The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates.

Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.

Defining the influence of Rad51 and Dmc1 lineage-specific amino acids on genetic recombination.

The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.

PRDM9 Methyltransferase Activity Is Essential for Meiotic DNA Double-Strand Break Formation at Its Binding Sites.

The programmed formation of hundreds of DNA double-strand breaks (DSBs) is essential for proper meiosis and fertility. In mice and humans, the location of these breaks is determined by the meiosis-specific protein PRDM9, through the DNA-binding specificity of its zinc-finger domain. PRDM9 also has methyltransferase activity. Here, we show that this activity is required for H3K4me3 and H3K36me3 deposition and for DSB formation at PRDM9-binding sites. By analyzing mice that express two PRDM9 variants with distinct DNA-binding specificities, we show that each variant generates its own set of H3K4me3 marks independently from the other variant. Altogether, we reveal several basic principles of PRDM9-dependent DSB site determination, in which an excess of sites are designated through PRDM9 binding and subsequent histone methylation, from which a subset is selected for DSB formation.

Mutational analysis of Mei5, a subunit of Mei5-Sae3 complex, in Dmc1-mediated recombination during yeast meiosis.

Interhomolog recombination in meiosis is mediated by the Dmc1 recombinase. The Mei5-Sae3 complex of Saccharomyces cerevisiae promotes Dmc1 assembly and functions with Dmc1 for homology-mediated repair of meiotic DNA double-strand breaks. How Mei5-Sae3 facilitates Dmc1 assembly remains poorly understood. In this study, we created and characterized several mei5 mutants featuring the amino acid substitutions of basic residues. We found that Arg97 of Mei5, conserved in its ortholog, SFR1 (complex with SWI5), RAD51 mediator, in humans and other organisms, is critical for complex formation with Sae3 for Dmc1 assembly. Moreover, the substitution of either Arg117 or Lys133 with Ala in Mei5 resulted in the production of a C-terminal truncated Mei5 protein during yeast meiosis. Notably, the shorter Mei5-R117A protein was observed in meiotic cells but not in mitotic cells when expressed, suggesting a unique regulation of Dmc1-mediated recombination by posttranslational processing of Mei5-Sae3.

Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences.

Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.

The Arabidopsis HOP2 gene has a role in preventing illegitimate connections between nonhomologous chromosome regions.

Meiotic homologous chromosomes synapse and undergo crossing over (CO). In many eukaryotes, both synapsis and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via homologous recombination. In these organisms, two key proteins are recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 assist RAD51 and DMC1 and also are required for synapsis and CO. We have investigated the hop2-1 phenotype in Arabidopsis during the segregation stages of both meiosis and mitosis. Despite a general lack of synapsis during prophase I, we observed extensive, stable interconnections between nonhomologous chromosomes in diploid hop2-1 nuclei in first and second meiotic divisions. Using γH2Ax as a marker of unrepaired DSBs, we detected γH2AX foci from leptotene through early pachytene but saw no foci from mid-pachytene onward. We conclude that the bridges seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Examining haploids, we found that wild type haploids produce only univalents, but hop2-1 haploids like hop2-1 diploids have illegitimate connections stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing repairs that use nonhomologous chromosomes during meiosis. We also found evidence that HOP2 plays a role in preventing illegitimate repair of radiation-induced DSBs in rapidly dividing petal cells. We conclude that HOP2 in Arabidopsis plays both a positive role in promoting synapsis and a separable role in preventing DSB repair using nonhomologous chromosomes. SIGNIFICANCE STATEMENT : The fidelity of homologous recombination (HR) during meiosis is essential to the production of viable gametes and for maintaining genome integrity in vegetative cells. HOP2 is an important protein for accurate meiotic HR in plants. We have found evidence of high levels of illegitimate repairs between nonhomologous chromosomes during meiosis and in irradiated petal cells in hop2-1 mutants, suggesting a role for HOP2 beyond its established role in synapsis and crossing over.

Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase.

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5'-to-3' polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein-protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.

Two auxiliary factors promote Dmc1-driven DNA strand exchange via stepwise mechanisms.

Homologous recombination (HR) is a universal mechanism operating in somatic and germ-line cells, where it contributes to the maintenance of genome stability and ensures the faithful distribution of genetic material, respectively. The ability to identify and exchange the strands of two homologous DNA molecules lies at the heart of HR and is mediated by RecA-family recombinases. Dmc1 is a meiosis-specific RecA homolog in eukaryotes, playing a predominant role in meiotic HR. However, Dmc1 cannot function without its two major auxiliary factor complexes, Swi5-Sfr1 and Hop2-Mnd1. Through biochemical reconstitutions, we demonstrate that Swi5-Sfr1 and Hop2-Mnd1 make unique contributions to stimulate Dmc1-driven strand exchange in a synergistic manner. Mechanistically, Swi5-Sfr1 promotes establishment of the Dmc1 nucleoprotein filament, whereas Hop2-Mnd1 defines a critical, rate-limiting step in initiating strand exchange. Following execution of this function, we propose that Swi5-Sfr1 then promotes strand exchange with Hop2-Mnd1. Thus, our findings elucidate distinct yet complementary roles of two auxiliary factors in Dmc1-driven strand exchange, providing mechanistic insights into some of the most critical steps in meiotic HR.

Recent advances in functional conservation and divergence of recombinase RAD51 and DMC1.

Meiosis is a specialized cell division that occurs in reproductive cells during sexual reproduction. It contains once DNA replication following nucleus division twice, thus producing haploid gametes. Fusion of male and female gametes restores genome to the diploid level, which not only ensures the genome stability between generations during sexual reproduction, but also leads to genetic diversity among offspring. Meiosis homologous recombination (HR) is one of the crucial events during meiotic prophase I, and it not only ensures the subsequently faithful segregation of homologous chromosomes (homologs), but also exchanges genetic information between homologs with greatly increasing the genetic diversity of progeny. RAD51 (RADiation sensitive 51) and DMC1 (disruption Meiotic cDNA 1) are essential recombinases for the HR process, and have certain commonalities and differences. In this review, we summarize and compare the conserved and differentiated features of RAD51 and DMC1 in terms of origin, evolution, structure, and function, we also provide an outlook on future research directions to further understand and study the molecular mechanisms in regulation of meiotic recombination.

SPO11.2 is essential for programmed double-strand break formation during meiosis in bread wheat (Triticum aestivum L.).

Meiotic recombination is initiated by formation of DNA double-strand breaks (DSBs). This involves a protein complex that includes in plants the two similar proteins, SPO11-1 and SPO11-2. We analysed the sequences of SPO11-2 in hexaploid bread wheat (Triticum aestivum), as well as in its diploid and tetraploid progenitors. We investigated its role during meiosis using single, double and triple mutants. The three homoeologous SPO11-2 copies of hexaploid wheat exhibit high nucleotide and amino acid similarities with those of the diploids, tetraploids and Arabidopsis. Interestingly, however, two nucleotides deleted in exon-2 of the A copy lead to a premature stop codon and suggest that it encodes a non-functional protein. Remarkably, the mutation was absent from the diploid A-relative Triticum urartu, but present in the tetraploid Triticum dicoccoides and in different wheat cultivars indicating that the mutation occurred after the first polyploidy event and has since been conserved. We further show that triple mutants with all three copies (A, B, D) inactivated are sterile. Cytological analyses of these mutants show synapsis defects, accompanied by severe reductions in bivalent formation and numbers of DMC1 foci, thus confirming the essential role of TaSPO11-2 in meiotic recombination in wheat. In accordance with its 2-nucleotide deletion in exon-2, double mutants for which only the A copy remained are also sterile. Notwithstanding, some DMC1 foci remain visible in this mutant, suggesting a residual activity of the A copy, albeit not sufficient to restore fertility.