The many facets of SC function during C. elegans meiosis

Sexually reproducing organisms rely on meiosis for the formation of haploid gametes. This is achieved through two consecutive rounds of cell division (meiosis I and II) after one round of DNA replication. During the meiotic divisions, chromosomes face several challenges to ultimately ensure proper chromosome segregation. Unique events unfold during meiosis I to overcome these challenges. Homologous chromosomes pair, synapse, and recombine. A remarkable feature throughout this process is the formation of an evolutionarily conserved tripartite proteinaceous structure known as the synaptonemal complex (SC). It is comprised of two lateral elements, assembled along each axis of a pair of homologous chromosomes, and a central region consisting of transverse filaments bridging the gap between lateral elements. While the presence of the SC during meiosis has been appreciated now for 50 years (Moses, Biophys Biochem Cytol 2:215–218, 1956; Fawcett, J Biophys Biochem Cytol 2:403–406, 1956), its role(s) remain a matter of intense investigation. This review concentrates on studies performed in Caenorhabditis elegans, a powerful system for investigating meiosis. Studies in this organism are contributing to the unraveling of the various processes leading to the formation of the SC and the various facets of the functions it exerts throughout meiosis.The synaptonemal complex-50 years     

The synaptonemal complex and meiotic recombination in humans: new approaches to old questions

Meiotic prophase serves as an arena for the interplay of two important cellular activities, meiotic recombination and synapsis of homologous chromosomes. Synapsis is mediated by the synaptonemal complex (SC), originally characterized as a structure linked to pairing of meiotic chromosomes (Moses (1958) J Biophys Biochem Cytol 4:633–638). In 1975, the first electron micrographs of human pachytene stage SCs were presented (Moses et al. (1975) Science 187:363–365) and over the next 15 years the importance of the SC to normal meiotic progression in human males and females was established (Jhanwar and Chaganti (1980) Hum Genet 54:405–408; Pathak and Elder (1980) Hum Genet 54:171–175; Solari (1980) Chromosoma 81:315–337; Speed (1984) Hum Genet 66:176–180; Wallace and Hulten (1985) Ann Hum Genet 49(Pt 3):215–226). Further, these studies made it clear that abnormalities in the assembly or maintenance of the SC were an important contributor to human infertility (Chaganti et al. (1980) Am J Hum Genet 32:833–848; Vidal et al. (1982) Hum Genet 60:301–304; Bojko (1983) Carlsberg Res Commun 48:285–305; Bojko (1985) Carlsberg Res Commun 50:43–72; Templado et al. (1984) Hum Genet 67:162–165; Navarro et al. (1986) Hum Reprod 1:523–527; Garcia et al. (1989) Hum Genet 2:147–53). However, the utility of these early studies was limited by lack of information on the structural composition of the SC and the identity of other SC-associated proteins. Fortunately, studies of the past 15 years have gone a long way toward remedying this problem. In this minireview, we highlight the most important of these advances as they pertain to human meiosis, focusing on temporal aspects of SC assembly, the relationship between the SC and meiotic recombination, and the contribution of SC abnormalities to human infertility.The synaptonemal complex–50 years     

The synaptonemal complex is assembled by a polySUMOylation-driven feedback mechanism in yeast

During meiotic prophase I, proteinaceous structures called synaptonemal complexes (SCs) connect homologous chromosomes along their lengths via polymeric arrays of transverse filaments (TFs). Thus, control of TF polymerization is central to SC formation. Using budding yeast, we show that efficiency of TF polymerization closely correlates with the extent of SUMO conjugation to Ecm11, a component of SCs. HyperSUMOylation of Ecm11 leads to highly aggregative TFs, causing frequent assembly of extrachromosomal structures. In contrast, hypoSUMOylation leads to discontinuous, fragmented SCs, indicative of defective TF polymerization. We further show that the N terminus of the yeast TF, Zip1, serves as an activator for Ecm11 SUMOylation. Coexpression of the Zip1 N terminus and Gmc2, a binding partner of Ecm11, is sufficient to induce robust polySUMOylation of Ecm11 in nonmeiotic cells. Because TF assembly is mediated through N-terminal head-to-head associations, our results suggest that mutual activation between TF assembly and Ecm11 polySUMOylation acts as a positive feedback loop that underpins SC assembly.     

