on residues known to be important for the recruitment of the Scc2-Scc4 cohesin regulator to kinetochores. We thus reveal a crucial determinant that links kinetochores to meiotic recombinational control. This work provides insight into localized control of meiotic recombination. Furthermore, our approach establishes a dCas9/CRISPR-based experimental platform that can be utilized to investigate and locally manipulate meiotic crossover control. This platform can easily be adapted in order to investigate other aspects of localized chromosome biology.

scholarly journals A dCas9-Based System Identifies a Central Role for Ctf19 in Kinetochore-Derived Suppression of Meiotic Recombination

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 395-408
Author(s):  
Lisa-Marie Kuhl ◽  
Vasso Makrantoni ◽  
Sarah Recknagel ◽  
Animish N. Vaze ◽  
Adele L. Marston ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Genome Stability ◽  
Meiotic Chromosome ◽  
Homologous Chromosomes ◽  
Experimental Platform ◽  
Chromosome Missegregation ◽  
Link Type ◽  
Insight Into

In meiosis, crossover (CO) formation between homologous chromosomes is essential for faithful segregation. However, misplaced meiotic recombination can have catastrophic consequences on genome stability. Within pericentromeres, COs are associated with meiotic chromosome missegregation. In organisms ranging from yeast to humans, pericentromeric COs are repressed. We previously identified a role for the kinetochore-associated Ctf19 complex (Ctf19c) in pericentromeric CO suppression. Here, we develop a dCas9/CRISPR-based system that allows ectopic targeting of Ctf19c-subunits. Using this approach, we query sufficiency in meiotic CO suppression, and identify Ctf19 as a mediator of kinetochore-associated CO control. The effect of Ctf19 is encoded in its NH2-terminal tail, and depends on residues important for the recruitment of the Scc2-Scc4 cohesin regulator. This work provides insight into kinetochore-derived control of meiotic recombination. We establish an experimental platform to investigate and manipulate meiotic CO control. This platform can easily be adapted in order to investigate other aspects of chromosome biology.

scholarly journals Genome Duplication Increases Meiotic Recombination Frequency: A Saccharomyces cerevisiae Model

2020 ◽  
Author(s):  
Ou Fang ◽  
Lin Wang ◽  
Yuxin Zhang ◽  
Jixuan Yang ◽  
Qin Tao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Genome Stability ◽  
Genome Duplication ◽  
Genetic Recombination ◽  
Recombination Frequency ◽  
Abiotic Factors ◽  
Strand Break ◽  
Homologous Chromosomes ◽  
Almost All

Abstract Genetic recombination characterized by reciprocal exchange of genes on paired homologous chromosomes is the most prominent event in meiosis of almost all sexually reproductive organisms. It contributes to genome stability by ensuring the balanced segregation of paired homologs in meiosis, and it is also the major driving factor in generating genetic variation for natural and artificial selection. Meiotic recombination is subjected to the control of a highly stringent and complex regulating process and meiotic recombination frequency (MRF) may be affected by biological and abiotic factors such as sex, gene density, nucleotide content, and chemical/temperature treatments, having motivated tremendous researches for artificially manipulating MRF. Whether genome polyploidization would lead to a significant change in MRF has attracted both historical and recent research interests; however, tackling this fundamental question is methodologically challenging due to the lack of appropriate methods for tetrasomic genetic analysis, thus has led to controversial conclusions in the literature. This article presents a comprehensive and rigorous survey of genome duplication-mediated change in MRF using Saccharomyces cerevisiae as a eukaryotic model. It demonstrates that genome duplication can lead to consistently significant increase in MRF and rate of crossovers across all 16 chromosomes of S. cerevisiae, including both cold and hot spots of MRF. This ploidy-driven change in MRF is associated with weakened recombination interference, enhanced double-strand break density, and loosened chromatin histone occupation. The study illuminates a significant evolutionary feature of genome duplication and opens an opportunity to accelerate response to artificial and natural selection through polyploidization.

