Processing and Visualization of Protec-Seq Data for the Generation of Calibrated, Genome-Wide Double Double-Strand Break (dDSB) Maps.

This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.

Exploring the removal of Spo11 and topoisomerases from DNA breaks in S. cerevisiae by human Tyrosyl DNA Phosphodiesterase 2.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by Spo11, a type-II topoisomerase-like protein that becomes covalently linked to DSB ends. Whilst Spo11 oligos-the products of nucleolytic removal by Mre11-have been detected in several organisms, the lifetime of the covalent Spo11-DSB precursor has not been determined and may be subject to alternative processing. Here, we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2-a protein known to repair DNA ends arising from abortive topoisomerase activity-on Spo11 DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 peptides from ssDNA oligos and dsDNA ends even in the presence of competitor genomic DNA. Interestingly, TDP2-processed DSB ends are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate HR at Spo11 DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro, TDP2 expression in meiotic cells was unable to remove Spo11 in vivo-contrasting its ability to aid repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher-order protein complexes at sites of meiotic recombination.

Transcriptomic analysis of meiotic genes during the mitosis-to-meiosis transition in Drosophila females.

Germline cells produce gametes, which are specialized cells essential for sexual reproduction. Germline cells first amplify through several rounds of mitosis before switching to the meiotic program, which requires specific sets of proteins for DNA recombination, chromosome pairing, and segregation. Surprisingly, we previously found that some proteins of the synaptonemal complex, a prophase I meiotic structure, are already expressed and required in the mitotic region of Drosophila females. Here, to assess if additional meiotic genes were expressed earlier than expected, we isolated mitotic and meiotic cell populations to compare their RNA content. Our transcriptomic analysis reveals that all known meiosis I genes are already expressed in the mitotic region; however, only some of them are translated. As a case study, we focused on mei-W68, the Drosophila homolog of Spo11, to assess its expression at both the mRNA and protein levels and used different mutant alleles to assay for a premeiotic function. We could not detect any functional role for Mei-W68 during homologous chromosome pairing in dividing germ cells. Our study paves the way for further functional analysis of meiotic genes expressed in the mitotic region.

The plant early recombinosome: a high security complex to break DNA during meiosis.

KEY MESSAGE: The formacion of numerous unpredictable DNA Double Strand Breaks (DSBs) on chromosomes iniciates meiotic recombination. In this perspective, we propose a 'multi-key lock' model to secure the risky but necesary breaks as well as a 'one per pair of cromatids' model for the topoisomerase-like early recombinosome. During meiosis, homologous chromosomes recombine at few sites of crossing-overs (COs) to ensure correct segregation. The initiation of meiotic recombination involves the formation of DNA double strand breaks (DSBs) during prophase I. Too many DSBs are dangerous for genome integrity: if these DSBs are not properly repaired, it could potentially lead to chromosomal fragmentation. Too few DSBs are also problematic: if the obligate CO cannot form between bivalents, catastrophic unequal segregation of univalents lead to the formation of sterile aneuploid spores. Research on the regulation of the formation of these necessary but risky DSBs has recently advanced in yeast, mammals and plants. DNA DSBs are created by the enzymatic activity of the early recombinosome, a topoisomerase-like complex containing SPO11. This opinion paper reviews recent insights on the regulation of the SPO11 cofactors necessary for the introduction of temporally and spatially controlled DSBs. We propose that a 'multi-key-lock' model for each subunit of the early recombinosome complex is required to secure the formation of DSBs. We also discuss the hypothetical implications that the established topoisomerase-like nature of the SPO11 core-complex can have in creating DSB in only one of the two replicated chromatids of early prophase I meiotic chromosomes. This hypothetical 'one per pair of chromatids' DSB formation model could optimize the faithful repair of the self-inflicted DSBs. Each DSB could use three potential intact homologous DNA sequences as repair template: one from the sister chromatid and the two others from the homologous chromosomes.

Elevated temperature affects the expression of signaling molecules in quail testes meiosis I prophase, but spermatogenesis remains normal.

