An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis

Meiosis is a defining feature of eukaryotes but its phylogenetic distribution has not been broadly determined, especially among eukaryotic microorganisms (i.e. protists)-which represent the majority of eukaryotic 'supergroups'. We surveyed genomes of animals, fungi, plants and protists for meiotic genes, focusing on the evolutionarily divergent parasitic protist Trichomonas vaginalis. We identified homologs of 29 components of the meiotic recombination machinery, as well as the synaptonemal and meiotic sister chromatid cohesion complexes. T. vaginalis has orthologs of 27 of 29 meiotic genes, including eight of nine genes that encode meiosis-specific proteins in model organisms. Although meiosis has not been observed in T. vaginalis, our findings suggest it is either currently sexual or a recent asexual, consistent with observed, albeit unusual, sexual cycles in their distant parabasalid relatives, the hypermastigotes. T. vaginalis may use meiotic gene homologs to mediate homologous recombination and genetic exchange. Overall, this expanded inventory of meiotic genes forms a useful "meiosis detection toolkit". Our analyses indicate that these meiotic genes arose, or were already present, early in eukaryotic evolution; thus, the eukaryotic cenancestor contained most or all components of this set and was likely capable of performing meiotic recombination using near-universal meiotic machinery.     

Arabidopsis SPO11-2 functions with SPO11-1 in meiotic recombination

The Spo11 protein is a eukaryotic homologue of the archaeal DNA topoisomerase VIA subunit (topo VIA). In archaea it is involved, together with its B subunit (topo VIB), in DNA replication. However, most eukaryotes, including yeasts, insects and vertebrates, instead have a single gene for Spo11/topo VIA and no homologues for topo VIB. In these organisms, Spo11 mediates DNA double-strand breaks that initiate meiotic recombination. Many plant species, in contrast to other eukaryotes, have three homologues for Spo11/topo VIA and one for topo VIB. The homologues in Arabidopsis, AtSPO11-1, AtSPO11-2 and AtSPO11-3, all share 20-30% sequence similarity with other Spo11/topo VIA proteins, but their functional relationship during meiosis or other processes is not well understood. Previous genetic evidence suggests that AtSPO11-1 is a true orthologue of Spo11 in other eukaryotes and is required for meiotic recombination, whereas AtSPO11-3 is involved in DNA endo-reduplication as a part of the topo VI complex. In this study, we show that plants homozygous for atspo11-2 exhibit a severe sterility phenotype. Both male and female meiosis are severely disrupted in the atspo11-2 mutant, and this is associated with severe defects in synapsis during the first meiotic division and reduced meiotic recombination. Further genetic analysis revealed that AtSPO11-1 and AtSPO11-2 genetically interact, i.e. plants heterozygous for both atspo11-1 and atspo11-2 are also sterile, suggesting that AtSPO11-1 and AtSPO11-2 have largely overlapping functions. Thus, the three Arabidopsis Spo11 homologues appear to function in two discrete processes, i.e. AtSPO11-1 and AtSPO11-2 in meiotic recombination and AtSPO11-3 in DNA replication.     

Compartmentalization of Spo11p in vegetative cells of yeast Saccharomyces cerevisiae

Homologous recombination is initiated in meiotic eukaryotic cells at DNA double-strand breaks, which are generated by several proteins, Spo11p playing a key role. The protein products of SPO11 orthologs are highly conserved, are found in most eukaryotes from plants to human, and are structurally similar to subunit A of archaeal DNA topoisomerase VI. Saccharomyces cerevisiae SPO11 is expressed in meiotic prophase I. Spo11p acts as topoisomerase II and is presumably active as a dimer. Experimental data on Spo11p compartmentalization in vegetative yeast cells are unavailable. The SPO11 coding region and its fragments were fused in frame with the egfp reporter and expressed in vegetative yeast cells. The Spo11p-EGFP fusion was localized in the nucleus, while cytoplasmic localization was observed for Spo11Δ-EGFP devoid of the 25 N-terminal residues. N-terminal Spo11p region 7–25 fused with EGFP ensured the nuclear targeting of the reporter protein and was assumed to harbor the nuclear localization signal.     

