Three Distinct Modes of Mec1/ATR and Tel1/ATM Activation Illustrate Differential Checkpoint Targeting during Budding Yeast Early Meiosis

Recombination and synapsis of homologous chromosomes are hallmarks of meiosis in many organisms. Meiotic recombination is initiated by Spo11-induced DNA double-strand breaks (DSBs), whereas chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). Previously, we proposed that budding yeast SC is assembled via noncovalent interactions between the axial SC protein Red1, SUMO chains or conjugates, and the central SC protein Zip1. Incomplete synapsis and unrepaired DNA are monitored by Mec1/Tel1-dependent checkpoint responses that prevent exit from the pachytene stage. Here, our results distinguished three distinct modes of Mec1/Tec1 activation during early meiosis that led to phosphorylation of three targets, histone H2A at S129 (γH2A), Hop1, and Zip1, which are involved, respectively, in DNA replication, the interhomolog recombination and chromosome synapsis checkpoint, and destabilization of homology-independent centromere pairing. γH2A phosphorylation is Red1 independent and occurs prior to Spo11-induced DSBs. DSB- and Red1-dependent Hop1 phosphorylation is activated via interaction of the Red1-SUMO chain/conjugate ensemble with the Ddc1-Rad17-Mec3 (9-1-1) checkpoint complex and the Mre11-Rad50-Xrs2 complex. During SC assembly, Zip1 outcompetes 9-1-1 from the Red1-SUMO chain ensemble to attenuate Hop1 phosphorylation. In contrast, chromosome synapsis cannot attenuate DSB-dependent and Red1-independent Zip1 phosphorylation. These results reveal how DNA replication, DSB repair, and chromosome synapsis are differentially monitored by the meiotic checkpoint network.     

Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis

Recombination between homologous chromosomes of different parental origin (homologs) is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs]) show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs) that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in the rate of inter-sister repair, combined with the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible.     

Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis

Yeast mutants lacking meiotic proteins Zip1, Zip2, Zip3, Mer3, and/or Msh5 (ZMMs) were analyzed for recombination, synaptonemal complex (SC), and meiotic progression. At 33 degrees C, recombination-initiating double-strand breaks (DSBs) and noncrossover products (NCRs) form normally while formation of single-end invasion strand exchange intermediates (SEIs), double Holliday junctions, crossover products (CRs), and SC are coordinately defective. Thus, during wild-type meiosis, recombinational interactions are differentiated into CR and NCR types very early, prior to onset of stable strand exchange and independent of SC. By implication, crossover interference does not require SC formation. We suggest that SC formation may require interference. Subsequently, CR-designated DSBs undergo a tightly coupled, ZMM-promoted transition that yields SEI-containing recombination complexes embedded in patches of SC. zmm mutant phenotypes differ strikingly at 33 degrees C and 23 degrees C, implicating higher temperature as a positive effector of recombination and identifying a checkpoint that monitors local CR-specific events, not SC formation, at late leptotene.     

The cohesion protein ORD is required for homologue bias during meiotic recombination

During meiosis, sister chromatid cohesion is required for normal levels of homologous recombination, although how cohesion regulates exchange is not understood. Null mutations in orientation disruptor (ord) ablate arm and centromeric cohesion during Drosophila meiosis and severely reduce homologous crossovers in mutant oocytes. We show that ORD protein localizes along oocyte chromosomes during the stages in which recombination occurs. Although synaptonemal complex (SC) components initially associate with synapsed homologues in ord mutants, their localization is severely disrupted during pachytene progression, and normal tripartite SC is not visible by electron microscopy. In ord germaria, meiotic double strand breaks appear and disappear with frequency and timing indistinguishable from wild type. However, Ring chromosome recovery is dramatically reduced in ord oocytes compared with wild type, which is consistent with the model that defects in meiotic cohesion remove the constraints that normally limit recombination between sisters. We conclude that ORD activity suppresses sister chromatid exchange and stimulates inter-homologue crossovers, thereby promoting homologue bias during meiotic recombination in Drosophila.     

