Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern.

During meiosis, chromosomes undergo a homology search in order to locate their homolog to form stable pairs and exchange genetic material. Early in prophase, chromosomes associate in mostly non-homologous pairs, tethered only at their centromeres. This phenomenon, conserved through higher eukaryotes, is termed centromere coupling in budding yeast. Both initiation of recombination and the presence of homologs are dispensable for centromere coupling (occurring in spo11 mutants and haploids induced to undergo meiosis) but the presence of the synaptonemal complex (SC) protein Zip1 is required. The nature and mechanism of coupling have yet to be elucidated. Here we present the first pairwise analysis of centromere coupling in an effort to uncover underlying rules that may exist within these non-homologous interactions. We designed a novel chromosome conformation capture (3C)-based assay to detect all possible interactions between non-homologous yeast centromeres during early meiosis. Using this variant of 3C-qPCR, we found a size-dependent interaction pattern, in which chromosomes assort preferentially with chromosomes of similar sizes, in haploid and diploid spo11 cells, but not in a coupling-defective mutant (spo11 zip1 haploid and diploid yeast). This pattern is also observed in wild-type diploids early in meiosis but disappears as meiosis progresses and homologous chromosomes pair. We found no evidence to support the notion that ancestral centromere homology plays a role in pattern establishment in S. cerevisiae post-genome duplication. Moreover, we found a role for the meiotic bouquet in establishing the size dependence of centromere coupling, as abolishing bouquet (using the bouquet-defective spo11 ndj1 mutant) reduces it. Coupling in spo11 ndj1 rather follows telomere clustering preferences. We propose that a chromosome size preference for centromere coupling helps establish efficient homolog recognition.

Centromere-like regions in the budding yeast genome.

Accurate chromosome segregation requires centromeres (CENs), the DNA sequences where kinetochores form, to attach chromosomes to microtubules. In contrast to most eukaryotes, which have broad centromeres, Saccharomyces cerevisiae possesses sequence-defined point CENs. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) reveals colocalization of four kinetochore proteins at novel, discrete, non-centromeric regions, especially when levels of the centromeric histone H3 variant, Cse4 (a.k.a. CENP-A or CenH3), are elevated. These regions of overlapping protein binding enhance the segregation of plasmids and chromosomes and have thus been termed Centromere-Like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of both point and regional CENs. CLR sequences are conserved among related budding yeasts. Many genomic features characteristic of CLRs are also associated with these conserved homologous sequences from closely related budding yeasts. These studies provide general and important insights into the origin and evolution of centromeres.

High throughput sequencing reveals alterations in the recombination signatures with diminishing Spo11 activity.

Spo11 is the topoisomerase-like enzyme responsible for the induction of the meiosis-specific double strand breaks (DSBs), which initiates the recombination events responsible for proper chromosome segregation. Nineteen PCR-induced alleles of SPO11 were identified and characterized genetically and cytologically. Recombination, spore viability and synaptonemal complex (SC) formation were decreased to varying extents in these mutants. Arrest by ndt80 restored these events in two severe hypomorphic mutants, suggesting that ndt80-arrested nuclei are capable of extended DSB activity. While crossing-over, spore viability and synaptonemal complex (SC) formation defects correlated, the extent of such defects was not predictive of the level of heteroallelic gene conversions (prototrophs) exhibited by each mutant. High throughput sequencing of tetrads from spo11 hypomorphs revealed that gene conversion tracts associated with COs are significantly longer and gene conversion tracts unassociated with COs are significantly shorter than in wild type. By modeling the extent of these tract changes, we could account for the discrepancy in genetic measurements of prototrophy and crossover association. These findings provide an explanation for the unexpectedly low prototroph levels exhibited by spo11 hypomorphs and have important implications for genetic studies that assume an unbiased recovery of prototrophs, such as measurements of CO homeostasis. Our genetic and physical data support previous observations of DSB-limited meioses, in which COs are disproportionally maintained over NCOs (CO homeostasis).

The synaptonemal complex protein, Zip1, promotes the segregation of nonexchange chromosomes at meiosis I.