Molecular characterization of homologues of both subunits A (SPO11) and B of the archaebacterial topoisomerase 6 in plants.

The Spo11 protein is an eukaryotic homologue of the topoisomerase 6 subunit A from archaebacteria. In yeast Spo11p has been found to bind covalently to double-strand breaks (DSBs) during meiosis. Single homologues of the SPO11 gene exist in various eukaryotes, except plants. Previously, we found in the Arabidopsis thaliana genome two ancient paralogs, AtSPO11-1 and 2. Here we report on the molecular characterization of a third one, AtSPO11-3. This puzzling finding might be explained by the fact that we detected additionally--for the first time outside of the archaebacterial kingdom--a homologue of the subunit B of topoisomerase 6, AtTOP6B. Both AtSPO11-3 and AtTOP6B are abundantly expressed in Arabidopsis and EST comparisons indicate the presence of both genes in various plant species. Via two hybrid studies we could demonstrate that full length AtTop6B is able to interact with AtSpo11-2 and 3 but not with AtSpo11-1. Our data suggest that plants possess in contrast to other eukaryotes an additional archaebacterial kind of topoisomerase.     

A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis

Sexual reproduction in eukaryotes is accomplished by meiosis, a complex and specialized process of cell division that results in haploid cells (e.g., gametes). The stereotypical reductive division in meiosis is a major evolutionary innovation in eukaryotic cells, and delineating its history is key to understanding the evolution of sex. Meiosis arose early in eukaryotic evolution, but when and how meiosis arose and whether all eukaryotes have meiosis remain open questions. The known phylogenetic distribution of meiosis comprises plants, animals, fungi, and numerous protists. Diplomonads including Giardia intestinalis (syn. G. lamblia) are not known to have a sexual cycle; these protists may be an early-diverging lineage and could represent a premeiotic stage in eukaryotic evolution. We surveyed the ongoing G. intestinalis genome project data and have identified, verified, and analyzed a core set of putative meiotic genes-including five meiosis-specific genes-that are widely present among sexual eukaryotes. The presence of these genes indicates that: (1) Giardia is capable of meiosis and, thus, sexual reproduction, (2) the evolution of meiosis occurred early in eukaryotic evolution, and (3) the conserved meiotic machinery comprises a large set of genes that encode a variety of component proteins, including those involved in meiotic recombination.     

Evolutionary conservation of recombination proteins and variability of meiosis-specific proteins of chromosomes

A comparison of amino acid sequences is performed for orthologs to the meiosis-specific proteins in humans and seven other species, including animals, fungi, and plants that serve as models for the study of molecular mechanisms of meiosis. It is demonstrated that the RAD51 recombination mediator protein is the most conserved of the studied proteins. Its meiotic homolog DMC1 is less conserved, like the MHL1 mismatch-repair protein. The meiosis-specific SPO11 endonuclease is the least conserved among the studied meiotic enzymes. Structural proteins of meiotic chromosomes are poorly conserved. REC8 meiotic cohesin has 6 times lower similarity in the organisms from different kingdoms than its somatic homolog RAD21. The intermediate conservation level is characteristic of the synaptonemal complex proteins containing HORMA domain. Two functional domains of SPO11 endonuclease and MutL Trans_MLH1 domain of MLH1 enzyme are equally or even less conserved than the whole proteins. HORMA functional domain of a number of synaptonemal complex proteins is only 2–3 times more conserved than the whole molecule. Thus, among the key meiotic proteins, the most conserved are proteins responsible for the accuracy of meiotic recombination. Cohesins, synaptonemal complex proteins, and meiosis-specific SPO11 endonuclease are less conserved even within their functional domains. Obviously, the meiosis-specific proteins have undergone independent evolution in different phylogenetic lineages of eukaryotes.     

