Robust designation of meiotic crossover sites by CDK-2 through phosphorylation of the MutSγ complex

SUMMARYCrossover formation is essential for proper segregation of homologous chromosomes during meiosis. Here we show that C. elegans Cyclin-dependent kinase 2 (CDK-2) forms a complex with cyclin-like protein COSA-1 and supports crossover formation by promoting conversion of meiotic double-strand breaks (DSBs) into crossover-specific recombination intermediates. Further, we identify MutSγ component MSH-5 as a CDK-2 phosphorylation target. MSH-5 has a disordered C-terminal tail that contains 13 potential CDK phosphosites and is required to concentrate crossover-promoting proteins at recombination sites. Phosphorylation of the MSH-5 tail appears dispensable in a wild- type background, but when MutSγ activity is partially compromised, crossover formation and retention of CDK-2/COSA-1 at recombination sites are exquisitely sensitive to phosphosite loss. Our data support a model in which robustness of crossover designation reflects a positive feedback mechanism involving CDK-2-mediated phosphorylation and scaffold-like properties of the MSH-5 C-terminal tail, features that combine to promote full recruitment and activity of crossover-promoting complexes.

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Assessment of the roles of SPO11-2 and SPO11-4 in meiosis in rice using CRISPR/Cas9 mutagenesis

Journal of Experimental Botany ◽

10.1093/jxb/eraa391 ◽

2020 ◽

Vol 71 (22) ◽

pp. 7046-7058 ◽

Cited By ~ 3

Author(s):

Ian Fayos ◽

Anne Cécile Meunier ◽

Aurore Vernet ◽

Sergi Navarro-Sanz ◽

Murielle Portefaix ◽

...

Keyword(s):

Double Strand Breaks ◽

Wild Type ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Pollen Mother Cells ◽

In The Wild ◽

Bivalent Formation ◽

Biallelic Mutations ◽

Mother Cells ◽

Mutant Lines

Abstract In Arabidopsis, chromosomal double-strand breaks at meiosis are presumably catalyzed by two distinct SPO11 transesterases, AtSPO11-1 and AtSPO11-2, together with M-TOPVIB. To clarify the roles of the SPO11 paralogs in rice, we used CRISPR/Cas9 mutagenesis to produce null biallelic mutants in OsSPO11-1, OsSPO11-2, and OsSPO11-4. Similar to Osspo11-1, biallelic mutations in the first exon of OsSPO11-2 led to complete panicle sterility. Conversely, all Osspo11-4 biallelic mutants were fertile. To generate segregating Osspo11-2 mutant lines, we developed a strategy based on dual intron targeting. Similar to Osspo11-1, the pollen mother cells of Osspo11-2 progeny plants showed an absence of bivalent formation at metaphase I, aberrant segregation of homologous chromosomes, and formation of non-viable tetrads. In contrast, the chromosome behavior in Osspo11-4 male meiocytes was indistinguishable from that in the wild type. While similar numbers of OsDMC1 foci were revealed by immunostaining in wild-type and Osspo11-4 prophase pollen mother cells (114 and 101, respectively), a surprisingly high number (85) of foci was observed in the sterile Osspo11-2 mutant, indicative of a divergent function between OsSPO11-1 and OsSPO11-2. This study demonstrates that whereas OsSPO11-1 and OsSPO11-2 are the likely orthologs of AtSPO11-1 and AtSPO11-2, OsSPO11-4 has no major role in wild-type rice meiosis.

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Translational control of oskar generates short OSK, the isoform that induces pole plasma assembly

Development ◽

10.1242/dev.121.11.3723 ◽

1995 ◽

Vol 121 (11) ◽

pp. 3723-3732 ◽

Cited By ~ 19

Author(s):

F.H. Markussen ◽

A.M. Michon ◽

W. Breitwieser ◽

A. Ephrussi

Keyword(s):

Positive Feedback ◽

Translational Control ◽

Feedback Mechanism ◽

Posterior Pole ◽

Start Codon ◽

Anterior Pole ◽

Wild Type ◽

Alternative Start Codon ◽

Positive Feedback Mechanism ◽

Pole Plasm

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.