Spermatogenesis in eukaryotes is a process that occurs within a very narrow temperature threshold, typically not exceeding 36 °C. SPO11 was isolated from the temperature-sensitive mutant receptor of Saccharomyces cerevisiae and is thought to be the only protein that functions during meiosis. This suggested that SPO11 may be the key protein that influenced the temperature of spermatogenesis not exceeding 36 °C. Elevated temperatures typically damage the spermatogenic cells. Birds have a core body temperature of 41-42 °C, and their testis are located inside their bodies, providing an alternative perspective to investigate the potential impact of temperature threshold on spermatogenesis. The objective of this study was to ascertain whether elevated ambient temperatures affect spermatogenesis in birds and whether SPO11 is the key gene affecting the temperature threshold for spermatogenesis. STRA8, SCP3, SPO11, γ-H2AX, and RAD51 were all crucial components in the process of meiotic initiation, synapsis, DNA double-strand break (DSB) induction, homologous chromosome crossover recombination, and repair of DSB. In this study, 39-day-old Japanese quail were subjected to heat stress (HS) at 38 °C for 8 h per day for 3 (3d HS) and 13 (13d HS) consecutive days and analyzed the expression of meiotic signaling molecules (STRA8, SCP3, SPO11, γ-H2AX, and RAD51) using molecular biology techniques, including Immunohistochemistry (IHC), Western Blot (WB), and Real-time Quantitative Polymerase Chain Reaction (qRT-PCR). We found that spermatogenesis was normal in both groups exposed to HS. Meiotic signaling molecules were expressed normally in the 3d HS group. All detected signaling molecules were normally expressed in the 13d HS group, except for SPO11, which showed a significant increase in expression, indicating that SPO11 was temperature-sensitive. We examined the localized expression of each meiotic signaling molecule in quail testis, explored the temperature sensitivity of SPO11, and determined that quail testis can undergo normal spermatogenesis at ambient temperatures exceeding 36 °C. This study concluded that SPO11 is not the key protein influencing spermatogenesis in birds. These findings enhance our understanding of avian spermatogenesis.

The MRE11-RAD50-NBS1 complex both starts and extends DNA end resection in mouse meiosis.

Nucleolytic resection of DNA ends is critical for homologous recombination, but its mechanism is not fully understood, particularly in mammalian meiosis. Here we examine roles of the conserved MRN complex (MRE11, RAD50, and NBS1) through genome-wide analysis of meiotic resection in mice with various MRN mutations, including several that cause chromosomal instability in humans. Meiotic DSBs form at elevated levels but remain unresected if Mre11 is conditionally deleted, thus MRN is required for both resection initiation and regulation of DSB numbers. Resection lengths are reduced to varying degrees in MRN hypomorphs or if MRE11 nuclease activity is attenuated in a conditional nuclease-dead Mre11 model. These findings unexpectedly establish that MRN is needed for longer-range extension of resection, not just resection initiation. Finally, resection defects are additively worsened by combining MRN and Exo1 mutations, and mice that are unable to initiate resection or have greatly curtailed resection lengths experience catastrophic spermatogenic failure. Our results elucidate multiple functions of MRN in meiotic recombination, uncover unanticipated relationships between short- and long-range resection, and establish the importance of resection for mammalian meiosis.

SPO-Seq: An Accessible Method for Efficient Evaluation of Spo11 Catalytic Activity and Profiling Meiotic DSB Hotspots in Saccharomyces cerevisiae.

Meiotic recombination is a key process facilitating the formation of crossovers and the exchange of genetic material between homologous chromosomes in early meiosis. This involves controlled double-strand breaks (DSBs) formation catalyzed by Spo11. DSBs exhibit a preferential location in specific genomic regions referred to as hotspots, and their variability is tied to varying Spo11 activity levels. We have refined a ChIP-Seq technique, called SPO-Seq, to map Spo11-specific DSB formation in Saccharomyces cerevisiae. The chapter describes our streamlined approach and the developed bioinformatic tools for processing data and comparing with existing DSB hotspot maps. Through this combined experimental and computational approach, we aim to enhance our understanding of meiotic recombination and genetic exchange processes in budding yeast, with the potential to expand this methodology to other organisms by applying a few modifications.

CC-seq: Nucleotide-Resolution Mapping of Spo11 DNA Double-Strand Breaks in S. cerevisiae Cells.

