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 Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis

2018 ◽  
Vol 115 (10) ◽  
pp. 2437-2442 ◽  
Author(s):  
Heïdi Serra ◽  
Christophe Lambing ◽  
Catherine H. Griffin ◽  
Stephanie D. Topp ◽  
Divyashree C. Nageswaran ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Homologous Chromosome ◽  
Crop Improvement ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Meiotic Dsbs ◽  
Competing Pathways

During meiosis, homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double-strand breaks (DSBs), which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of ∼100–200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as noncrossovers. To bias DSB repair toward crossovers, we simultaneously increased dosage of the procrossover E3 ligase gene HEI10 and introduced mutations in the anticrossovers helicase genes RECQ4A and RECQ4B. As HEI10 and recq4a recq4b increase interfering and noninterfering crossover pathways, respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases distally towards the subtelomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

scholarly journals Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis

2017 ◽  
Author(s):  
Heïdi Serra ◽  
Christophe Lambing ◽  
Catherine H. Griffin ◽  
Stephanie D. Topp ◽  
Mathilde Séguéla-Arnaud ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Homologous Chromosome ◽  
Crop Improvement ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Meiotic Dsbs ◽  
Competing Pathways

AbstractDuring meiosis homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double strand breaks, which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of ∼100–200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as non-crossovers. In order to bias DSB repair towards crossovers, we simultaneously increased dosage of the pro-crossover E3 ligase gene HEI10 and introduced mutations in the anti-crossover helicase genes RECQ4A and RECQ4B. As HEI10 and recq4a recq4b increase interfering and non-interfering crossover pathways respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect of HEI10 on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases towards the sub-telomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover-suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

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 SPO16 binds SHOC1 to promote homologous recombination and crossing-over in meiotic prophase I

2019 ◽  
Vol 5 (1) ◽  
pp. eaau9780 ◽  
Author(s):  
Qianting Zhang ◽  
Shu-Yan Ji ◽  
Kiran Busayavalasa ◽  
Chao Yu
Keyword(s):  
Homologous Recombination ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Crossing Over ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Recombination Nodules ◽  
Evolutionarily Conserved

Segregation of homologous chromosomes in meiosis I is tightly regulated by their physical links, or crossovers (COs), generated from DNA double-strand breaks (DSBs) through meiotic homologous recombination. In budding yeast, three ZMM (Zip1/2/3/4, Mer3, Msh4/5) proteins, Zip2, Zip4, and Spo16, form a “ZZS” complex, functioning to promote meiotic recombination via a DSB repair pathway. Here, we identified the mammalian ortholog of Spo16, termed SPO16, which interacts with the mammalian ortholog of Zip2 (SHOC1/MZIP2), and whose functions are evolutionarily conserved to promote the formation of COs. SPO16 localizes to the recombination nodules, as SHOC1 and TEX11 do. SPO16 is required for stabilization of SHOC1 and proper localization of other ZMM proteins. The DSBs formed in SPO16-deleted meiocytes were repaired without COs formation, although synapsis is less affected. Therefore, formation of SPO16-SHOC1 complex–associated recombination intermediates is a key step facilitating meiotic recombination that produces COs from yeast to mammals.

scholarly journals Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis

2008 ◽  
Vol 180 (4) ◽  
pp. 673-679 ◽  
Author(s):  
Fang Yang ◽  
Sigrid Eckardt ◽  
N. Adrian Leu ◽  
K. John McLaughlin ◽  
Peijing Jeremy Wang
Keyword(s):  
Meiotic Recombination ◽  
Strand Break ◽  
Male Meiosis ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Recombination Proteins ◽  
Meiotic Chromosomes ◽  
Dna Double Strand Break

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination.

scholarly journals MEIOK21: a new component of meiotic recombination bridges required for spermatogenesis