Human Synaptonemal Complex Protein 1 (SCP1): Isolation and Characterization of the cDNA and Chromosomal Localization of the Gene

Synaptonemal complexes (SCs) are structures that are formed between homologous chromosomes (homologs) during meiotic prophase. They consist of two proteinaceous axes, one along each homolog, that are connected along their length by numerous transverse filaments (TFs). The cDNA encoding one major component of TFs of SCs of the rat, rnSCP1, has recently been isolated and characterized. In this paper we describe the isolation and characterization of the cDNA encoding the human protein homologous to rnSCP1, hsSCP1. hsSCP1 and rnSCP1 have 75% amino acid identity. The most prominent structural features and amino acid sequence motifs of rnSCP1 have been conserved in hsSCP1. Most probably, hsSCP1 is functionally homologous to rnSCP1. The hsSCP1 gene was assigned to human chromosome 1p12–p13 by fluorescencein situhybridization.     

Interplay between synaptonemal complex, homologous recombination, and centromeres during mammalian meiosis

The intimate synapsis of homologous chromosome pairs (homologs) by synaptonemal complexes (SCs) is an essential feature of meiosis. In many organisms, synapsis and homologous recombination are interdependent: recombination promotes SC formation and SCs are required for crossing-over. Moreover, several studies indicate that initiation of SC assembly occurs at sites where crossovers will subsequently form. However, recent analyses in budding yeast and fruit fly imply a special role for centromeres in the initiation of SC formation. In addition, in budding yeast, persistent SC–dependent centromere-association facilitates the disjunction of chromosomes that have failed to become connected by crossovers. Here, we examine the interplay between SCs, recombination, and centromeres in a mammal. In mouse spermatocytes, centromeres do not serve as SC initiation sites and are invariably the last regions to synapse. However, centromeres are refractory to de-synapsis during diplonema and remain associated by short SC fragments. Since SC–dependent centromere association is lost before diakinesis, a direct role in homolog segregation seems unlikely. However, post–SC disassembly, we find evidence of inter-centromeric connections that could play a more direct role in promoting homolog biorientation and disjunction. A second class of persistent SC fragments is shown to be crossover-dependent. Super-resolution structured-illumination microscopy (SIM) reveals that these structures initially connect separate homolog axes and progressively diminish as chiasmata form. Thus, DNA crossing-over (which occurs during pachynema) and axis remodeling appear to be temporally distinct aspects of chiasma formation. SIM analysis of the synapsis and crossover-defective mutant Sycp1^−/− implies that SCs prevent unregulated fusion of homolog axes. We propose that SC fragments retained during diplonema stabilize nascent bivalents and help orchestrate local chromosome reorganization that promotes centromere and chiasma function.     

Protein SYCP2 provides a link between transverse filaments and lateral elements of mammalian synaptonemal complexes

Synaptonemal complexes (SCs) are evolutionarily conserved meiosis-specific nuclear structures critically involved in synapsis, recombination, and segregation of homologous chromosomes. SCs are proteinaceous structures composed of (a) two lateral elements (LEs), to which the chromatin of the homologs is attached, (b) numerous transverse filaments (TFs) that link the LEs, and (c) a central element (CE). Major protein components of mammalian SCs are the TF protein SYCP1 and the LE proteins SYCP2 and SCYP3. How SCs become assembled is presently poorly understood, in particular, it is not known how TFs assemble at the plane of LEs to interconnect the homologous chromosomes. Therefore, we have investigated possible interactions between SYCP1 and other SC proteins. In immunoprecipitation experiments we could find that SYCP1 and SYCP2 interact in extracts of meiotic cells. Using the yeast two-hybrid system, we were able to demonstrate that the C-terminus of SYCP1 directly interacts with SYCP2. These results were confirmed by different interaction traps. Furthermore, we could narrow down the interacting domain of the SYCP2 molecule to its C-terminal region. We propose that SYCP2 acts as a linker between SYCP1 and SYCP3 and therefore would be the missing connecting link between LEs and TFs essential for proper chromosome synapsis.     

The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis,Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.     

The CSN/COP9 Signalosome Regulates Synaptonemal Complex Assembly during Meiotic Prophase I of Caenorhabditis elegans

The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism C. elegans. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in csn mutants that specifically eliminates nuclei with aggregated SC proteins. csn mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.     