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The Yeast Motor Protein, Kar3p, Is Essential for Meiosis I

The Journal of Cell Biology ◽

10.1083/jcb.139.2.459 ◽

1997 ◽

Vol 139 (2) ◽

pp. 459-467 ◽

Cited By ~ 29

Author(s):

Carol A. Bascom-Slack ◽

Dean S. Dawson

Keyword(s):

Synaptonemal Complex ◽

Meiotic Recombination ◽

Molecular Motor ◽

Motor Protein ◽

Double Strand Breaks ◽

Wild Type ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Low Levels ◽

Meiosis I

The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown. We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type. These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.

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

10.1101/2021.02.01.429210 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.26.8.2913-2923.2006 ◽

2006 ◽

Vol 26 (8) ◽

pp. 2913-2923 ◽

Cited By ~ 31

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.

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Saccharomyces cerevisiae Mer3 Is a DNA Helicase Involved in Meiotic Crossing Over

Molecular and Cellular Biology ◽

10.1128/mcb.22.10.3281-3291.2002 ◽

2002 ◽

Vol 22 (10) ◽

pp. 3281-3291 ◽

Cited By ~ 39

Author(s):

Takuro Nakagawa ◽

Richard D. Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Helicase ◽

Double Strand Breaks ◽

Crossing Over ◽

Helicase Activity ◽

Wild Type ◽

Rna Helicases ◽

Conserved Residues ◽

Strand Breaks ◽

Homologous Chromosomes

ABSTRACT Crossing over is regulated to occur at least once per each pair of homologous chromosomes during meiotic prophase to ensure proper segregation of chromosomes at the first meiotic division. In a mer3 deletion mutant of Saccharomyces cerevisiae, crossing over is decreased, and the distribution of the crossovers that occur is random. The predicted Mer3 protein contains seven motifs characteristic of the DExH box type of DNA/RNA helicases. The mer3G166D and the mer3K167A mutation, amino acid substitutions of conserved residues in a putative nucleotide-binding domain of the helicase motifs caused a defect in the transition of meiosis-specific double-strand breaks to later intermediates, decreased crossing over, and reduced crossover interference. The purified Mer3 protein was found to have DNA helicase activity. This helicase activity was reduced by the mer3GD mutation to <1% of the wild-type activity, even though binding of the mutant protein to single- and double-strand DNA was unaffected. The mer3KA mutation eliminated the ATPase activity of the wild-type protein. These results demonstrate that Mer3 is a DNA helicase that functions in meiotic crossing over.

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The Zip4 protein directly couples meiotic crossover formation to synaptonemal complex assembly

Genes & Development ◽

10.1101/gad.348973.121 ◽

2021 ◽

Author(s):

Alexandra Pyatnitskaya ◽

Jessica Andreani ◽

Raphaël Guérois ◽

Arnaud De Muyt ◽

Valérie Borde

Keyword(s):

Synaptonemal Complex ◽

Central Element ◽

Underlying Mechanism ◽

Double Strand Breaks ◽

General Mechanism ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Evolutionarily Conserved ◽

Meiotic Crossover ◽

Chromosome Axis

Meiotic recombination is triggered by programmed double-strand breaks (DSBs), a subset of these being repaired as crossovers, promoted by eight evolutionarily conserved proteins, named ZMM. Crossover formation is functionally linked to synaptonemal complex (SC) assembly between homologous chromosomes, but the underlying mechanism is unknown. Here we show that Ecm11, a SC central element protein, localizes on both DSB sites and sites that attach chromatin loops to the chromosome axis, which are the starting points of SC formation, in a way that strictly requires the ZMM protein Zip4. Furthermore, Zip4 directly interacts with Ecm11, and point mutants that specifically abolish this interaction lose Ecm11 binding to chromosomes and exhibit defective SC assembly. This can be partially rescued by artificially tethering interaction-defective Ecm11 to Zip4. Mechanistically, this direct connection ensuring SC assembly from CO sites could be a way for the meiotic cell to shut down further DSB formation once enough recombination sites have been selected for crossovers, thereby preventing excess crossovers. Finally, the mammalian ortholog of Zip4, TEX11, also interacts with the SC central element TEX12, suggesting a general mechanism.