During meiosis, Spo11 generates DNA double-strand breaks to induce recombination, becoming covalently attached to the 5' ends on both sides of the break during this process. Such Spo11 "covalent complexes" are transient in wild-type cells, but accumulate in nuclease mutants unable to initiate repair. The CC-seq method presented here details how to map the location of these Spo11 complexes genome-wide with strand-specific nucleotide-resolution accuracy in synchronized Saccharomyces cerevisiae meiotic cells.

Recent advances in mechanisms ensuring the pairing, synapsis and segregation of XY chromosomes in mice and humans.

Sex chromosome aneuploidies are among the most common variations in human whole chromosome copy numbers, with an estimated prevalence in the general population of 1:400 to 1:1400 live births. Unlike whole-chromosome aneuploidies of autosomes, those of sex chromosomes, such as the 47, XXY aneuploidy that causes Klinefelter Syndrome (KS), often originate from the paternal side, caused by a lack of crossover (CO) formation between the X and Y chromosomes. COs must form between all chromosome pairs to pass meiotic checkpoints and are the product of meiotic recombination that occurs between homologous sequences of parental chromosomes. Recombination between male sex chromosomes is more challenging compared to both autosomes and sex chromosomes in females, as it is restricted within a short region of homology between X and Y, called the pseudo-autosomal region (PAR). However, in normal individuals, CO formation occurs in PAR with a higher frequency than in any other region, indicating the presence of mechanisms that promote the initiation and processing of recombination in each meiotic division. In recent years, research has made great strides in identifying genes and mechanisms that facilitate CO formation in the PAR. Here, we outline the most recent and relevant findings in this field. XY chromosome aneuploidy in humans has broad-reaching effects, contributing significantly also to Turner syndrome, spontaneous abortions, oligospermia, and even infertility. Thus, in the years to come, the identification of genes and mechanisms beyond XY aneuploidy is expected to have an impact on the genetic counseling of a wide number of families and adults affected by these disorders.

Genetic and physical interactions reveal overlapping and distinct contributions to meiotic double-strand break formation in C. elegans.

Double-strand breaks (DSBs) are the most deleterious lesions experienced by our genome. Yet, DSBs are intentionally induced during gamete formation to promote the exchange of genetic material between homologous chromosomes. While the conserved topoisomerase-like enzyme Spo11 catalyzes DSBs, additional regulatory proteins-referred to as "Spo11 accessory factors"- regulate the number, timing, and placement of DSBs during early meiotic prophase ensuring that SPO11 does not wreak havoc on the genome. Despite the importance of the accessory factors, they are poorly conserved at the sequence level suggesting that these factors may adopt unique functions in different species. In this work, we present a detailed analysis of the genetic and physical interactions between the DSB factors in the nematode Caenorhabditis elegans providing new insights into conserved and novel functions of these proteins. This work shows that HIM-5 is the determinant of X-chromosome-specific crossovers and that its retention in the nucleus is dependent on DSB-1, the sole accessory factor that interacts with SPO-11. We further provide evidence that HIM-5 coordinates the actions of the different accessory factors sub-groups, providing insights into how components on the DNA loops may interact with the chromosome axis.

Essential roles of the ANKRD31-REC114 interaction in meiotic recombination and mouse spermatogenesis.

Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that "anchors" other proteins together and to meiotic chromosomes. To determine whether and why the REC114 interaction is important for ANKRD31 function, we generated mice with Ankrd31 mutations that either reduced (missense mutation) or eliminated (C-terminal truncation) the ANKRD31-REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31-REC114 interaction mimicked an Ankrd31 null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31-REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114 and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction.

The proper interplay between the expression of Spo11 splice isoforms and the structure of the pseudoautosomal region promotes XY chromosomes recombination.

XY chromosome missegregation is relatively common in humans and can lead to sterility or the generation of aneuploid spermatozoa. A leading cause of XY missegregation in mammals is the lack of formation of double-strand breaks (DSBs) in the pseudoautosomal region (PAR), a defect that may occur in mice due to faulty expression of Spo11 splice isoforms. Using a knock-in (ki) mouse that expresses only the single Spo11ß splice isoform, here we demonstrate that by varying the genetic background of mice, the length of chromatin loops extending from the PAR axis and the XY recombination proficiency varies. In spermatocytes of C57Spo11ßki/- mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11ßki/- males where PAR loops are relatively long, formation of DSBs in the PAR (more frequently the Y-PAR) and XY synapsis fails at a high rate, and mice produce sperm with sex-chromosomal aneuploidy. However, if the entire set of Spo11 splicing isoforms is expressed by a wild type allele in the C57/129 background, XY recombination and synapsis is recovered. By generating a Spo11αki mouse model, we prove that concomitant expression of SPO11ß and SPO11α isoforms, boosts DSB formation in the PAR. Based on these findings, we propose that SPO11 splice isoforms cooperate functionally in promoting recombination in the PAR, constraining XY asynapsis defects that may arise due to differences in the conformation of the PAR between mouse strains.