The Central Element Protein ZEP1 of the Synaptonemal Complex Regulates the Number of Crossovers during Meiosis in Rice

ZEP1, a transverse filament (TF) protein, is the rice (Oryza sativa) homolog of Arabidopsis thaliana ZYP1. In the Tos17-insertional zep1 mutants, homologous chromosomes align along the entire length of the chromosome, but the synaptonemal complex is not assembled in early prophase I. Crossovers are well formed, and 12 bivalents could be detected from diakinesis to metaphase I, which leads to equal chromosomal segregation in anaphase I. Moreover, the number of crossovers has a tendency to be increased compared with that in the wild type. These phenomena are different from the TF mutants identified so far in other organisms. Chiasma terminalization of the bivalent, which occurs frequently in the wild type, seldom occurred in zep1. Transmission electron micrographs and immunodetection using an antibody against ZEP1 showed that ZEP1 is the central element of the synaptonemal complex. Although PAIR2 and MER3 were loaded normally in zep1, their dissociation was delayed severely compared with the wild type. In addition, ZEP1 is reloaded onto chromosomes in early microspores as the chromosome decondense, suggesting that ZEP1 might have other biological functions during this process.     

Rapid telomere movement in meiotic prophase is promoted by NDJ1, MPS3, and CSM4 and is modulated by recombination

Haploidization of the genome in meiosis requires that chromosomes be sorted exclusively into pairs stabilized by synaptonemal complexes (SCs) and crossovers. This sorting and pairing is accompanied by active chromosome positioning in meiotic prophase in which telomeres cluster near the spindle pole to form the bouquet before dispersing around the nuclear envelope. We now describe telomere-led rapid prophase movements (RPMs) that frequently exceed 1 microm/s and persist throughout meiotic prophase. Bouquet formation and RPMs depend on NDJ1, MPS3, and a new member of this pathway, CSM4, which encodes a meiosis-specific nuclear envelope protein required specifically for telomere mobility. RPMs initiate independently of recombination but differ quantitatively in mutants that fail to complete recombination, suggesting that RPMs respond to recombination status. Together with recombination defects described for ndj1, our observations suggest that RPMs and SCs balance the disruption and stabilization of recombinational interactions, respectively, to regulate crossing over.     

Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1''s strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51''s strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1''s chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1''s dominance in promoting strand exchange between homologs involves repression of Rad51''s strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.     

The Synaptonemal complex component C(2)M regulates meiotic crossing over in Drosophila

BACKGROUND: The synaptonemal complex (SC) is a proteinaceous structure that forms between homologously paired meiotic chromosomes. Previous studies have suggested that the SC is required for meiotic crossing over in Drosophila. However, only one component of this structure, C(3)G, has been identified in Drosophila. RESULTS: Mutations in c(2)M cause a reduced frequency of meiotic crossing over due, in part, to how recombination events are resolved. Cytological evidence suggests that C(2)M is a component of the SC and is required for the assembly of C(3)G (a putative transverse filament of the SC) along the chromosomes. Additionally, C(2)M localizes along the chromosomes in the absence of C(3)G. Despite having a defect in C(3)G localization, c(2)M mutants unexpectedly affect crossing over less severely than a c(3)G mutant. There is virtually no crossing over in a c(3)G mutant, but c(2)M or c(2)M; c(3)G double mutants produce a substantial number of crossovers. The appearance of C(3)G-independent crossovers in c(2)M mutants suggests that C(2)M prevents recombination in the absence of complete SC formation. CONCLUSIONS: We have identified a new Drosophila SC component, C(2)M, that promotes the formation of crossovers. Furthermore, the appearance of C(3)G-independent crossovers in c(2)M mutants suggests a novel role in preventing recombination in the absence of complete SC.     