scholarly journals CRISPR targeting of MEIOTIC-TOPOISOMERASE VIB-dCas9 to a recombination hotspot is insufficient to increase crossover frequency in Arabidopsis

2021 ◽  
Author(s):  
Nataliya E. Yelina ◽  
Sabrina Gonzalez-Jorge ◽  
Dominique Hirsz ◽  
Ziyi Yang ◽  
Ian R. Henderson
Keyword(s):  
Meiotic Recombination ◽  
Crop Improvement ◽  
Double Strand Breaks ◽  
Crossover Frequency ◽  
Dna Double Strand Breaks ◽  
Crop Breeding ◽  
Catalytic Subunits ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Meiotic Crossover

AbstractDuring meiosis, homologous chromosomes pair and recombine, which can result in reciprocal crossovers that increase genetic diversity. Crossovers are unevenly distributed along eukaryote chromosomes and show repression in heterochromatin and the centromeres. Within the chromosome arms crossovers are often concentrated in hotspots, which are typically in the kilobase range. The uneven distribution of crossovers along chromosomes, together with their low number per meiosis, creates a limitation during crop breeding, where recombination can be beneficial. Therefore, targeting crossovers to specific genome locations has the potential to accelerate crop improvement. In plants, meiotic crossovers are initiated by DNA double strand breaks (DSBs) that are catalysed by SPO11 complexes, which consist of two catalytic (SPO11-1 and SPO11-2) and two non-catalytic subunits (MTOPVIB). We used the model plant Arabidopsis thaliana to target a dCas9-MTOPVIB fusion protein to the 3a crossover hotspot via CRISPR. We observed that this was insufficient to significantly change meiotic crossover frequency or pattern within 3a. We discuss the implications of our findings for targeting meiotic recombination within plant genomes.

scholarly journals Mammalian BLM helicase is critical for integrating multiple pathways of meiotic recombination

2010 ◽  
Vol 188 (6) ◽  
pp. 779-789 ◽  
Author(s):  
J. Kim Holloway ◽  
Meisha A. Morelli ◽  
Peter L. Borst ◽  
Paula E. Cohen
Keyword(s):  
Meiotic Recombination ◽  
Genome Stability ◽  
Autosomal Recessive Disorder ◽  
Dna Structures ◽  
Homologous Chromosomes ◽  
Recq Helicases ◽  
Meiotic Progression ◽  
Meiotic Chromosomes ◽  
Conditional Mutant ◽  
Blm Helicase

Bloom’s syndrome (BS) is an autosomal recessive disorder characterized by growth retardation, cancer predisposition, and sterility. BS mutated (Blm), the gene mutated in BS patients, is one of five mammalian RecQ helicases. Although BLM has been shown to promote genome stability by assisting in the repair of DNA structures that arise during homologous recombination in somatic cells, less is known about its role in meiotic recombination primarily because of the embryonic lethality associated with Blm deletion. However, the localization of BLM protein on meiotic chromosomes together with evidence from yeast and other organisms implicates a role for BLM helicase in meiotic recombination events, prompting us to explore the meiotic phenotype of mice bearing a conditional mutant allele of Blm. In this study, we show that BLM deficiency does not affect entry into prophase I but causes severe defects in meiotic progression. This is exemplified by improper pairing and synapsis of homologous chromosomes and altered processing of recombination intermediates, resulting in increased chiasmata. Our data provide the first analysis of BLM function in mammalian meiosis and strongly argue that BLM is involved in proper pairing, synapsis, and segregation of homologous chromosomes; however, it is dispensable for the accumulation of recombination intermediates.

scholarly journals Mnd1/Hop2 Facilitates Dmc1-Dependent Interhomolog Crossover Formation in Meiosis of Budding Yeast

2006 ◽  
Vol 26 (8) ◽  
pp. 2913-2923 ◽  
Author(s):  
Jill M. Henry ◽  
Raymond Camahort ◽  
Douglas A. Rice ◽  
Laurence Florens ◽  
Selene K. Swanson ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Double Mutant ◽  
Repetitive Sequences ◽  
Double Strand Breaks ◽  
Genomic Integrity ◽  
Dna Interactions ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Meiotic Crossover ◽  
Strand Invasion