Crossing over establishes connections between homologous chromosomes that promote their proper segregation at the first meiotic division. However, there exists a backup system to ensure the correct segregation of those chromosome pairs that fail to cross over. We have found that, in budding yeast, a mutation eliminating the synaptonemal complex protein, Zip1, increases the meiosis I nondisjunction rate of nonexchange chromosomes (NECs). The centromeres of NECs become tethered during meiotic prophase, and this tethering is disrupted by the zip1 mutation. Furthermore, the Zip1 protein often colocalizes to the centromeres of the tethered chromosomes, suggesting that Zip1 plays a direct role in holding NECs together. Zip3, a protein involved in the initiation of synaptonemal complex formation, is also important for NEC segregation. In the absence of Zip3, both the tethering of NECs and the localization of Zip1 to centromeres are impaired. A mutation in the MAD3 gene, which encodes a component of the spindle checkpoint, also increases the nondisjunction of NECs. Together, the zip1 and mad3 mutations have an additive effect, suggesting that these proteins act in parallel pathways to promote NEC segregation. We propose that Mad3 promotes the segregation of NECs that are not tethered by Zip1 at their centromeres.

The meiosis-specific zip4 protein regulates crossover distribution by promoting synaptonemal complex formation together with zip2.

We have characterized Zip4 (a.k.a. Spo22), a meiosis-specific protein essential for chromosome synapsis in budding yeast. In the absence of Zip4, the synaptonemal complex protein Zip1 fails to polymerize along chromosomes. Zip2 and Zip3 are previously characterized components of the synapsis initiation complex. Zip4 forms a functional unit with Zip2 that is distinct from Zip3. Zip2 and Zip4 are mutually dependent for their chromosomal localization; in polycomplexes, the pattern of Zip2/Zip4 localization is distinct from that of Zip3. Crossing-over is decreased in the zip4 mutant (as in zip1, zip2, and zip3); the remaining crossovers are largely dependent on a parallel pathway utilizing Mms4. zip4 displays a novel phenotype: negative crossover interference, meaning that crossovers tend to cluster. This clustering depends on Zip1. Our results suggest an interaction between crossover pathways such that a protein (Zip1) acting in one pathway influences the distribution of crossovers promoted by a parallel (Mms4-dependent) pathway.

A Role for SUMO in meiotic chromosome synapsis.

During meiotic prophase, homologous chromosomes engage in a complex series of interactions that ensure their proper segregation at meiosis I. A central player in these interactions is the synaptonemal complex (SC), a proteinaceous structure elaborated along the lengths of paired homologs. In mutants that fail to make SC, crossing over is decreased, and chromosomes frequently fail to recombine; consequently, many meiotic products are inviable because of aneuploidy. Here, we have investigated the role of the small ubiquitin-like protein modifier (SUMO) in SC formation during meiosis in budding yeast. We show that SUMO localizes specifically to synapsed regions of meiotic chromosomes and that this localization depends on Zip1, a major building block of the SC. A non-null allele of the UBC9 gene, which encodes the SUMO-conjugating enzyme, impairs Zip1 polymerization along chromosomes. The Ubc9 protein localizes to meiotic chromosomes, coincident with SUMO staining. In the zip1 mutant, SUMO localizes to discrete foci on chromosomes. These foci coincide with axial associations, where proteins involved in synapsis initiation are located. Our data suggest a model in which SUMO modification of chromosomal proteins promotes polymerization of Zip1 along chromosomes. The ubc9 mutant phenotype provides the first evidence for a cause-and-effect relationship between sumoylation and synapsis.

The importance of genetic recombination for fidelity of chromosome pairing in meiosis.

In budding yeast, absence of the Hop2 protein leads to extensive synaptonemal complex (SC) formation between nonhomologous chromosomes, suggesting a crucial role for Hop2 in the proper alignment of homologous chromosomes during meiotic prophase. Genetic analysis indicates that Hop2 acts in the same pathway as the Rad51 and Dmc1 proteins, two homologs of E. coli RecA. Thus, the hop2 mutant phenotype demonstrates the importance of the recombination machinery in promoting accurate chromosome pairing. We propose that the Dmc1/Rad51 recombinases require Hop2 to distinguish homologous from nonhomologous sequences during the homology search process. Thus, when Hop2 is absent, interactions between nonhomologous sequences become inappropriately stabilized and can initiate SC formation. Overexpression of RAD51 largely suppresses the meiotic defects of the dmc1 and hop2 mutants. We conclude that Rad51 is capable of carrying out a homology search independently, whereas Dmc1 requires additional factors such as Hop2.

