scholarly journals The Mitotic DNA Damage Checkpoint Proteins Rad17 and Rad24 Are Required for Repair of Double-Strand Breaks During Meiosis in Yeast

Genetics ◽  
2003 ◽  
Vol 164 (3) ◽  
pp. 855-865 ◽  
Author(s):  
Miki Shinohara ◽  
Kazuko Sakai ◽  
Tomoko Ogawa ◽  
Akira Shinohara
Keyword(s):  
Dna Damage ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Dna Damage Checkpoint ◽  
Wild Type ◽  
Checkpoint Proteins ◽  
Strand Breaks ◽  
Meiotic Dsbs ◽  
Γ Ray ◽  
Checkpoint Genes

Abstract We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products. These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and cause a delay in the disappearance of Rad51 foci, but not of Dmc1. These observations imply that RAD17 and RAD24 promote efficient repair of meiotic DSBs by facilitating proper assembly of the meiotic recombination complex containing Rad51. Consistent with this proposal, extra copies of RAD51 and RAD54 substantially suppress not only the spore inviability of the rad24 mutant, but also the γ-ray sensitivity of the mutant. Unexpectedly, the entry into meiosis I (metaphase I) is delayed in the checkpoint single mutants compared to wild type. The control of the cell cycle in response to meiotic DSBs is also discussed.

scholarly journals Coordination of the Initiation of Recombination and the Reductional Division in Meiosis in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Kai Jiao ◽  
Steven A Bullard ◽  
Laura Salem ◽  
Robert E Malone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
The Other ◽  
Wild Type ◽  
Strand Breaks ◽  
Proper Timing ◽  
Meiotic Dsbs ◽  
Reductional Division

Abstract Early exchange (EE) genes are required for the initiation of meiotic recombination in Saccharomyces cerevisiae. Cells with mutations in several EE genes undergo an earlier reductional division (MI), which suggests that the initiation of meiotic recombination is involved in determining proper timing of the division. The different effects of null mutations on the timing of reductional division allow EE genes to be assorted into three classes: mutations in RAD50 or REC102 that confer a very early reductional division; mutations in REC104 or REC114 that confer a division earlier than that of wild-type (WT) cells, but later than that of mutants of the first class; and mutations in MEI4 that do not significantly alter the timing of MI. The very early mutations are epistatic to mutations in the other two classes. We propose a model that accounts for the epistatic relationships and the communication between recombination initiation and the first division. Data in this article indicate that double-strand breaks (DSBs) are not the signal for the normal delay of reductional division; these experiments also confirm that MEI4 is required for the formation of meiotic DSBs. Finally, if a DSB is provided by the HO endonuclease, recombination can occur in the absence of MEI4 and REC104.

scholarly journals Meiotic Recombination Initiated by a Double-Strand Break in rad50Δ Yeast Cells Otherwise Unable to Initiate Meiotic Recombination

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 741-754 ◽  
Author(s):  
A Malkova ◽  
L Ross ◽  
D Dawson ◽  
Merl F Hoekstra ◽  
J E Haber
Keyword(s):  
Meiotic Recombination ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Yeast Cells ◽  
Meiotic Cell ◽  
Wild Type ◽  
Strand Breaks ◽  
Meiotic Dsbs ◽  
The Times ◽  
Rad50 Gene

Abstract Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad+ strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad+ and in rad50Δ cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50Δ cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50Δ diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination.

scholarly journals Association of Rad9 with Double-Strand Breaks through a Mec1-Dependent Mechanism

2004 ◽  
Vol 24 (8) ◽  
pp. 3277-3285 ◽  
Author(s):  
Takahiro Naiki ◽  
Tatsushi Wakayama ◽  
Daisuke Nakada ◽  
Kunihiro Matsumoto ◽  
Katsunori Sugimoto
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Dna Damage Checkpoint ◽  
Dependent Manner ◽  
Wild Type ◽  
Strand Breaks ◽  
Dependent Mechanism ◽  
In Cells

ABSTRACT Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Δ or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Δ mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.

scholarly journals Escherichia coli MutM Suppresses Illegitimate Recombination Induced by Oxidative Stress

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Masaaki Onda ◽  
Katsuhiro Hanada ◽  
Hirokazu Kawachi ◽  
Hideo Ikeda
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Recombination Analysis ◽  
Wild Type ◽  
Strand Breaks ◽  
In The Wild

