Recombinational DNA double-strand breaks in mice precede synapsis (original) (raw)
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Meiotic Chromosome Synapsis in Yeast Can Occur Without Spo11-Induced DNA Double-Strand Breaks
Genetics, 2004
Proper chromosome segregation and formation of viable gametes depend on synapsis and recombination between homologous chromosomes during meiosis. Previous reports have shown that the synaptic structures, the synaptonemal complexes (SCs), do not occur in yeast cells with the SPO11 gene removed. The Spo11 enzyme makes double-strand breaks (DSBs) in the DNA and thereby initiates recombination. The view has thus developed that synapsis in yeast strictly depends on the initiation of recombination. Synapsis in some other species (Drosophila melanogaster and Caenorhabditis elegans) is independent of recombination events, and SCs are found in spo11 mutants. This difference between species led us to reexamine spo11 deletion mutants of yeast. Using antibodies against Zip1, a SC component, we found that a small fraction (1%) of the spo11 null mutant cells can indeed form wild-type-like SCs. We further looked for synapsis in a spo11 mutant strain that accumulates pachytene cells (spo11Δ ndt80Δ)...
DNA Repair, 2018
Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiationinduced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11 YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/ Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11 YF/YF mice, 140-160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11 YF/YF compared to Spo11 +/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11 YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes.
Genes to Cells, 1997
Background: When Saccharomyces cerevisiae cells that have begun meiosis are transferred to mitotic growth conditions ('return-to-growth', RTG), they can complete recombination at high meiotic frequencies, but undergo mitotic cell division and remain diploid. It was not known how meiotic recombination intermediates are repaired following RTG. Using molecular and cytological methods, we investigated whether the usual meiotic apparatus could repair meiotically induced DSBs during RTG, or whether other mechanisms are invoked when the developmental context changes.
Meiotic recombination: Breaking the genome to save it
Current Biology, 2001
Meiosis produces the faithful and efficient division of a replicated diploid genome into four equal parts. All other cell divisions separate sister chromatids; in the first meiotic division (meiosis I), sister chromatids remain associated and homologous chromosomes (homologs) separate. The first meiotic division is preceded by an extended prophase, featuring progressive homolog condensation and colocalization ( ). This culminates at pachytene, where homolog axes are aligned end-to-end and tightly juxtaposed in synaptonemal complexes (reviewed in [1]). Between S phase and pachytene are leptotene, with visible axes but no obvious pairing, and zygotene, where homolog juxtaposition is apparent and synaptonemal complexes begin to form. After pachytene, chromosomes become diffuse. They then recondense in preparation for separation (diplotene/diakinesis), with homologs connected only at points of crossover, known as chiasmata.
Genome-Wide Redistribution of Meiotic Double-Strand Breaks in Saccharomyces cerevisiae
Molecular and Cellular Biology, 2007
Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. DSBs are not randomly distributed along chromosomes. To better understand factors that control the distribution of DSBs in budding yeast, we have examined the genome-wide binding and cleavage properties of the Gal4 DNA binding domain (Gal4BD)-Spo11 fusion protein. We found that Gal4BD-Spo11 cleaves only a subset of its binding sites, indicating that the association of Spo11 with chromatin is not sufficient for DSB formation. In centromere-associated regions, the centromere itself prevents DSB cleavage by tethered Gal4BD-Spo11 since its displacement restores targeted DSB formation. In addition, we observed that new DSBs introduced by Gal4BD-Spo11 inhibit surrounding DSB formation over long distances (up to 60 kb), keeping constant the number of DSBs per chromosomal region. Together, these results demonstrate that the targeting of Spo11 to new chromosomal locations leads to both local stimulation and genome-wide redistribution of recombination initiation and that some chromosomal regions are inherently cold regardless of the presence of Spo11.