A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene

Obligatory homologous recombination (HR) is required for chiasma formation and chromosome segregation in meiosis I. Meiotic HR is initiated by DNA double-strand breaks (DSBs), generated by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Rad50, Mre11 and Com1/Sae2 are essential to process an intermediate of the cleavage reaction consisting of Spo11 covalently linked to the 5' termini of DNA. While Rad50 and Mre11 also confer genome stability to vegetative cells and are well conserved in evolution, Com1/Sae2 was believed to be fungal-specific. Here, we identify COM1/SAE2 homologues in all eukaryotic kingdoms. Arabidopsis thaliana Com1/Sae2 mutants are sterile, accumulate AtSPO11-1 during meiotic prophase and fail to form AtRAd51 foci despite the presence of unrepaired DSBs. Furthermore, DNA fragmentation in AtCom1 is suppressed by eliminating AtSPO11-1. In addition, AtCOM1 is specifically required for mitomycin C resistance. Interestingly, we identified CtIP, an essential protein interacting with the DNA repair machinery, as the mammalian homologue of Com1/Sae2, with important implications for the molecular role of CtIP.     

AtPRD1 is required for meiotic double strand break formation in Arabidopsis thaliana

The initiation of meiotic recombination by the formation of DNA double-strand breaks (DSBs) catalysed by the Spo11 protein is strongly evolutionary conserved. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation, but, unlike Spo11, few of these proteins seem to be conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we have isolated a new gene, AtPRD1, whose mutation affects meiosis in Arabidopsis thaliana. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination markers (e.g., DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11-1 interact in a yeast two-hybrid assay, suggesting that AtPRD1 could be a partner of AtSPO11-1. Moreover, our study reveals similarity between AtPRD1 and the mammalian protein Mei1, suggesting that AtPRD1 could be a Mei1 functional homologue.     

Both conserved and non-conserved regions of Spo11 are essential for meiotic recombination initiation in yeast

DNA double-strand breaks (DSBs) are the initiators of most meiotic recombination events. In Saccharomyces cerevisiae, at least ten genes are necessary for meiotic DSB formation. However, the molecular roles of these proteins are not clearly understood. The meiosis-specific Spo11 protein, which shows sequence similarity with a subunit of an archaeal topoisomerase, is believed to catalyze the meiotic DSB formation. Spo11 is also required for induction of meiotic DSBs at long inverted repeats and at large trinucleotide repeat tracts. Here we report the isolation and characterization of temperature-sensitive spo11-mutant alleles to better understand how Spo11 functions, and how meiotic DSBs are generated at various recombination hotspots. Analysis of mutation sites of isolated spo11-mutant alleles indicated that both N-terminal and C-terminal non-conserved residues of Spo11 are essential for the protein’s function, possibly for interaction with other meiotic DSB enzymes. Several of the mutation sites within the conserved region are predicted to lie on the surface of the protein, suggesting that this region is required for activation of the meiotic initiation complex via protein-protein interaction. In addition to the conditional mutants, we isolated partially recombination-defective mutants; analysis of one of these mutants indicated that Ski8, as observed previously, interacts with Spo11 via the latter’s C-terminal residues.     

Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision

In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein-protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics.     

Compartmentalization of Spo11p in vegetative cells of yeast Saccharomyces cerevisiae

Double-stranded DNA breaks are currently thought to initiate homologous DNA recombination during meiosis. These breaks are mediated by several proteins, the key protein is Spol1p. Spo11 proteins being encoded by the highly conserved orthologs of SPO11 are present in most eukaryotes ranging from plants to man and are structurally similar to the subunit A of the archaea topoisomerase VI. The SPO11 of S. cerevisiae is currently known to be expressed during prophase I. It encodes a topoisomerase II that is apparently active as a dimer. Neither its localization in the native cells nor its nuclear localisation signals have been described in the literature. We report the expression of the coding region of SPO11 and its truncated variants C-terminally tagged by the egfp reporter in yeast. As judged by the EGFP fluorescence, the Spo11 p-EGFP fusion was localized in vegetative yeast nuclei whereas Spo11pdelta-EGFP lacking 25 N-terminal amino acids of Spollp was localized in cytoplasm. Nineteen N-terminal amino acids of Spo11p fused to EGFP made some reporter to be localized in the nucleus. Thus, we conclude that N-terminal part of Spo11p is a nuclear localization signal that is not specific for prophase I and is used to import proteins in vegetative yeast cells.     