Meiotic chromosome synapsis-promoting proteins antagonize the anti-crossover activity of sgs1.

Sgs1, the budding yeast homolog of the mammalian BLM helicase, has been implicated in preventing excess recombination during both vegetative growth and meiosis. Most meiotic crossover (CO) recombination requires full function of a set of yeast proteins (Zip1, Zip2, Zip3, Zip4/Spo22, Mer3, Msh4, and Msh5, termed the SIC or ZMM proteins) that are also required for homologous chromosome synapsis. We report here genetic and molecular assays showing that sgs1 single mutants display relatively modest increases in CO recombination (less than 1.6-fold relative to wild-type). In contrast, a much greater CO increase is seen when an sgs1 mutation is introduced into the CO- and synapsis-deficient zip1, zip2, zip3, mer3, or msh4 mutants (2- to 8-fold increase). Furthermore, close juxtaposition of the axes of homologous chromosomes is restored. CO restoration in the mutants is not accompanied by significant changes in noncrossover (NCO) recombinant frequencies. These findings show that Sgs1 has potent meiotic anti-CO activity, which is normally antagonized by SIC/ZMM proteins. Our data reinforce previous proposals for an early separation of meiotic processes that form CO and NCO recombinants.

SSP2 and OSW1, two sporulation-specific genes involved in spore morphogenesis in Saccharomyces cerevisiae.

Spore formation in Saccharomyces cerevisiae requires the synthesis of prospore membranes (PSMs) followed by the assembly of spore walls (SWs). We have characterized extensively the phenotypes of mutants in the sporulation-specific genes, SSP2 and OSW1, which are required for spore formation. A striking feature of the osw1 phenotype is asynchrony of spore development, with some spores displaying defects in PSM formation and others spores in the same ascus blocked at various stages in SW development. The Osw1 protein localizes to spindle pole bodies (SPBs) during meiotic nuclear division and subsequently to PSMs/SWs. We propose that Osw1 performs a regulatory function required to coordinate the different stages of spore morphogenesis. In the ssp2 mutant, nuclei are surrounded by PSMs and SWs; however, PSMs and SWs often also encapsulate anucleate bodies both inside and outside of spores. In addition, the SW is not as thick as in wild type. The ssp2 mutant defect is partially suppressed by overproduction of either Spo14 or Sso1, both of which promote the fusion of vesicles at the outer plaque of the SPB early in PSM formation. We propose that Ssp2 plays a role in vesicle fusion during PSM formation.

A novel nonnull ZIP1 allele triggers meiotic arrest with synapsed chromosomes in Saccharomyces cerevisiae.

During meiotic prophase, assembly of the synaptonemal complex (SC) brings homologous chromosomes into close apposition along their lengths. The Zip1 protein is a major building block of the SC in Saccharomyces cerevisiae. In the absence of Zip1, SC fails to form, cells arrest or delay in meiotic prophase (depending on strain background), and crossing over is reduced. We created a novel allele of ZIP1, zip1-4LA, in which four leucine residues in the central coiled-coil domain have been replaced by alanines. In the zip1-4LA mutant, apparently normal SC assembles with wild-type kinetics; however, crossing over is delayed and decreased compared to wild type. The zip1-4LA mutant undergoes strong checkpoint-induced arrest in meiotic prophase; the defect in cell cycle progression is even more severe than that of the zip1 null mutant. When the zip1-4LA mutation is combined with the pch2 checkpoint mutation, cells sporulate with wild-type efficiency and crossing over occurs at wild-type levels. This result suggests that the zip1-4LA defect in recombination is an indirect consequence of cell cycle arrest. Previous studies have suggested that the Pch2 protein acts in a checkpoint pathway that monitors chromosome synapsis. We hypothesize that the zip1-4LA mutant assembles aberrant SC that triggers the synapsis checkpoint.

The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over.