Meiotic chromosome synapsis in yeast can occur without spo11-induced DNA double-strand breaks

Proper chromosome segregation and formation of viable gametes depend on synapsis and recombination between homologous chromosomes during meiosis. Previous reports have shown that the synaptic structures, the synaptonemal complexes (SCs), do not occur in yeast cells with the SPO11 gene removed. The Spo11 enzyme makes double-strand breaks (DSBs) in the DNA and thereby initiates recombination. The view has thus developed that synapsis in yeast strictly depends on the initiation of recombination. Synapsis in some other species (Drosophila melanogaster and Caenorhabditis elegans) is independent of recombination events, and SCs are found in spo11 mutants. This difference between species led us to reexamine spo11 deletion mutants of yeast. Using antibodies against Zip1, a SC component, we found that a small fraction (1%) of the spo11 null mutant cells can indeed form wild-type-like SCs. We further looked for synapsis in a spo11 mutant strain that accumulates pachytene cells (spo11Delta ndt80Delta), and found that the frequency of cells with apparently complete SC formation was 10%. Other phenotypic criteria, such as spore viability and homologous chromosome juxtaposition measured by FISH labeling of chromosomal markers, agree with several previous reports of the spo11 mutant. Our results demonstrate that although the Spo11-induced DSBs obviously promote synapsis in yeast, the presence of Spo11 is not an absolute requirement for synapsis.     

Synaptonemal complex-dependent centromeric clustering and the initiation of synapsis in Drosophila oocytes

The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.     

Two distinct surveillance mechanisms monitor meiotic chromosome metabolism in budding yeast

Meiotic recombination is initiated by Spo11-generated DNA double-strand breaks (DSBs) . A fraction of total DSBs is processed into crossovers (CRs) between homologous chromosomes, which promote their accurate segregation at meiosis I (MI) . The coordination of recombination-associated events and MI progression is governed by the "pachytene checkpoint", which in budding yeast requires Rad17, a component of a PCNA clamp-like complex, and Pch2, a putative AAA-ATPase . We show that two genetically separable pathways monitor the presence of distinct meiotic recombination-associated lesions: First, delayed MI progression in the presence of DNA repair intermediates is suppressed when RAD17 or SAE2, encoding a DSB-end processing factor , is deleted. Second, delayed MI progression in the presence of aberrant synaptonemal complex (SC) is suppressed when PCH2 is deleted. Importantly, ZIP1, encoding the central element of the SC , is required for PCH2-dependent checkpoint activation. Analysis of the rad17Deltapch2Delta double mutant revealed a redundant function regulating interhomolog CR formation. These findings suggest a link between the surveillance of distinct recombination-associated lesions, control of CR formation kinetics, and regulation of MI timing. A PCH2-ZIP1-dependent checkpoint in meiosis is likely conserved among synaptic organisms from yeast to human .     

Meiotic checkpoints and the interchromosomal effect on crossing over in Drosophila females

During prophase of meiosis I, genetic recombination is initiated with a Spo11-dependent DNA double-strand break (DSB). Repair of these DSBs can generate crossovers, which become chiasmata and are important for the process of chromosome segregation. To ensure at least one chiasma per homologous pair of chromosomes, the number and distribution of crossovers is regulated. One system contributing to the distribution of crossovers is the pachytene checkpoint, which requires the conserved gene pch2 that encodes an AAA+ATPase family member. Pch2-dependent pachytene checkpoint function causes delays in pachytene progression when there are defects in processes required for crossover formation, such as mutations in DSB-repair genes and when there are defects in the structure of the meiotic chromosome axis. Thus, the pachytene checkpoint appears to monitor events leading up to the generation of crossovers. Interestingly, heterozygous chromosome rearrangements cause Pch2-dependent pachytene delays and as little as two breaks in the continuity of the paired chromosome axes are sufficient to evoke checkpoint activity. These chromosome rearrangements also cause an interchromosomal effect on recombination whereby crossing over is suppressed between the affected chromosomes but is increased between the normal chromosome pairs. We have shown that this phenomenon is also due to pachytene checkpoint activity.     

Yeast axial-element protein,Red1, binds SUMO chains to promote meiotic interhomologue recombination and chromosome synapsis

The synaptonemal complex (SC) is a tripartite protein structure consisting of two parallel axial elements (AEs) and a central region. During meiosis, the SC connects paired homologous chromosomes, promoting interhomologue (IH) recombination. Here, we report that, like the CE component Zip1, Saccharomyces cerevisiae axial-element structural protein, Red1, can bind small ubiquitin-like modifier (SUMO) polymeric chains. The Red1–SUMO chain interaction is dispensable for the initiation of meiotic DNA recombination, but it is essential for Tel1- and Mec1-dependent Hop1 phosphorylation, which ensures IH recombination by preventing the inter-sister chromatid DNA repair pathway. Our results also indicate that Red1 and Zip1 may directly sandwich the SUMO chains to mediate SC assembly. We suggest that Red1 and SUMO chains function together to couple homologous recombination and Mec1–Tel1 kinase activation with chromosome synapsis during yeast meiosis.     