ABSTRACT During meiosis, each chromosome must pair with its homolog and undergo meiotic crossover recombination in order to segregate properly at the first meiotic division. Recombination in meiosis in Saccharomyces cerevisiae relies on two Escherichia coli recA homologs, Rad51 and Dmc1, as well as the more recently discovered heterodimer Mnd1/Hop2. Meiotic recombination in S. cerevisiae mnd1 and hop2 single mutants is initiated via double-strand breaks (DSBs) but does not progress beyond this stage; heteroduplex DNA, joint molecules, and crossovers are not detected. Whereas hop2 and mnd1 single mutants are profoundly recombination defective, we show that mnd1 rad51, hop2 rad51, and mnd1 rad17 double mutants are able to carry out crossover recombination. Interestingly, noncrossover recombination is absent, indicating a role for Mnd1/Hop2 in the designation of DSBs for noncrossover recombination. We demonstrate that in the rad51 mnd1 double mutant, recombination is more likely to occur between repetitive sequences on nonhomologous chromosomes. Our results support a model in which Mnd1/Hop2 is required for DNA-DNA interactions that help ensure Dmc1-mediated stable strand invasion between homologous chromosomes, thereby preserving genomic integrity.

scholarly journals Meiotic MCM proteins promote and inhibit crossovers during meiotic recombination

2018 ◽  
Author(s):  
Michaelyn Hartmann ◽  
Kathryn P. Kohl ◽  
Jeff Sekelsky ◽  
Talia Hatkevich
Keyword(s):  
Meiotic Recombination ◽  
Protein Complexes ◽  
Class Ii ◽  
Specific Protein ◽  
Class I ◽  
Homologous Chromosomes ◽  
N Terminus ◽  
Mcm Complex ◽  
Mcm Proteins ◽  
Insight Into

AbstractCrossover formation as a result of meiotic recombination is vital for proper segregation of homologous chromosomes at the end of meiosis I. In many organisms, crossovers are generated through two crossover pathways: Class I and Class II. To ensure accurate crossover formation, meiosis-specific protein complexes regulate the degree in which each pathway is used. One such complex is the mei-MCM complex, which contains MCM (mini-chromosome maintenance) and MCM-like proteins REC (ortholog of Mcm8), MEI-217, and MEI-218, collectively called the mei-MCM complex. The mei-MCM complex genetically promotes Class I crossovers and inhibits Class II crossovers in Drosophila, but it is unclear how individual mei-MCM proteins contribute to crossover regulation. In this study, we perform genetic analyses to understand how specific regions and motifs of mei-MCM proteins contribute to Class I and II crossover formation and distribution. Our analyses show that the long, disordered N-terminus of MEI-218 is dispensable for crossover formation, and that mutations that disrupt REC’s Walker A and B motifs differentially affect Class I and Class II crossover formation. In Rec Walker A mutants, Class I crossovers exhibit no change, but Class II crossovers are increased. However, in rec Walker B mutants, Class I crossovers are severely impaired, and Class II crossovers are increased. These results suggest that REC may form multiple complexes that exhibit differential REC-dependent ATP binding and hydrolyzing requirements. These results provide genetic insight into the mechanisms through which mei-MCM proteins promote Class I crossovers and inhibit Class II crossovers.

scholarly journals A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability

2019 ◽  
Author(s):  
Diedre Reitz ◽  
Jennifer Grubb ◽  
Douglas K. Bishop
Keyword(s):  
Meiotic Recombination ◽  
Genome Rearrangements ◽  
Accessory Protein ◽  
Filament Formation ◽  
Single Stranded Dna ◽  
Homologous Chromosomes ◽  
Sister Chromatids ◽  
Accessory Factor ◽  
Accessory Factors ◽  
Insight Into