BACKGROUND: In budding yeast, Sgs1 is the sole member of the RecQ family of DNA helicases. Like the human Bloom syndrome helicase (BLM), Sgs1 functions during both vegetative growth and meiosis. The sgs1 null mutant sporulates poorly and displays reduced spore viability. RESULTS: We have identified novel functions for Sgs1 in meiosis. Loss of Sgs1 increases the number of axial associations, which are connections between homologous chromosomes that serve as initiation sites for synaptonemal complex formation. In addition, mutation of SGS1 increases the number of synapsis initiation complexes and increases the rate of chromosome synapsis. Loss of Sgs1 also increases the number of meiotic crossovers without changing the frequency of gene conversion. The sgs1 defect in sporulation is due to checkpoint-induced arrest/delay at the pachytene stage of meiotic prophase. A non-null allele of SGS1 that specifically deletes the helicase domain is defective in the newly described meiotic functions of Sgs1, but wild-type for most vegetative functions and for spore formation. CONCLUSIONS: We have shown that the helicase domain of Sgs1 serves as a negative regulator of meiotic interchromosomal interactions. The activity of the wild-type Sgs1 protein reduces the numbers of axial associations, synapsis initiation complexes, and crossovers, and decreases the rate of chromosome synapsis. Our data argue strongly that axial associations marked by synapsis initiation complexes correspond to sites of reciprocal exchange. We propose that the Sgs1 helicase prevents a subset of recombination intermediates from becoming crossovers, and this distinction is made at an early stage in meiotic prophase.

Saccharomyces cerevisiae Mer2, Mei4 and Rec114 form a complex required for meiotic double-strand break formation.

In budding yeast, at least 10 proteins are required for formation of the double-strand breaks (DSBs) that initiate meiotic recombination. Spo11 is the enzyme responsible for cleaving DNA and is found in a complex that also contains Ski8, Rec102, and Rec104. The Mre11/Rad50/Xrs2 complex is required for both DSB formation and DSB processing. In this article we investigate the functions of the remaining three proteins--Mer2, Mei4, and Rec114--with particular emphasis on Mer2. The Mer2 protein is present in vegetative cells, but it increases in abundance and becomes phosphorylated specifically during meiotic prophase. Mer2 localizes to distinct foci on meiotic chromosomes, with foci maximally abundant prior to the formation of synaptonemal complex. If DSB formation is blocked (e.g., by a spo11 mutation), dephosphorylation of Mer2 and its dissociation from chromosomes are delayed. We have also found that the Mei4 and Rec114 proteins localize to foci on chromosomes and these foci partially colocalize with each other and with Mer2. Furthermore, the three proteins co-immunoprecipitate. Mer2 does not show significant colocalization with Mre11 or Rec102 and Mer2 does not co-immunoprecipitate with Rec102. We propose that Mer2, Mei4, and Rec114 form a distinct complex required for DSB formation.

Centromere-proximal crossovers are associated with precocious separation of sister chromatids during meiosis in Saccharomyces cerevisiae.

In most organisms, meiotic chromosome segregation is dependent on crossovers (COs), which enable pairs of homologous chromosomes to segregate to opposite poles at meiosis I. In mammals, the majority of meiotic chromosome segregation errors result from a lack of COs between homologs. Observations in Homo sapiens and Drosophila melanogaster have revealed a second class of exceptional events in which a CO occurred near the centromere of the missegregated chromosome. We show that in wild-type strains of Saccharomyces cerevisiae, most spore inviability is due to precocious separation of sister chromatids (PSSC) and that PSSC is often associated with centromere-proximal crossing over. COs, as opposed to nonreciprocal recombination events (NCOs), are preferentially associated with missegregation. Strains mutant for the RecQ homolog, SGS1, display reduced spore viability and increased crossing over. Much of the spore inviability in sgs1 results from PSSC, and these events are often associated with centromere-proximal COs, just as in wild type. When crossing over in sgs1 is reduced by the introduction of a nonnull allele of SPO11, spore viability is improved, suggesting that the increased PSSC is due to increased crossing over. We present a model for PSSC in which a centromere-proximal CO promotes local loss of sister-chromatid cohesion.

The Mnd1 protein forms a complex with hop2 to promote homologous chromosome pairing and meiotic double-strand break repair.