Essential roles of the ANKRD31-REC114 interaction in meiotic recombination and mouse spermatogenesis.

Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that "anchors" other proteins together and to meiotic chromosomes. To determine if and why the REC114 interaction is important for ANKRD31 function, we generated mice with Ankrd31 mutations that either reduced (missense mutation) or eliminated (C-terminal truncation) the ANKRD31-REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31-REC114 interaction mimicked an Ankrd31 null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31-REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114, and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction.

The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots.

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11ß and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

Orchestrating recombination initiation in mice and men.

Recent discoveries have advanced our understanding of recombination initiation beyond the placement of double-stranded DNA breaks (DSBs) from germline replication timing to the dynamic reorganization of chromatin, and defined critical players of recombination initiation. This article focuses on recombination initiation in mammals utilizing the PRDM9 protein to orchestrate crucial stages of meiotic recombination initiation by interacting with the local DNA environment and several protein complexes. The Pioneer Complex with the SNF2-type chromatin remodeling enzyme HELLS, exposes PRDM9-bound DNA. At the same time, a Compass-Complex containing EWSR1, CXXC1, CDYL, EHMT2 and PRDM9 facilitates the association of putative hotspot sites in DNA loops with the chromosomal axis where DSB-promoting complexes are located, and DSBs are catalyzed by the SPO11/TOPOVIBL complex. Finally, homology search is facilitated at PRDM9-directed sites by ANKRD31. The Reader-Writer system consists of PRDM9 writing characteristic histone methylation signatures, which are read by ZCWPW1, promoting efficient homology engagement.

Tracing the evolution of the plant meiotic molecular machinery.

Meiosis is a highly conserved specialised cell division in sexual life cycles of eukaryotes, forming the base of gene reshuffling, biological diversity and evolution. Understanding meiotic machinery across different plant lineages is inevitable to understand the lineage-specific evolution of meiosis. Functional and cytogenetic studies of meiotic proteins from all plant lineage representatives are nearly impossible. So, we took advantage of the genomics revolution to search for core meiotic proteins in accumulating plant genomes by the highly sensitive homology search approaches, PSI-BLAST, HMMER and CLANS. We could find that most of the meiotic proteins are conserved in most of the lineages. Exceptionally, Arabidopsis thaliana ASY4, PHS1, PRD2, PRD3 orthologs were mostly not detected in some distant algal lineages suggesting their minimal conservation. Remarkably, an ancestral duplication of SPO11 to all eukaryotes could be confirmed. Loss of SPO11-1 in Chlorophyta and Charophyta is likely to have occurred, suggesting that SPO11-1 and SPO11-2 heterodimerisation may be a unique feature in land plants of Viridiplantae. The possible origin of the meiotic proteins described only in plants till now, DFO and HEIP1, could be traced and seems to occur in the ancestor of vascular plants and Streptophyta, respectively. Our comprehensive approach is an attempt to provide insights about meiotic core proteins and thus the conservation of meiotic pathways across plant kingdom. We hope that this will serve the meiotic community a basis for further characterisation of interesting candidates in future.

Chromosome-dependent aneuploid formation in Spo11-less meiosis.