Meiotic checkpoints and the interchromosomal effect on crossing over in Drosophila females

During prophase of meiosis I, genetic recombination is initiated with a Spo11-dependent DNA double-strand break (DSB). Repair of these DSBs can generate crossovers, which become chiasmata and are important for the process of chromosome segregation. To ensure at least one chiasma per homologous pair of chromosomes, the number and distribution of crossovers is regulated. One system contributing to the distribution of crossovers is the pachytene checkpoint, which requires the conserved gene pch2 that encodes an AAA+ATPase family member. Pch2-dependent pachytene checkpoint function causes delays in pachytene progression when there are defects in processes required for crossover formation, such as mutations in DSB-repair genes and when there are defects in the structure of the meiotic chromosome axis. Thus, the pachytene checkpoint appears to monitor events leading up to the generation of crossovers. Interestingly, heterozygous chromosome rearrangements cause Pch2-dependent pachytene delays and as little as two breaks in the continuity of the paired chromosome axes are sufficient to evoke checkpoint activity. These chromosome rearrangements also cause an interchromosomal effect on recombination whereby crossing over is suppressed between the affected chromosomes but is increased between the normal chromosome pairs. We have shown that this phenomenon is also due to pachytene checkpoint activity.     

Dot1-Dependent Histone H3K79 Methylation Promotes the Formation of Meiotic Double-Strand Breaks in the Absence of Histone H3K4 Methylation in Budding Yeast

Epigenetic marks such as histone modifications play roles in various chromosome dynamics in mitosis and meiosis. Methylation of histones H3 at positions K4 and K79 is involved in the initiation of recombination and the recombination checkpoint, respectively, during meiosis in the budding yeast. Set1 promotes H3K4 methylation while Dot1 promotes H3K79 methylation. In this study, we carried out detailed analyses of meiosis in mutants of the SET1 and DOT1 genes as well as methylation-defective mutants of histone H3. We confirmed the role of Set1-dependent H3K4 methylation in the formation of double-strand breaks (DSBs) in meiosis for the initiation of meiotic recombination, and we showed the involvement of Dot1 (H3K79 methylation) in DSB formation in the absence of Set1-dependent H3K4 methylation. In addition, we showed that the histone H3K4 methylation-defective mutants are defective in SC elongation, although they seem to have moderate reduction of DSBs. This suggests that high levels of DSBs mediated by histone H3K4 methylation promote SC elongation.     

Involvement of Sir2/4 in silencing of DNA breakage and recombination on mouse YACs during yeast meiosis

Yeast artificial chromosomes (YACs) that contain human DNA backbone undergo DNA double-strand breaks (DSBs) and recombination during yeast meiosis at rates similar to the yeast native chromosomes. Surprisingly, YACs containing DNA covering a recombination hot spot in the mouse major histocompatibility complex class III region do not show meiotic DSBs and undergo meiotic recombination at reduced levels. Moreover, segregation of these YACs during meiosis is seriously compromised. In meiotic yeast cells carrying the mutations sir2 or sir4, but not sir3, these YACs show DSBs, suggesting that a unique chromatin structure of the YACs, involving Sir2 and Sir4, protects the YACs from the meiotic recombination machinery. We speculate that the paucity of DSBs and recombination events on these YACs during yeast meiosis may reflect the refractory nature of the corresponding region in the mouse genome.     

Characterisation of a new reporter system allowing high throughput in planta screening for recombination events before and after controlled DNA double strand break induction

DNA double strand breaks (DSBs) are created either by DNA damaging reagents or in a programmed manner, for example during meiosis. Homologous recombination (HR) can be used to repair DSBs, a process vital both for cell survival and for genetic rearrangement during meiosis. In order to easily quantify this mechanism, a new HR reporter gene that is suitable for the detection of rare recombination events in high-throughput screens was developed in Arabidopsis thaliana. This reporter, pPNP, is composed of two mutated Pat genes and has also one restriction site for the meganuclease I-SceI. A functional Pat gene can be reconstituted by an HR event giving plants which are resistant to the herbicide glufosinate. The basal frequency of intra-chromosomal recombination is very low (10^?5) and can be strongly increased by the expression of I-SceI which creates a DSB. Expression of I-SceI under the control of the 35S CaMV promoter dramatically increases HR frequency (10,000 fold); however the measured recombinant events are in majority somatic. In contrast only germinal recombination events were measured when the meganuclease was expressed from a floral-specific promoter. Finally, the reporter was used to test a dexamethasone inducible I-SceI which could produce up to 200× more HR events after induction. This novel inducible I-SceI should be useful in fundamental studies of the mechanism of repair of DSBs and for biotechnological applications.     