The hop2 mutant of Saccharomyces cerevisiae arrests in meiosis with extensive synaptonemal complex (SC) formation between nonhomologous chromosomes. A screen for multicopy suppressors of a hop2-ts allele identified the MND1 gene. The mnd1-null mutant arrests in meiotic prophase, with most double-strand breaks (DSBs) unrepaired. A low level of mature recombinants is produced, and the Rad51 protein accumulates at numerous foci along chromosomes. SC formation is incomplete, and homolog pairing is severely reduced. The Mnd1 protein localizes to chromatin throughout meiotic prophase, and this localization requires Hop2. Unlike recombination enzymes such as Rad51, Mnd1 localizes to chromosomes even in mutants that fail to initiate meiotic recombination. The Hop2 and Mnd1 proteins coimmunoprecipitate from meiotic cell extracts. These results suggest that Hop2 and Mnd1 work as a complex to promote meiotic chromosome pairing and DSB repair. The identification of Hop2 and Mnd1 homologs in other organisms suggests that the function of this complex is conserved among eukaryotes.

The budding yeast mei5 and sae3 proteins act together with dmc1 during meiotic recombination.

Here we provide evidence that the Mei5 and Sae3 proteins of budding yeast act together with Dmc1, a meiosis-specific, RecA-like recombinase. The mei5 and sae3 mutations reduce sporulation, spore viability, and crossing over to the same extent as dmc1. In all three mutants, these defects are largely suppressed by overproduction of Rad51. In addition, mei5 and sae3, like dmc1, suppress the cell-cycle arrest phenotype of the hop2 mutant. The Mei5, Sae3, and Dmc1 proteins colocalize to foci on meiotic chromosomes, and their localization is mutually dependent. The localization of Rad51 to chromosomes is not affected in either mei5 or sae3. Taken together, these observations suggest that the Mei5 and Sae3 proteins are accessory factors specific to Dmc1. We speculate that Mei5 and Sae3 are necessary for efficient formation of Dmc1-containing nucleoprotein filaments in vivo.

A role for DNA polymerase delta in gene conversion and crossing over during meiosis in Saccharomyces cerevisiae.

A screen for mutants of budding yeast defective in meiotic gene conversion identified a novel allele of the POL3 gene. POL3 encodes the catalytic subunit of DNA polymerase delta, an essential DNA polymerase involved in genomic DNA replication. The new allele, pol3-ct, specifies a protein missing the last four amino acids. pol3-ct shows little or no defect in DNA replication, but displays a reduction in the length of meiotic gene conversion tracts and a decrease in crossing over. We propose a model in which DNA synthesis determines the length of strand exchange intermediates and influences their resolution toward crossing over.

Cdc14 Early Anaphase Release, FEAR, Is Limited to the Nucleus and Dispensable for Efficient Mitotic Exit.

Cdc14 phosphatase is a key regulator of exit from mitosis, acting primarily through antagonism of cyclin-dependent kinase, and is also thought to be important for meiosis. Cdc14 is released from its sequestration site in the nucleolus in two stages, first by the non-essential Cdc Fourteen Early Anaphase Release (FEAR) pathway and later by the essential Mitotic Exit Network (MEN), which drives efficient export of Cdc14 to the cytoplasm. We find that Cdc14 is confined to the nucleus during early mitotic anaphase release, and during its meiosis I release. Proteins whose degradation is directed by Cdc14 as a requirement for mitotic exit (e.g. the B-type cyclin, Clb2), remain stable during mitotic FEAR, a result consistent with Cdc14 being restricted to the nucleus and not participating directly in mitotic exit. Cdc14 released by the FEAR pathway has been proposed to have a wide variety of activities, all of which are thought to promote passage through anaphase. Proposed functions of FEAR include stabilization of anaphase spindles, resolution of the rDNA to allow its segregation, and priming of the MEN so that mitotic exit can occur promptly and efficiently. We tested the model for FEAR functions using the FEAR-deficient mutation net1-6cdk. Our cytological observations indicate that, contrary to the current model, FEAR is fully dispensable for timely progression through a series of anaphase landmarks and mitotic exit, although it is required for timely rDNA segregation. The net1-6cdk mutation suppresses temperature-sensitive mutations in MEN genes, suggesting that rather than activating mitotic exit, FEAR either inhibits the MEN or has no direct effect upon it. One interpretation of this result is that FEAR delays MEN activation to ensure that rDNA segregation occurs before mitotic exit. Our findings clarify the distinction between FEAR and MEN-dependent Cdc14 activities and will help guide emerging quantitative models of this cell cycle transition.