Wild-Type Levels of Spo11Induced DSBs Are Required for Normal Single-Strand Resection during Meiosis
Molecular Cell, 2002
homolog as repair template (e.g., Hunter and Kleckner, 2001; Schwacha and Kleckner, 1997; Thompson and Stahl, 1999; Zenvirth et al., 1997; and references within). The coordinated nature of chromosome pairing, synapsis, and recombination suggests that these processes are Biotechnology mechanistically linked. This view is supported by a num-The University of Sheffield ber of observations. Mutations in various yeast and Sheffield, S10 2TN mouse recombination genes affect chromosome pairing United Kingdom and/or synapsis (see Burgess, 2002; Hunter and Kleck-2 Department of Human Biology ner, 2001; Zickler and Kleckner, 1998). Nonmutational University of Kaiserslautern loss of allelic recombination between divergent chromo-D-67663 Kaiserslautern somes is also sufficient to reduce the effectiveness of Germany meiotic chromosome pairing (Goldman and Lichten, 2000). On the other hand, genes whose products are structural components of the SC are required for a nor-Summary mal recombination frequency and normal crossover distribution (see Zickler and Kleckner, 1998). The coordina-We have studied the repair of a DNA-DSB created by tion of recombination with chromosome organization the VMA1-derived endonuclease in mutants that have involves checkpoint-type monitoring (Lydall et al., 1996; different levels of Spo11-DSBs: WT (sae2), few (hop1), Roeder and Bailis, 2000). Interruption of DSB repair durand none (spo11-Y135F). In spo11-Y135F and hop1 cells, ing meiosis leads to checkpoint-dependent arrest. Esintrachromosomal repair is more frequent than in WT tablishing arrest requires surveillance of the recombinaand sae2 cells. In spo11-Y135F cells there was no tion process and thus requires Spo11-DSB formation chromosome pairing or synapsis and a faster turnover (Lydall et al., 1996; McKee and Kleckner, 1997a; Roeder of resected DNA. Compared to WT and sae2 cells, and Bailis, 2000; and references within). spo11-Y135F and hop1 cells have a greater proportion It is possible that Spo11-DSBs also create a signal that of long resection tracts. The data suggest that high stimulates interchromosomal repair. Genetic studies of levels of Spo11-DSBs are required for normal regulameiotic recombination at HO-induced DSBs show that tion of resection, even at a DSB created by another repair is substantially normal when Spo11-DSBs are abprotein. WT control over resection could be important sent but that gene conversion rates are reduced and for directing repair to be interchromosomal, increasconversion tracts are lengthened (Malkova et al., 1996, ing the chance of creating interhomolog connections 2000). We have tested the idea that Spo11-DSBs stimuessential to meiotic segregation. late wild-type (WT) interchromosomal DSB repair during meiosis using genetic and molecular analyses of a Introduction Spo11-independent DSB induced by the VMA1-derived endonuclease (VDE, otherwise known as PI-SceI; Bremer Specialized functions are required to segregate homoloet al., 1992; Gimble and Thorner, 1993). The strains used gous chromosomes during the meiotic reductional diviexhibited three different levels of Spo11-DSB formation: sion; these include chromosome pairing, synapsis, and WT, up to 12% of WT, or no breaks. We have found that recombination (Zickler and Kleckner, 1998). During pairrepair of the VDE-DSB during meiosis is substantially ing, homologous chromosomes align and move close modified when few or no Spo11-DSBs are formed. The to each other, eventually permitting synapsis when a modifications include the increased presence of longer tripartite proteinaceous structure (the synaptonemal resection tracts and shorter life span of resected intercomplex, SC) forms between them (Zickler and Kleckmediates. These changes show a positive correlation with more frequent repair using flanking repeated DNA. ner, 1998). To avoid nondisjunction, the homologs are Overall, the data suggest that Spo11-DSBs have a negaheld together as bivalents until first anaphase (Uhlmann, tive regulatory role during DSB processing, which influ-2001). In most organisms, this is achieved by interchroences the outcome of DSB repair. mosomal recombination creating crossovers that link nonsister chromatids together. The progression of interchromosomal recombination from induction to com-Results pletion is concomitant with chromosome pairing and synapsis. Meiotic recombination is induced by double-We set out to determine whether Spo11-DSBs are restrand DNA breaks (DSBs) catalyzed by the Spo11 proquired to stimulate normal meiotic DSB repair of a tein (Keeney et al., 1997). Genetic control, ensuring re-Spo11-independent DSB induced by VDE. We initially combination in meiosis is interchromosomal, is indistudied repair in a heterozygous strain expressing both cated by mutants that show reduced bias for using the WT SPO11 and mutant spo11-Y135F-HA3His6 (referred to as WT when no other mutation is present) and in a homozygous strain expressing only spo11-Y135F-3 Correspondence: a.goldman@shef.ac.uk HA3His6 (referred to as spo11f ). The spo11f allele has 4 Present address: MP1 Mol. Genetics, Dept. Ropers, D-14195, Berlin, Germany. lost the active tyrosine that normally catalyzes formation Signal patterns in spread nuclei were investigated using a Zeiss Axioskop epifluorescence microscope equipped with a double-We would like to thank S. Burgess, N. Hollingsworth, C. Price, and band-pass filter for simultaneous observation of red and green fluo-A. Villeneuve for discussions and comments on an early version of rescence, or single-band-pass filters for excitation of red, green, and the manuscript. We are also grateful to