Drosophila PCH2 is required for a pachytene checkpoint that monitors double-strand-break-independent events leading to meiotic crossover formation

During meiosis, programmed DNA double-strand breaks (DSBs) are repaired to create at least one crossover per chromosome arm. Crossovers mature into chiasmata, which hold and orient the homologous chromosomes on the meiotic spindle to ensure proper segregation at meiosis I. This process is usually monitored by one or more checkpoints that ensure that DSBs are repaired prior to the meiotic divisions. We show here that mutations in Drosophila genes required to process DSBs into crossovers delay two important steps in meiotic progression: a chromatin-remodeling process associated with DSB formation and the final steps of oocyte selection. Consistent with the hypothesis that a checkpoint has been activated, the delays in meiotic progression are suppressed by a mutation in the Drosophila homolog of pch2. The PCH2-dependent delays also require proteins thought to regulate the number and distribution of crossovers, suggesting that this checkpoint monitors events leading to crossover formation. Surprisingly, two lines of evidence suggest that the PCH2-dependent checkpoint does not reflect the accumulation of unprocessed recombination intermediates: the delays in meiotic progression do not depend on DSB formation or on mei-41, the Drosophila ATR homolog, which is required for the checkpoint response to unrepaired DSBs. We propose that the sites and/or conditions required to promote crossovers are established independently of DSB formation early in meiotic prophase. Furthermore, the PCH2-dependent checkpoint is activated by these events and pachytene progression is delayed until the DSB repair complexes required to generate crossovers are assembled. Interestingly, PCH2-dependent delays in prophase may allow additional crossovers to form.     

A pathway for synapsis initiation during zygotene in Drosophila oocytes

Formation of the synaptonemal complex (SC), or synapsis, between homologs in meiosis is essential for crossing over and chromosome segregation [1-4]. How SC assembly initiates is poorly understood but may have a critical role in ensuring synapsis between homologs and regulating double-strand break (DSB) and crossover formation. We investigated the genetic requirements for synapsis in Drosophila and found that there are three temporally and genetically distinct stages of synapsis initiation. In "early zygotene" oocytes, synapsis is only observed at the centromeres. We also found that nonhomologous centromeres are clustered during this process. In "mid-zygotene" oocytes, SC initiates at several euchromatic sites. The centromeric and first euchromatic SC initiation sites depend on the cohesion protein ORD. In "late zygotene" oocytes, SC initiates at many more sites that depend on the Kleisin-like protein C(2)M. Surprisingly, late zygotene synapsis initiation events are independent of the earlier mid-zygotene events, whereas both mid and late synapsis initiation events depend on the cohesin subunits SMC1 and SMC3. We propose that the enrichment of cohesion proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly independent of DSBs. Furthermore, the early euchromatic SC initiation events at mid-zygotene may be required for DSBs to be repaired as crossovers.     

A novel recombination pathway initiated by the Mre11/Rad50/Nbs1 complex eliminates palindromes during meiosis in Schizosaccharomyces pombe

DNA palindromes are rare in humans but are associated with meiosis-specific translocations. The conserved Mre11/Rad50/Nbs1 (MRN) complex is likely directly involved in processing palindromes through the homologous recombination pathway of DNA repair. Using the fission yeast Schizosaccharomyces pombe as a model system, we show that a 160-bp palindrome (M-pal) is a meiotic recombination hotspot and is preferentially eliminated by gene conversion. Importantly, this hotspot depends on the MRN complex for full activity and reveals a new pathway for generating meiotic DNA double-strand breaks (DSBs), separately from the Rec12 (ortholog of Spo11) pathway. We show that MRN-dependent DSBs are formed at or near the M-pal in vivo, and in contrast to the Rec12-dependent breaks, they appear early, during premeiotic replication. Analysis of mrn mutants indicates that the early DSBs are generated by the MRN nuclease activity, demonstrating the previously hypothesized MRN-dependent breakage of hairpins during replication. Our studies provide a genetic and physical basis for frequent translocations between palindromes in human meiosis and identify a conserved meiotic process that constantly selects against palindromes in eukaryotic genomes.     