Sex-specific crossover patterns in Zebrafish (Danio rerio)

Some species display intersex variation in their rate of meiotic recombination, where recombination is usually suppressed in the heterogametic sex. Although no heteromorphic sex chromosomes have been detected in zebrafish (Danio rerio), genetic analysis has indicated a lower frequency of recombination in males relative to females. Our study of the meiotic recombination pattern in female zebrafish indicates that adult females have only a few meiotic oocytes that are found in groups in the ventral zone of the ovarian surface. We used antibody staining of human mutL homolog 1 (MLH1) protein to mark the sites of putative chiasmata to seek a physical basis for the pattern of recombination and its relative frequency in both sexes. We report that MLH1 foci are found mostly in distal regions of the synaptonemal complexes (SCs) in males, but tend to be more evenly distributed in females. Our cytological analysis yields a ratio of MLH1 foci per chromosome in males versus females of 1:1.55. This lower level of recombination in males is in general agreement with previously published results from linkage map analysis. However, the similar ratio of MLH1 foci per unit length of SCs in both sexes demonstrates a correlation between SC length and the frequency of recombination rather than a mechanism that suppresses recombination in males. Thus, chiasma interference seems to provide similar expression in males and females in agreement with the situation in humans, where oocytes with longer SCs display a higher level of recombination that is not a consequence of more closely spaced crossovers along the SCs.     

Crossing over is coupled to late meiotic prophase bivalent differentiation through asymmetric disassembly of the SC

Homologous chromosome pairs (bivalents) undergo restructuring during meiotic prophase to convert a configuration that promotes crossover recombination into one that promotes bipolar spindle attachment and localized cohesion loss. We have imaged remodeling of meiotic chromosome structures after pachytene exit in Caenorhabditis elegans. Chromosome shortening during diplonema is accompanied by coiling of chromosome axes and highly asymmetric departure of synaptonemal complex (SC) central region proteins SYP-1 and SYP-2, which diminish over most of the length of each desynapsing bivalent while becoming concentrated on axis segments distal to the single emerging chiasma. This and other manifestations of asymmetry along chromosomes are lost in synapsis-proficient crossover-defective mutants, which often retain SYP-1,2 along the full lengths of coiled diplotene axes. Moreover, a gamma-irradiation treatment that restores crossovers in the spo-11 mutant also restores asymmetry of SYP-1 localization. We propose that crossovers or crossover precursors serve as symmetry-breaking events that promote differentiation of subregions of the bivalent by triggering asymmetric disassembly of the SC.     

Inhibition of the Smc5/6 Complex during Meiosis Perturbs Joint Molecule Formation and Resolution without Significantly Changing Crossover or Non-crossover Levels

Meiosis is a specialized cell division used by diploid organisms to form haploid gametes for sexual reproduction. Central to this reductive division is repair of endogenous DNA double-strand breaks (DSBs) induced by the meiosis-specific enzyme Spo11. These DSBs are repaired in a process called homologous recombination using the sister chromatid or the homologous chromosome as a repair template, with the homolog being the preferred substrate during meiosis. Specific products of inter-homolog recombination, called crossovers, are essential for proper homolog segregation at the first meiotic nuclear division in budding yeast and mice. This study identifies an essential role for the conserved Structural Maintenance of Chromosomes (SMC) 5/6 protein complex during meiotic recombination in budding yeast. Meiosis-specific smc5/6 mutants experience a block in DNA segregation without hindering meiotic progression. Establishment and removal of meiotic sister chromatid cohesin are independent of functional Smc6 protein. smc6 mutants also have normal levels of DSB formation and repair. Eliminating DSBs rescues the segregation block in smc5/6 mutants, suggesting that the complex has a function during meiotic recombination. Accordingly, smc6 mutants accumulate high levels of recombination intermediates in the form of joint molecules. Many of these joint molecules are formed between sister chromatids, which is not normally observed in wild-type cells. The normal formation of crossovers in smc6 mutants supports the notion that mainly inter-sister joint molecule resolution is impaired. In addition, return-to-function studies indicate that the Smc5/6 complex performs its most important functions during joint molecule resolution without influencing crossover formation. These results suggest that the Smc5/6 complex aids primarily in the resolution of joint molecules formed outside of canonical inter-homolog pathways.     