The catalytically active tyrosine residues of both SPO11-1 and SPO11-2 are required for meiotic double-strand break induction in Arabidopsis

SPO11, a homolog of the subunit A of the archaebacterial topoisomerase VI, is essential for double-strand break (DSB)-induced initiation of meiotic recombination. In contrast with single homologs in animals and yeasts, three homologs are present in Arabidopsis thaliana and other higher plants. Whereas At SPO11-3 is involved in somatic endoreduplication, At SPO11-1 and, as recently shown, At SPO11-2 are essential for the initiation of meiotic recombination. Further defining the role of At SPO11-2, we were able to demonstrate that it is required for proper chromosome segregation, as its loss resulted in aneuploidy in the surviving progeny. The double mutant spo11-1 spo11-2 does not differ phenotypically from the single mutants, indicating that both proteins are required for the same step. Contrary to the observations for the At rad51-1 single mutant, the combination of spo11-2 and rad51-1 did not lead to chromosome fragmentation, indicating that SPO11-2, like SPO11-1, is required for DSB induction. As the meiotic phenotype of both single SPO11 mutants can be reversed by complementation using the full-length genes but not the same constructs mutated in their respective catalytically active Tyr, both proteins seem to participate directly in the DNA breakage reaction. The active involvement of two SPO11 homologs for DSB formation reveals a striking difference between plants and other eukaryotes in meiosis.     

The mouse Spo11 gene is required for meiotic chromosome synapsis

The Spo11 protein initiates meiotic recombination by generating DNA double-strand breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, Spo11 homologs are dispensable for synapsis in C. elegans and Drosophila yet required for meiotic recombination. Disruption of mouse Spo11 results in infertility. Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Spo11 localizes to discrete foci during leptotene and to homologously synapsed chromosomes. Other mouse mutants that arrest during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, and Morc(-/-)) showed altered Spo11 protein localization and expression. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis.     

Molecular characterisation of two paralogous SPO11 homologues in Arabidopsis thaliana

The Spo11 protein of yeast has been found to be covalently bound to double-strand breaks in meiosis, demonstrating a unique role of the protein in the formation of these breaks. Homologues of the SPO11 gene have been found in various eukaryotes, indicating that the machinery involved in meiotic recombination is conserved in eukaryotes. Here we report on SPO11 homologues in plants. In contrast to what is known from other eukaryotes, Arabidopsis thaliana carries in its genome at least two SPO11 homologues, AtSPO11-1 and AtSPO11-2. Both genes are not more closely related to each other than to other eukaryotic SPO11 homologues, indicating that they did not arise via a recent duplication event during higher plant evolution. For both genes three different polyadenylation sites were found. AtSPO11-1 is expressed not only in generative but also to a lesser extent in somatic tissues. We were able to detect in different organs various AtSPO11-1 cDNAs in which introns were differently spliced—a surprising phenomenon also reported for SPO11 homologues in mammals. In the case of AtSPO11-2 we found that the 3′ end of the mRNA is overlapping with a mRNA produced by a gene located in inverse orientation next to it. This points to a possible antisense regulation mechanism. Our findings hint to the intriguing possibility that, at least for plants, Spo11-like proteins might have more and possibly other biological functions than originally anticipated for yeast.     

The Arabidopsis ROCK-N-ROLLERS gene encodes a homolog of the yeast ATP-dependent DNA helicase MER3 and is required for normal meiotic crossover formation

Recent studies in Saccharomyces cerevisiae have unveiled that meiotic recombination crossovers are formed by two genetically distinct pathways: a major interference-sensitive pathway and a minor interference-insensitive pathway. Several proteins, including the MSH4/MSH5 heterodimer and the MER3 DNA helicase, are indispensable for the interference-sensitive pathway. MSH4 homologs have been identified in mice and Arabidopsis and shown to be required for normal levels of crossovers, suggesting that the function of MSH4 may be conserved among major eukaryotic kingdoms. However, it is not known whether an MER3-like function is also required for meiosis in animals and plants. We have identified an Arabidopsis gene that encodes a putative MER3 homolog and is preferentially expressed in meiocytes. T-DNA insertional mutants of this gene exhibit defects in fertility and meiosis. Detailed cytological studies indicate that the mutants are defective in homolog synapsis and crossover formation, resulting in a reduction of bivalents and in the formation of univalents at late prophase I. We have named this gene ROCK-N-ROLLERS (RCK) to reflect the mutant phenotype of chromosomes undergoing the meiotic 'dance' either in pairs or individually. Our results demonstrate that an MER3-like function is required for meiotic crossover in plants and provide further support for the idea that Arabidopsis, like the budding yeast, possesses both interference-sensitive and insensitive pathways for crossover formation.     