T. Allers, V. Borde, R. Borts, blue (Chroma Technologies). Digital images were obtained using a L. Gilbertson, J. Haber, S. Keeney, M. Lichten, A. Malkova, and P. cooled gray-scale CCD camera (Hamamatsu) controlled by the ISIS Sudbery for discussions and/or technical advice. Yeast strains and/ fluorescence image analysis system (MetaSystems). For all time or plasmids were generously provided by R. Borts, N. Hollingsworth, points and probe combinations, more than 100 nuclei of randomized S. Keeney, and M. Lichten. This work was supported by BBSRC preparations were examined directly in the microscope for single grant 50/G12839 to A.S.H.G.; H.S. was supported by the DFG (SCHE or double FISH signals of the various probes. Only nuclei containing 350/8-3). signals of both probes were scored. Spores of dAG206 were dissected to determine the repair pattern of the VDE-DSB. Spore colonies were replica plated to synthetic References medium lacking arginine (SC-Arg) or uracil (SC-Ura) to check to see if they were: (1) arginine and uracil prototrophs (meaning there had Allers, T., and Lichten, M. (2001). Differential timing and control of been a gene conversion event of arg4-vde to ARG4), (2) arginine noncrossover and crossover recombination during meiosis. Cell and uracil auxotrophs (meaning the spore contained the prominent 106, 47-57. class of deletion product to ura3::Ty), and (3) arginine auxotrophs Arbel, A., Zenvirth, D., and Simchen, G. (1999). Sister chromatidbut uracil prototrophs (meaning the spore contained either parental based DNA repair is mediated by RAD54, not by DMC1 or TID1. arg4-bgl, gene conversion of arg4-vde to arg4-bgl, unchanged EMBO J. 18, 2648-2658. arg4-vde, or rare deletion to URA3). The spore colonies from class 3 Bergerat, A., de Massy, B., Gadelle, D., Varoutas, P.C., Nicolas, A., were further tested by mating in patches with arg4-nsp and arg4-bgl and Forterre, P. (1997). An atypical topoisomerase II from Archaea tester strains on YEPD. Matings were replica plated to SC-Arg mewith implications for meiotic recombination. Nature 386, 414-417. dium and exposed to a pulse of UV light to stimulate recombination. Recombination between arg4-bgl and arg4-nsp alleles yielded Arg ϩ Bishop, D.K. (1994). RecA homologs Dmc1 and Rad51 interact to papillae. No such papillae could arise from an arg4-bgl and arg4-bgl form multiple nuclear complexes prior to meiotic chromosome synmating. Spore colonies with uncut arg4-vde were able to produce apsis. Cell 79, 1081-1092. Arg ϩ papillae when mated with both tester strains. Spore colonies Bremer, M.C., Gimble, F.S., Thorner, J., and Smith, C.L. (1992). VDE containing a URA3 deletion product were unable to form papillae endonuclease cleaves Saccharomyces cerevisiae genomic DNA at with either tester strain. a single site: physical mapping of the VMA1 gene. Nucleic Acids Res. 20, 5484. Return to Growth Burgess, S.M. (2002). Homologous chromosome associations and Aliquots of synchronously sporulating cells were serial diluted in nuclear order in meiotic and mitotically dividing cells of budding water and plated onto YEPD and SC-Arg medium to assess the yeast. Adv. Genet. 46, 49-90. proportion of Arg ϩ prototrophs. Cha, R.S., Weiner, B.M., Keeney, S., Dekker, J., and Kleckner, N. (2000). Progression of meiotic DNA replication is modulated by inter-DNA Isolation and Southern and Slot Blot Analyses chromosomal interaction proteins, negatively by Spo11p and posi-Twenty-five milliliter samples of sporulating culture were removed at intervals and processed for storage and DNA isolation according tively by Rec8p. Genes Dev. 14, 493-503.
Cell, 2011
Meiotic recombination between homologous chromosomes initiates via programmed DNA doublestrand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.
Genetics, 1996
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 rad50 delta 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 rad50 delta 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 ...
Genetics, 1996
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 SP013 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 rad5OA cells that cannot initiate normal meiotic DSBs. We find that the RtlDSO gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad5OA 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 rad5OA diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination.
International Journal of Molecular Sciences, 2020
Homologous recombination is essential for chromosome segregation during meiosis I. Meiotic recombination is initiated by the introduction of double-strand breaks (DSBs) at specific genomic locations called hotspots, which are catalyzed by Spo11 and its partners. DSB hotspots during meiosis are marked with Set1-mediated histone H3K4 methylation. The Spo11 partner complex, Rec114-Mer2-Mei4, essential for the DSB formation, localizes to the chromosome axes. For efficient DSB formation, a hotspot with histone H3K4 methylation on the chromatin loops is tethered to the chromosome axis through the H3K4 methylation reader protein, Spp1, on the axes, which interacts with Mer2. In this study, we found genetic interaction of mutants in a histone modification protein complex called PAF1C with the REC114 and MER2 in the DSB formation in budding yeast Saccharomyces cerevisiae. Namely, the paf1c mutations rtf1 and cdc73 showed synthetic defects in meiotic DSB formation only when combined with a wild-type-like tagged allele of either the REC114 or MER2. The synthetic defect of the tagged REC114 allele in the DSB formation was seen also with the set1, but not with spp1 deletion. These results suggest a novel role of histone modification machinery in DSB formation during meiosis, which is independent of Spp1-mediated loop-axis tethering.