Meiotic Transverse Filament Proteins: Essential for Crossing Over

Meiosis is a specialized set of two nuclear divisions, meiosis I and II, by which a diploid cell produces four haploid daughters. After premeiotic DNA replication, homologous chromosomes pair and recombine, and then disjoin at meiosis I. Subsequently, at meiosis II, the sister chromatids of each chromosome segregate. In nearly all eukaryotes, meiotic chromosome pairing culminates in the formation of a ladderlike supramolecular protein structure, the synaptonemal complex (SC) (Page and Hawley, 2004). The rungs of the ladder are known as transverse filaments (TFs). Genes encoding TF proteins have been identified in a limited number of organisms, and their function has been studied by mutational analysis. Although TF proteins show little amino acid sequence conservation, their structure and function are largely conserved. In all analyzed species, TF proteins are required for meiotic reciprocal recombination (crossing over).     

Synaptonemal complex assembly and H3K4Me3 demethylation determine DIDO3 localization in meiosis

Synapsis of homologous chromosomes is a key meiotic event, mediated by a large proteinaceous structure termed the synaptonemal complex. Here, we describe a role in meiosis for the murine death-inducer obliterator (Dido) gene. The Dido gene codes for three proteins that recognize trimethylated histone H3 lysine 4 through their amino-terminal plant homeodomain domain. DIDO3, the largest of the three isoforms, localizes to the central region of the synaptonemal complex in germ cells. DIDO3 follows the distribution of the central region protein SYCP1 in Sycp3−/− spermatocytes, which lack the axial elements of the synaptonemal complex. This indicates that synapsis is a requirement for DIDO3 incorporation. Interestingly, DIDO3 is missing from the synaptonemal complex in Atm mutant spermatocytes, which form synapses but show persistent trimethylation of histone H3 lysine 4. In order to further address a role of epigenetic modifications in DIDO3 localization, we made a mutant of the Dido gene that produces a truncated DIDO3 protein. This truncated protein, which lacks the histone-binding domain, is incorporated in the synaptonemal complex irrespective of histone trimethylation status. DIDO3 protein truncation in Dido mutant mice causes mild meiotic defects, visible as gaps in the synaptonemal complex, but allows for normal meiotic progression. Our results indicate that histone H3 lysine 4 demethylation modulates DIDO3 localization in meiosis and suggest epigenetic regulation of the synaptonemal complex.     

A component of C. elegans meiotic chromosome axes at the interface of homolog alignment, synapsis, nuclear reorganization, and recombination

A universal feature of meiotic prophase is the pairing of homologous chromosomes, a fundamental prerequisite for the successful completion of all subsequent meiotic events. HIM-3 is a Caenorhabditis elegans meiosis-specific non-cohesin component of chromosome axes that is required for synapsis. Our characterization of new him-3 alleles reveals previously unknown functions for the protein. HIM-3 is required for the establishment of initial contacts between homologs, for the nuclear reorganization characteristic of early meiotic prophase, and for the coordination of these events with synaptonemal complex (SC) assembly. Despite the absence of homolog alignment, we find that recombination is initiated efficiently, indicating that initial pairing is not a prerequisite for early steps of the recombination pathway. Surprisingly, RAD-51-marked recombination intermediates disappear with apparent wild-type kinetics in him-3 null mutants in which homologs are spatially unavailable for recombination, raising the possibility that HIM-3's presence at chromosome axes inhibits the use of sister chromatids as templates for repair. We propose that HIM-3 is a molecular link between multiple landmark events of meiotic prophase; it is critical for establishing chromosome identity by configuring homologs to facilitate their recognition while simultaneously imposing structural constraints that later promote the formation of the crossover essential for proper segregation.     

Msh4 and Msh5 Function in SC-Independent Chiasma Formation During the Streamlined Meiosis of Tetrahymena

ZMM proteins have been defined in budding yeast as factors that are collectively involved in the formation of interfering crossovers (COs) and synaptonemal complexes (SCs), and they are a hallmark of the predominant meiotic recombination pathway of most organisms. In addition to this so-called class I CO pathway, a minority of crossovers are formed by a class II pathway, which involves the Mus81-Mms4 endonuclease complex. This is the only CO pathway in the SC-less meiosis of the fission yeast. ZMM proteins (including SC components) were always found to be co-occurring and hence have been regarded as functionally linked. Like the fission yeast, the protist Tetrahymena thermophila does not possess a SC, and its COs are dependent on Mus81-Mms4. Here we show that the ZMM proteins Msh4 and Msh5 are required for normal chiasma formation, and we propose that they have a pro-CO function outside a canonical class I pathway in Tetrahymena. Thus, the two-pathway model is not tenable as a general rule.