AtSPO11-1 is necessary for efficient meiotic recombination in plants

The Saccharomyces cerevisiae Spo11 protein catalyses DNA double-strand breaks (DSBs) that initiate meiotic recombination. The model plant Arabidopsis thaliana possesses at least three SPO11 homologues. T-DNA and ethyl-methane sulfonate mutagenesis allowed us to show that meiotic progression is altered in plants in which the AtSPO11-1 gene is disrupted. Both male and female meiocytes formed very few bivalents. Furthermore, no fully synapsed chromosomes were observed during prophase I. Later, in meiosis I, we observed that chromosomes segregated randomly, leading to the production of a large proportion of non-functional gametes. These meiotic aberrations were associated with a drastic reduction in meiotic recombination. Thus, our data show that initiation of meiotic recombination by SPO11- induced DSBs is a mechanism conserved in plants. Furthermore, unlike Drosophila and Caenorhabditis elegans, but like fungi, SPO11 is necessary for normal synapsis in plants.     

An archaebacterial topoisomerase homolog not present in other eukaryotes is indispensable for cell proliferation of plants

Plants, in contrast to other eukaryotes, possess not only homologs of subunit A (AtSPO11-1, 2, 3) but also of subunit B (AtTOP6B) of the archaebacterial topoisomerase VI. AtTOP6B and AtSPO11-3 are strongly expressed in somatic tissue of Arabidopsis and are able to interact with each other in vitro. A T-DNA insertion in AtTOP6B results in deficient cell proliferation; plants stop growing at the rosette stage, have small crinkled leaves, and die about 4 weeks after germination. Cultured root cells die after a limited number of cell divisions. The mitotic index of the root meristems is strongly reduced. Flow cytometric analysis demonstrates that endoreplication in mutant plants is stopped at the 8C stage; the last cycle is not completed in most cases. Mutant plants show a significant increase in nuclear DNA strand breaks. A T-DNA insertion mutant of AtSPO11-3 has a phenotype that is almost to that of AtTOP6B and the double mutant. Thus, both genes seem to act in vivo as subunits of a functional entity. A loss of this function most likely results in a defect in DNA replication, leading directly, or via the activation of a DNA damage checkpoint, to an arrest of cell division and endoreduplication. The dependence on an archaebacterial topoisomerase VI homolog distinguishes plants from the other eukaryotic kingdoms.     

Peculiarities of chromosome organization in meiosis

Meiotic and mitotic chromosomes have a complex of differences. (1) At the early prophase I of meiosis, chromosomes acquire protein axial elements (AEs) that were absent in mitosis; in addition to somatic cohesins, AEs contain the meiosis-specific cohesins REC8, SMC1β, and STAG3. (2) At the middle prophase I, protein lateral elements (LEs) of synaptonemal complexes (SCs) are formed on the basis of AEs. The LE proteins are not conserved, but in Saccharomyces cerevisiae and Arabidopsis thaliana they contain functional domains with conserved secondary structures. Among the almost 679 thousand proteins of primitive eukaryotes that we studied by bioinformatics methods, in green and brown algae, some lower fungi, and Coelenterata, we revealed proteins or functional domains similar to SC proteins. (3) During the pachytene and diplotene stages of meiosis, chromosomes of spermatocytes and mother pollen cells acquire a general structure resembling the structure of amphibian and avian lampbrush chromosomes in miniature. Lateral chromatin loops with sizes of 90, 160, and even over 480 Kb were observed in human spermatocytes during the diplotene stage. In combination, all these observations confirm the considerable conservation of the scheme of molecular and ultrastructural organization of meiotic chromosomes in a large variety of eukaryotic organisms.