2020 ◽  
Vol 48 (12) ◽  
pp. 6624-6639
Author(s):  
Yongliang Shang ◽  
Tao Huang ◽  
Hongbin Liu ◽  
Yanlei Liu ◽  
Heng Liang ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Strand Exchange ◽  
Homology Search ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Chromosome Localization ◽  
Strand Breaks ◽  
Homologous Chromosomes

Abstract Repair of DNA double-strand breaks (DSBs) with homologous chromosomes is a hallmark of meiosis that is mediated by recombination ‘bridges’ between homolog axes. This process requires cooperation of DMC1 and RAD51 to promote homology search and strand exchange. The mechanism(s) regulating DMC1/RAD51-ssDNA nucleoprotein filament and the components of ‘bridges’ remain to be investigated. Here we show that MEIOK21 is a newly identified component of meiotic recombination bridges and is required for efficient formation of DMC1/RAD51 foci. MEIOK21 dynamically localizes on chromosomes from on-axis foci to ‘hanging foci’, then to ‘bridges’, and finally to ‘fused foci’ between homolog axes. Its chromosome localization depends on DSBs. Knockout of Meiok21 decreases the numbers of HSF2BP and DMC1/RAD51 foci, disrupting DSB repair, synapsis and crossover recombination and finally causing male infertility. Therefore, MEIOK21 is a novel recombination factor and probably mediates DMC1/RAD51 recruitment to ssDNA or their stability on chromosomes through physical interaction with HSF2BP.

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 Four-pronged negative feedback of DSB machinery in meiotic DNA-break control in mice

2021 ◽  
Author(s):  
Ihsan Dereli ◽  
Marcello Stanzione ◽  
Fabrizio Olmeda ◽  
Frantzeskos Papanikos ◽  
Marek Baumann ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Protein Complexes ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Damage Response ◽  
Auxiliary Protein ◽  
Genomic Locations ◽  
Spatiotemporal Control

Abstract In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.

scholarly journals End-resection at DNA double-strand breaks in the three domains of life

2013 ◽  
Vol 41 (1) ◽  
pp. 314-320 ◽  
Author(s):  
John K. Blackwood ◽  
Neil J. Rzechorzek ◽  
Sian M. Bray ◽  
Joseph D. Maman ◽  
Luca Pellegrini ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Domains Of Life ◽  
Strand Invasion ◽  
End Resection

During DNA repair by HR (homologous recombination), the ends of a DNA DSB (double-strand break) must be resected to generate single-stranded tails, which are required for strand invasion and exchange with homologous chromosomes. This 5′–3′ end-resection of the DNA duplex is an essential process, conserved across all three domains of life: the bacteria, eukaryota and archaea. In the present review, we examine the numerous and redundant helicase and nuclease systems that function as the enzymatic analogues for this crucial process in the three major phylogenetic divisions.

scholarly journals Epigenetic activation of meiotic recombination in Arabidopsis centromeres via loss of H3K9me2 and non-CG DNA methylation

2017 ◽  
Author(s):  
Charles J. Underwood ◽  
Kyuha Choi ◽  
Christophe Lambing ◽  
Xiaohui Zhao ◽  
Heïdi Serra ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Pericentromeric Heterochromatin ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Histone 3 ◽  
Differential Control

AbstractEukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis centromeres are suppressed for crossovers, as recombination in these regions can cause chromosome mis-segregation. Plant centromeres are surrounded by repetitive, transposon-dense heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. Here we show that disruption of Arabidopsis H3K9me2 and non-CG DNA methylation pathways increases meiotic DNA double strand breaks (DSBs) within centromeres, whereas crossovers increase within pericentromeric heterochromatin. Increased pericentromeric crossovers in H3K9me2/non-CG mutants occurs in both inbred and hybrid backgrounds, and involves the interfering crossover repair pathway. Epigenetic activation of recombination may also account for the curious tendency of maize transposon Ds to disrupt CHROMOMETHYLASE3 when launched from proximal loci. Thus H3K9me2 and non-CG DNA methylation exert differential control of meiotic DSB and crossover formation in centromeric and pericentromeric heterochromatin.

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