Deficiency in meiotic recombination leads to aberrant chromosome disjunction during meiosis, often resulting in the lethality of gametes or genetic disorders due to aneuploidy formation. Budding yeasts lacking Spo11, which is essential for initiation of meiotic recombination, produce many inviable spores in meiosis, while very rarely all sets of 16 chromosomes are coincidentally assorted into gametes to form viable spores. We induced meiosis in a spo11∆ diploid, in which homolog pairs can be distinguished by single nucleotide polymorphisms and determined whole-genome sequences of their exceptionally viable spores. We detected no homologous recombination in the viable spores of spo11∆ diploid. Point mutations were fewer in spo11∆ than in wild-type. We observed spo11∆ viable spores carrying a complete diploid set of homolog pairs or haploid spores with a complete haploid set of homologs but with aneuploidy in some chromosomes. In the latter, we found the chromosome-dependence in the aneuploid incidence, which was positively and negatively influenced by the chromosome length and the impact of dosage-sensitive genes, respectively. Selection of aneuploidy during meiosis II or mitosis after spore germination was also chromosome dependent. These results suggest a pathway by which specific chromosomes are more prone to cause aneuploidy, as observed in Down syndrome.

DNA/RNA helicase DHX36 is required for late stages of spermatogenesis.

Spermatogenesis is a highly complex developmental process that typically consists of mitosis, meiosis, and spermiogenesis. DNA/RNA helicase DHX36, a unique guanine-quadruplex (G4) resolvase, plays crucial roles in a variety of biological processes. We previously showed that DHX36 is highly expressed in male germ cells with the highest level in zygotene spermatocytes. Here, we deleted Dhx36 in advanced germ cells with Stra8-GFPCre and found that a Dhx36 deficiency in the differentiated spermatogonia leads to meiotic defects and abnormal spermiogenesis. These defects in late stages of spermatogenesis arise from dysregulated transcription of G4-harboring genes, which are required for meiosis. Thus, this study reveals that Dhx36 plays crucial roles in late stages of spermatogenesis.

Identification, characterization, and rescue of CRISPR/Cas9 generated wheat SPO11-1 mutants.

Increasing crop yields through plant breeding is time consuming and laborious, with the generation of novel combinations of alleles being limited by chromosomal linkage blocks and linkage-drag. Meiotic recombination is essential to create novel genetic variation via the reshuffling of parental alleles. The exchange of genetic information between homologous chromosomes occurs at crossover (CO) sites but CO frequency is often low and unevenly distributed. This bias creates the problem of linkage-drag in recombination 'cold' regions, where undesirable variation remains linked to useful traits. In plants, programmed meiosis-specific DNA double-strand breaks, catalysed by the SPO11 complex, initiate the recombination pathway, although only ~5% result in the formation of COs. To study the role of SPO11-1 in wheat meiosis, and as a prelude to manipulation, we used CRISPR/Cas9 to generate edits in all three SPO11-1 homoeologues of hexaploid wheat. Characterization of progeny lines shows plants deficient in all six SPO11-1 copies fail to undergo chromosome synapsis, lack COs and are sterile. In contrast, lines carrying a single copy of any one of the three wild-type homoeologues are phenotypically indistinguishable from unedited plants both in terms of vegetative growth and fertility. However, cytogenetic analysis of the edited plants suggests that homoeologues differ in their ability to generate COs and in the dynamics of synapsis. In addition, we show that the transformation of wheat mutants carrying six edited copies of SPO11-1 with the TaSPO11-1B gene, restores synapsis, CO formation, and fertility and hence opens a route to modifying recombination in this agronomically important crop.

Regulation Mechanisms of Meiotic Recombination Revealed from the Analysis of a Fission Yeast Recombination Hotspot ade6-M26.

Meiotic recombination is a pivotal event that ensures faithful chromosome segregation and creates genetic diversity in gametes. Meiotic recombination is initiated by programmed double-strand breaks (DSBs), which are catalyzed by the conserved Spo11 protein. Spo11 is an enzyme with structural similarity to topoisomerase II and induces DSBs through the nucleophilic attack of the phosphodiester bond by the hydroxy group of its tyrosine (Tyr) catalytic residue. DSBs caused by Spo11 are repaired by homologous recombination using homologous chromosomes as donors, resulting in crossovers/chiasmata, which ensure physical contact between homologous chromosomes. Thus, the site of meiotic recombination is determined by the site of the induced DSB on the chromosome. Meiotic recombination is not uniformly induced, and sites showing high recombination rates are referred to as recombination hotspots. In fission yeast, ade6-M26, a nonsense point mutation of ade6 is a well-characterized meiotic recombination hotspot caused by the heptanucleotide sequence 5'-ATGACGT-3' at the M26 mutation point. In this review, we summarize the meiotic recombination mechanisms revealed by the analysis of the fission ade6-M26 gene as a model system.