Fpr3 and Zip3 ensure that initiation of meiotic recombination precedes chromosome synapsis in budding yeast.

BACKGROUND: Homolog pairing, synaptonemal complex (SC) assembly (chromosome synapsis), and crossover recombination are essential for successful meiotic chromosome segregation. A distinguishing feature of meiosis in budding yeast and mammals is that synapsis between homologs depends upon recombination; however, the molecular basis for this contingency is not understood. RESULTS: We show here that the yeast proline isomerase Fpr3 and the small ubiquitin-like modifier (SUMO) ligase Zip3 ensure that SC assembly is dependent upon recombination initiation. When Fpr3 and Zip3 are absent, synapsis occurs even in a mutant that fails to initiate recombination and homolog pairing. Fpr3 and Zip3 appear to specifically prevent synapsis initiation at centromeric sites. This result is consistent with previous observations of SC proteins localizing to centromeres prior to and independent of meiotic recombination initiation. Finally, we show that without Fpr3 and Zip3 activities, the synapsis initiation components Zip2 and Zip4 are dispensable for chromosome synapsis. CONCLUSIONS: Fpr3 and Zip3 represent parallel pathways that function in a checkpoint-like manner to ensure that chromosome synapsis is contingent on the initiation of recombination. We propose that, during normal meiosis, Zip2 and Zip4 act downstream of recombination signals to oppose Fpr3- and Zip3-mediated inhibitions to initiating SC assembly at centromeres. These data suggest a role for centromeres in coordinating major meiotic chromosomal events and draw an interesting parallel between yeast centromeres and C. elegans pairing centers.

Initiation of meiotic chromosome synapsis at centromeres in budding yeast.

Previous studies of synaptonemal complex assembly in budding yeast have suggested that chromosome synapsis initiates at the sites of crossing over. The data presented here, however, indicate that centromeric regions are preferred sites for synapsis initiation. At early times during meiosis in wild type, the Zip1 protein (a major building block of the synaptonemal complex) localizes specifically to centromeric regions. As synapsis progresses and linear stretches of Zip1 are formed, the majority of stretches are associated with a centromere, as expected if the Zip1 protein present at the centromere polymerized outward along the chromosome arm. In many cases, the centromere is present at one end of a linear stretch, suggesting that synapsis is often unidirectional. Furthermore, the Zip2 protein, a protein that promotes Zip1 polymerization, is often present at the opposite end from the centromere, implying that Zip2 and associated proteins move at the leading edge of Zip1 polymerization. Surprisingly, synapsis initiation at centromeres is independent of the Zip3 protein, which plays a major role in synapsis initiation events at noncentromeric locations. Our data provide evidence for two classes of synapsis initiation events that differ in location, timing, genetic requirements, and relationship to meiotic recombination.

Hed1 regulates Rad51-mediated recombination via a novel mechanism.

Two RecA orthologs, Rad51 and Dmc1, mediate homologous recombination in meiotic cells. During budding yeast meiosis, Hed1 coordinates the actions of Rad51 and Dmc1 by down-regulating Rad51 activity. It is thought that Hed1-dependent attenuation of Rad51 facilitates formation of crossovers that are necessary for the correct segregation of chromosomes at the first meiotic division. We purified Hed1 in order to elucidate its mechanism of action. Hed1 binds Rad51 with high affinity and specificity. We show that Hed1 does not adversely affect assembly of the Rad51 presynaptic filament, but it specifically prohibits interaction of Rad51 with Rad54, a Swi2/Snf2-like factor that is indispensable for Rad51-mediated recombination. In congruence with the biochemical results, Hed1 prevents the recruitment of Rad54 to a site-specific DNA double-strand break in vivo but has no effect on the recruitment of Rad51. These findings shed light on the function of Hed1 and, importantly, unveil a novel mechanism for the regulation of homologous recombination.