Pulsed-field gel analysis of the pattern of DNA double-strand breaks in the Saccharomyces genome during meiosis.

Pulsed-field gel electrophoresis (PFGE) has been used to study the timing, frequency, and distribution of double-strand breaks (DSBs) in chromosomal-sized DNA during meiosis in yeast. It has previously been shown that DSBs are associated with some genetic hotspots during recombination, and it is important to know whether meiotic recombination events routinely initiate via DSBs. Two strains have been studied here--a high-sporulating homothallic wild type and a congenic mutant strain carrying a rad50S mutation. This mutant has previously been reported to accumulate broken molecules in meiosis to much higher frequencies than wild type and to abolish the characteristic wild-type processing of DNA that has been observed at the break sites. When whole chromosomes are resolved by PFGE, both strains show some broken molecules starting at the time that cells commit to genetic recombination. Breakage has been assessed primarily on Chromosome III and Chr. XV, using Southern hybridization to identify these chromosomes and their fragments. At any one time, break frequency in wild type is much lower than the cumulative frequency of recombination events that occur during meiosis. However, there is suggestive evidence that each break is short-lived, and it is therefore difficult to estimate the total number of breaks that may occur. In rad50S, chromosome breaks accumulate to much higher levels, which are probably broadly consistent with the estimated number of recombination events in wild type. However, since rad50S is substantially defective in completing recombination, it is not known for certain if it initiates events at wild-type frequencies. A surprising feature of the data is that a strong banding pattern is observed in the fragment distribution from broken chromosomes in both strains, implying that at least much of the breakage occurs at specific sites or within short regions. However, with the exception of the rDNA region on Chr. XII, assessment of the genetic map indicates that recombination can occur almost anywhere in the genome, although some regions are much hotter than others. Possible reasons for this apparent paradox are discussed. It may in part result from breakage levels too low for adequate detection in cold regions but may also imply that recombination events are localized more than previously realized. Alternatively, there may be a more indirect relationship between break sites and the associated recombination events.     

ATM controls meiotic DNA double-strand break formation and recombination and affects synaptonemal complex organization in plants

Meiosis is a specialized cell division that gives rise to genetically distinct gametic cells. Meiosis relies on the tightly controlled formation of DNA double-strand breaks (DSBs) and their repair via homologous recombination for correct chromosome segregation. Like all forms of DNA damage, meiotic DSBs are potentially harmful and their formation activates an elaborate response to inhibit excessive DNA break formation and ensure successful repair. Previous studies established the protein kinase ATM as a DSB sensor and meiotic regulator in several organisms. Here we show that Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover (CO) formation and synaptonemal complex (SC) organization, all vital for the successful completion of meiosis. We developed a single-molecule approach to quantify meiotic breaks and determined that ATM is essential to limit the number of meiotic DSBs. Local and genome-wide recombination screens showed that ATM restricts the number of interference-insensitive COs, while super-resolution STED nanoscopy of meiotic chromosomes revealed that the kinase affects chromatin loop size and SC length and width. Our study extends our understanding of how ATM functions during plant meiosis and establishes it as an integral factor of the meiotic program.

Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover formation and synaptonemal complex organization, all vital for the successful completion of meiosis.     

An essential role of DmRad51/SpnA in DNA repair and meiotic checkpoint control

Rad51 is a conserved protein essential for recombinational repair of double-stranded DNA breaks (DSBs) in somatic cells and during meiosis in germ cells. Yeast Rad51 mutants are viable but show meiosis defects. In the mouse, RAD51 deletions cause early embryonic death, suggesting that in higher eukaryotes Rad51 is required for viability. Here we report the identification of SpnA as the Drosophila Rad51 gene, whose sequence among the five known Drosophila Rad51-like genes is most closely related to the Rad51 homologs of human and yeast. DmRad51/spnA null mutants are viable but oogenesis is disrupted by the activation of a meiotic recombination checkpoint. We show that the meiotic phenotypes result from an inability to effectively repair DSBs. Our study further demonstrates that in Drosophila the Rad51-dependent homologous recombination pathway is not essential for DNA repair in the soma, unless exposed to DNA damaging agents. We therefore propose that under normal conditions a second, Rad51-independent, repair pathway prevents the lethal effects of DNA damage.     

A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.