AbstractDuring meiosis, homologous recombination repairs programmed DNA double-stranded breaks (DSBs). Meiotic recombination physically links the homologous chromosomes (“homologs”), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1’s activity is regulated by its accessory factors Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. We also provide evidence that Mei5-Sae3 promotes Dmc1 filament formation specifically on single-stranded DNA. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister (IS) joint molecules intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1 (Dmc1-WT). Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filaments lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.Author SummaryDuring meiosis, two rounds of division follow a single round of DNA replication to create the gametes for biparental reproduction. The first round of division requires that the homologous chromosomes become physically linked to one another to create the tension that is necessary for their segregation. This linkage is achieved through DNA recombination between the two homologous chromosomes, followed by resolution of the recombination intermediate into a crossover (CO). Central to this process is the meiosis-specific recombinase Dmc1, and its accessory factors, which provide important regulatory functions to ensure that recombination is accurate, efficient, and occurs predominantly between homologous chromosomes, and not sister chromatids. To gain insight into the regulation of Dmc1 by its accessory factors, we mutated Dmc1 such that it was no longer dependent on its accessory factor Mei5-Sae3. Our analysis reveals that Dmc1 accessory factors Mei5-Sae3 and Rad51 have independent roles in stabilizing Dmc1 filaments. Furthermore, we find that although Rad51 is required for promoting recombination between homologous chromosomes, Mei5-Sae3 is not. Lastly, we show that our Dmc1 mutant forms abnormally long filaments, and high levels of aberrant recombination intermediates and products. These findings suggest that filaments are actively maintained at short lengths to prevent deleterious genome rearrangements.

scholarly journals Separable functions of the PHD finger protein Spp1 in the Set1 and the meiotic DSB forming complexes cooperate for meiotic DSB formation

2017 ◽  
Author(s):  
Céline Adam ◽  
Raphaël Guérois ◽  
Anna Citarella ◽  
Laura Verardi ◽  
Florine Adolphe ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Budding Yeast ◽  
Gene Promoters ◽  
H3k4 Methylation ◽  
Functional Link ◽  
Phd Finger ◽  
Homologous Chromosomes ◽  
Separable Functions ◽  
Recombination Hotspots ◽  
Histone H3k4

AbstractHistone H3K4 methylation is a feature of meiotic recombination hotspots shared by many organisms including plants and mammals. Meiotic recombination is initiated by programmed double-strand break (DSB) formation that in budding yeast takes place in gene promoters and is promoted by histone H3K4 di/trimethylation. This histone modification is recognized by Spp1, a PHD-finger containing protein that belongs to the conserved histone H3K4 methyltransferase Set1 complex. During meiosis, Spp1 binds H3K4me3 and interacts with a DSB protein, Mer2, to promote DSB formation close to gene promoters. How Set1 complex- and Mer2- related functions of Spp1 are connected is not clear. Here, combining genome-wide localization analyses, biochemical approaches and the use of separation of function mutants, we show that Spp1 is present within two distinct complexes in meiotic cells, the Set1 and the Mer2 complexes. Disrupting the Spp1-Set1 interaction mildly decreases H3K4me3 levels and does not affect meiotic recombination initiation. Conversely, the Spp1-Mer2 interaction is required for normal meiotic recombination initiation, but dispensable for Set1 complex-mediated histone H3K4 methylation. Finally, we evidence that Spp1 preserves normal H3K4me3 levels independently of the Set1 complex. We propose a model where the three populations of Spp1 work sequentially to promote recombination initiation: first by depositing histone H3K4 methylation (Set1 complex), next by “reading” and protecting histone H3K4 methylation, and finally by making the link with the chromosome axis (Mer2-Spp1 complex). This work deciphers the precise roles of Spp1 in meiotic recombination and opens perspectives to study its functions in other organisms where H3K4me3 is also present at recombination hotspots.Author summaryMeiotic recombination is a conserved pathway of sexual reproduction that is required to faithfully segregate homologous chromosomes and produce viable gametes. Recombination events between homologous chromosomes are triggered by the programmed formation of DNA breaks, which occur preferentially at places called hotspots. In many organisms, these hotspots are located close to a particular chromatin modification, the methylation of lysine 4 of histone H3 (H3K4me3). It was previously shown in the budding yeast model that one protein, Spp1, plays an important function in this process. We further explored the functional link between Spp1 and its interacting partners, and show that Spp1 shows genetically separable functions, by depositing the H3K4me3 mark on the chromatin, “reading” and protecting it, and linking it to the recombination proteins. We provide evidence that Spp1 is in three independent complexes to perform these functions. This work opens perspectives for understanding the process in other eukaryotes such as mammals, where most of the proteins involved are conserved.