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The Zip4 protein directly couples meiotic crossover formation to synaptonemal complex assembly

10.1101/2021.08.13.456249 ◽

2021 ◽

Author(s):

Alexandra Pyatnitskaya ◽

Jessica Andreani ◽

Raphael Guerois ◽

Arnaud De Muyt ◽

Valerie Borde

Keyword(s):

Synaptonemal Complex ◽

Central Element ◽

Underlying Mechanism ◽

Double Strand Breaks ◽

General Mechanism ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Evolutionarily Conserved ◽

Meiotic Crossover ◽

Chromosome Axis

Meiotic recombination is triggered by programmed double-strand breaks (DSBs), a subset of these being repaired as crossovers, promoted by eight evolutionarily conserved proteins, named ZMM. Crossover formation is functionally linked to synaptonemal complex (SC) assembly between homologous chromosomes, but the underlying mechanism is unknown. Here we show that Ecm11, a SC central element protein, localizes on both DSB sites and sites that attach chromatin loops to the chromosome axis, which are the starting points of SC formation, in a way that strictly requires the ZMM protein Zip4. Furthermore, Zip4 directly interacts with Ecm11 and point mutants that specifically abolish this interaction lose Ecm11 binding to chromosomes and exhibit defective SC assembly. This can be partially rescued by artificially tethering interaction-defective Ecm11 to Zip4. Mechanistically, this direct connection ensuring SC assembly from CO sites could be a way for the meiotic cell to shut down further DSB formation once enough recombination sites have been selected for crossovers, thereby preventing excess crossovers. Finally, the mammalian ortholog of Zip4, TEX11, also interacts with the SC central element TEX12, suggesting a general mechanism.

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Reduction of insulin/IGF-1 receptor rejuvenates immunity via positive feedback circuit

10.1101/795781 ◽

2019 ◽

Author(s):

Yujin Lee ◽

Dae-Eun Jeong ◽

Wooseon Hwang ◽

Seokjin Ham ◽

Hae-Eun H. Park ◽

...

Keyword(s):

Transcription Factor ◽

Positive Feedback ◽

Feedback Mechanism ◽

Mitogen Activated Protein Kinase ◽

Bzip Transcription Factor ◽

C Elegans ◽

Positive Feedback Circuit ◽

Mitogen Activated Protein ◽

Positive Feedback Mechanism ◽

Transcription Factor 1

SummaryImmunosenescence is considered an inevitable decline in immune function during aging. Here we show that genetic inhibition of the DAF-2/insulin/IGF-1 receptor drastically delays immunosenescence and rejuvenates immunity in C. elegans. We find that p38 mitogen-activated protein kinase 1 (PMK-1), a key determinant of immunosenescence, is dispensable for this rejuvenated immunity. Instead, we demonstrate that longevity-promoting DAF-16/FOXO and heat-shock transcription factor 1 (HSF-1) increase immunocompetence in old daf-2(-) animals. The upregulation of DAF-16/FOXO and HSF-1 decreases the expression of the zip-10/bZIP transcription factor, which in turn downregulates INS-7, an agonistic insulin-like peptide, resulting in further reduction of insulin/IGF-1 signaling (IIS). Thus, reduced IIS bypasses immunosenescence and rejuvenates immunity via the upregulation of anti-aging transcription factors that modulate an endocrine insulin-like peptide through a positive feedback mechanism. Because many functions of IIS are conserved across phyla, our study may lead to the development of strategies for human immune rejuvenation.

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Coordinated and Independent Roles for MLH Subunits in DNA Repair

Cells ◽

10.3390/cells10040948 ◽

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.

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