Abstract DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of λbio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of λbio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total λbio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

scholarly journals Numerical and Spatial Patterning of Yeast Meiotic DNA Breaks by Tel1

2016 ◽  
Author(s):  
Neeman Mohibullah ◽  
Scott Keeney
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Deep Sequencing ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Spatial Patterning ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Context Dependent

AbstractThe Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to examine the contribution of the DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM) to this regulation in Saccharomyces cerevisiae. A tel1Δ mutant had globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tell mutation also increased Spo11-oligonucleotide levels, but mutating known Tel1 phosphotargets on Hop1 and Rec114 did not. Deep sequencing of Spo11 oligonucleotides from tel1Δ mutants demonstrated that Tel1 shapes the nonrandom DSB distribution in ways that are distinct but partially overlapping with previously described contributions of the recombination regulator Zip3. Finally, we uncover a context-dependent role for Tel1 in hotspot competition, in which an artificial DSB hotspot inhibits nearby hotspots. Evidence for Tel1-dependent competition involving strong natural hotspots is also provided.

scholarly journals Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, induces accumulation of DNA double-strand breaks

2020 ◽  
Vol 61 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Makoto Ihara ◽  
Kazuko Shichijo ◽  
Satoshi Takeshita ◽  
Takashi Kudo
Keyword(s):  
Dna Damage ◽  
Specific Inhibitor ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Phosphatidylinositol 3 Kinase ◽  
Strand Breaks ◽  
Γ Ray ◽  
Scid Cells ◽  
Phosphatidylinositol 3

Abstract Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.

scholarly journals Tex19.1 Regulates Meiotic DNA Double Strand Break Frequency in Mouse Spermatocytes

2017 ◽  
Author(s):  
James H. Crichton ◽  
Christopher J. Playfoot ◽  
Marie MacLennan ◽  
David Read ◽  
Howard J. Cooke ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Meiotic Recombination ◽  
Homologous Chromosome ◽  
E3 Ubiquitin Ligase ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Genetic Pathway ◽  
Strand Breaks ◽  
Chromosome Synapsis ◽  
Meiotic Dsbs

AbstractMeiosis relies on the SPO11 endonuclease to generate the recombinogenic DNA double strand breaks (DSBs) required for homologous chromosome synapsis and segregation. The number of meiotic DSBs needs to be sufficient to allow chromosomes to search for and find their homologs, but not excessive to the point of causing genome instability. Here we report that meiotic DSB frequency in mouse spermatocytes is regulated by the mammal-specific gene Tex19.1. We show that the chromosome asynapsis previously reported in Tex19.1-/- spermatocytes is preceded by reduced numbers of recombination foci in leptotene and zygotene. Tex19.1 is required for the generation of normal levels of Spo11-dependent DNA damage during leptotene, but not for upstream events such as MEI4 foci formation or accumulation of H3K4me3 at recombination hotspots. Furthermore, we show that mice carrying mutations in the E3 ubiquitin ligase UBR2, a TEX19.1-interacting partner, phenocopy the Tex19.1-/- recombination defects. These data show that Tex19.1 and Ubr2 are required for mouse spermatocytes to generate sufficient meiotic DSBs to ensure that homology search is consistently successful, and reveal a hitherto unknown genetic pathway regulating meiotic DSB frequency in mammals.Author SummaryMeiosis is a specialised type of cell division that occurs during sperm and egg development to reduce chromosome number prior to fertilisation. Recombination is a key step in meiosis as it facilitates the pairing of homologous chromosomes prior to their reductional division, and generates new combinations of genetic alleles for transmission in the next generation. Regulating the amount of recombination is key for successful meiosis: too much will likely cause mutations, chromosomal re-arrangements and genetic instability, whereas too little causes defects in homologous chromosome pairing prior to the meiotic divisions. This study identifies a genetic pathway requiredto generate robust meiotic recombination in mouse spermatocytes. We show that male mice with mutations in Tex19.1 or Ubr2, which encodes an E3 ubiquitin ligase that interacts with TEX19.1, have defects in generating normal levels of meiotic recombination. We show that the defects in these mutants impact on the recombination process at the stage when programmed DNA double strand breaks are being made. This defect likely contributes to the chromosome synapsis and meiotic progression phenotypes previously described in these mutant mice. This study has implications for our understanding of how this fundamental aspect of genetics and inheritance is controlled.

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.

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