scholarly journals Coordinated and Independent Roles for MLH Subunits in DNA Repair

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 948
Author(s):  
Gianno Pannafino ◽  
Eric Alani
Keyword(s):  
Meiotic Recombination ◽  
Baker’S Yeast ◽  
Double Strand Breaks ◽  
Baker's Yeast ◽  
Genomic Integrity ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Dna Mismatch ◽  
Meiotic Crossover ◽  
Mmr Proteins

The MutL family of DNA mismatch repair proteins (MMR) acts to maintain genomic integrity in somatic and meiotic cells. In baker’s yeast, the MutL homolog (MLH) MMR proteins form three heterodimeric complexes, MLH1-PMS1, MLH1-MLH2, and MLH1-MLH3. The recent discovery of human PMS2 (homolog of baker’s yeast PMS1) and MLH3 acting independently of human MLH1 in the repair of somatic double-strand breaks questions the assumption that MLH1 is an obligate subunit for MLH function. Here we provide a summary of the canonical roles for MLH factors in DNA genomic maintenance and in meiotic crossover. We then present the phenotypes of cells lacking specific MLH subunits, particularly in meiotic recombination, and based on this analysis, propose a model for an independent early role for MLH3 in meiosis to promote the accurate segregation of homologous chromosomes in the meiosis I division.

scholarly journals Meiotic crossover patterning in the absence of ATR: Loss of interference and assurance but not the centromere effect

2017 ◽  
Author(s):  
Morgan M. Brady ◽  
Susan McMahan ◽  
Jeff Sekelsky
Keyword(s):  
Spatial Distribution ◽  
Birth Defects ◽  
Meiotic Recombination ◽  
Maternal Effect ◽  
Crossover Frequency ◽  
Wild Type ◽  
Homologous Chromosomes ◽  
Meiotic Crossover ◽  
Null Mutants ◽  
Meiosis I

ABSTRACTMeiotic crossovers must be properly patterned to ensure accurate disjunction of homologous chromosomes during meiosis I. Disruption of the spatial distribution of crossovers can lead to nondisjunction, aneuploidy, gamete dysfunction, miscarriage, or birth defects. One of the earliest identified genes in involved proper crossover patterning is textitDrosophila mei-41, which encodes the ortholog of the checkpoint kinase ATR. Analysis of hypomorphic mutants suggested the existence of crossover patterning defects, but it was not possible to assess this in null mutants because of maternal-effect embryonic lethality. To overcome this lethality, we constructed mei-41 null mutants in which we expressed wild-type Mei-41 in the germline after completion of meiotic recombination, allowing progeny to survive. We find that crossovers are decreased to about one third of wild-type levels, but the reduction is not uniform, being less severe in the proximal regions of 2L than in medial or distal 2L or on the X chromosome. None of the crossovers formed in the absence of Mei-41 require Mei-9, the presumptive meiotic resolvase, suggesting that Mei-41 functions everywhere, despite the differential effects on crossover frequency. Interference appears to be significantly reduced or absent in mei-41 mutants, but the reduction in crossover density in centromere-proximal regions is largely intact. We propose that crossover patterning is achieved in a stepwise manner, with the crossover suppression related to proximity to the centromere occurring prior to and independently of crossover designation and enforcement of interference. In this model, Mei-41 has an essential after the centromere effect is established but before crossover designation and interference occur.

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