The Budding Yeast Msh4 Protein Functions in Chromosome Synapsis and the Regulation of Crossover Distribution

Corresponding author: G. Shirleen Roeder, Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103., shirleen.roeder{at}yale.edu (E-mail)

Communicating editor: M. LICHTEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.

REDUCTIONAL chromosome segregation is unique to the first division of meiosis. Sister chromatids remain associated throughout this division, while homologous chromosomes segregate to opposite poles of the spindle apparatus. The chromosome content of a diploid cell is thereby reduced to the haploid number of chromosomes. A prerequisite to proper chromosome segregation at meiosis I is meiotic recombination. Crossing over establishes chromatin bridges between homologs, called chiasmata, that ensure the proper orientation of chromosomes on the meiosis I spindle (ROEDER 1997 ). The proper segregation of chromosomes in meiosis also depends on formation of the synaptonemal complex (SC), an elaborate proteinaceous structure that holds homologous chromosomes close together along their lengths during the pachytene stage of meiotic prophase (ROEDER 1997 ). Mutations in genes encoding structural components of the SC lead to homolog nondisjunction at meiosis I and precocious separation of sister chromatids (ROEDER 1997 ).

Two budding yeast proteins involved in meiotic recombination are Msh4 and Msh5 (ROSS-MACDONALD and ROEDER 1994 ; HOLLINGSWORTH et al. 1995 ), which are homologs of the bacterial MutS mismatch repair protein. However, Msh4 and Msh5 play no role in mismatch correction; instead, they are required specifically for wild-type levels of meiotic crossing over. In the absence of Msh4 or Msh5, meiotic gene conversion occurs at approximately wild-type levels, but crossing over is reduced two- to threefold. The MSH4 and MSH5 genes are expressed specifically in meiotic cells (ROSS-MACDONALD and ROEDER 1994 ; CHU et al. 1998 ). The proteins directly interact to form a complex (POCHART et al. 1997 ), and they colocalize to foci on meiotic chromosomes (J. E. NOVAK, unpublished data). In the absence of Msh4 (and presumably also Msh5), chromosomes sometimes fail to undergo crossing over (ROSS-MACDONALD and ROEDER 1994 ). Because nonrecombinant chromosomes often missegregate at meiosis I, the msh4 and msh5 mutants display reduced spore viability.

The recombination phenotype of msh4 and msh5 is not unique to these mutants. Mutations in a number of other yeast genes also reduce the frequency of meiotic crossing over without decreasing the frequency of non-Mendelian segregation. These genes include ZIP1, ZIP2, ZIP3, MER3, MLH1, MLH3, and EXO1. The three ZIP genes are involved in SC formation. The Zip1 protein is present along the lengths of synapsed meiotic chromosomes and serves as a major structural component of the SC (SYM et al. 1993 ; SYM and ROEDER 1995 ; TUNG and ROEDER 1998 ; DONG and ROEDER 2000 ). Zip2 and Zip3 are present on meiotic chromosomes at discrete foci that correspond to the sites where synapsis initiates, and these proteins are required for the normal polymerization of Zip1 along chromosomes (CHUA and ROEDER 1998 ; AGARWAL and ROEDER 2000 ). The MER3 gene is expressed specifically in meiotic cells and encodes a putative helicase (NAKAGAWA and OGAWA 1999 ). The two MutL homologs, Mlh1 and Mlh3, form a heterodimer specifically involved in meiotic crossing over (HUNTER and BORTS 1997 ; WANG et al. 1999 ); Mlh1 also functions in mismatch repair both in vegetative and meiotic cells (KOLODNER and MARSISCHKY 1999 ). The Exo1 protein is a 5' to 3' exonuclease specific for double-stranded DNA (HUANG and SYMINGTON 1993 ; FIORENTINI et al. 1997 ). In addition to its role in meiotic crossing over (KHAZANEHDARI and BORTS 2000 ; KIRKPATRICK et al. 2000 ; TSUBOUCHI and OGAWA 2000 ), Exo1 is involved in mismatch repair and recombination in vegetative cells (FIORENTINI et al. 1997 ; TISHKOFF et al. 1997 ).

Meiotic crossovers are nonrandomly distributed along chromosomes such that two crossovers rarely occur close together—a phenomenon known as crossover interference. Interference is generally assumed to involve the transmission of an inhibitory signal from one crossover site to nearby potential sites of crossing over. In budding yeast, mutations in three different genes—ZIP1, NDJ1 (a.k.a. TAM1), and MER3—have been shown to reduce or abolish crossover interference (SYM and ROEDER 1994 ; CHUA and ROEDER 1997 ; NAKAGAWA and OGAWA 1999 ). A zip1 null mutation abolishes SC formation (SYM et al. 1993 ), while an ndj1 null mutation causes a substantial delay in SC formation (CHUA and ROEDER 1997 ; CONRAD et al. 1997 ). mer3 has not been tested for its effect on synapsis. The observed correlation between impaired synapsis and decreased interference in mutants of yeast and other organisms (MOENS 1969 ; HAVEKES et al. 1994 ) is consistent with the hypothesis that the SC is involved in transmission of the inhibitory signal responsible for interference (EGEL 1978 ; MAGUIRE 1988 ). The observation that Schizosaccharomyces pombe and Aspergillus nidulans lack both SC and interference further supports this idea (STRICKLAND 1958 ; OLSON and ZIMMERMANN 1978 ; EGEL-MITANI et al. 1982 ; BAHLER et al. 1993 ).

Meiotic crossovers are nonrandomly distributed not only along chromosomes, but also among chromosomes. In most meioses, every chromosome pair, no matter how small, sustains at least one crossover—a so-called obligate crossover or obligate chiasma. Obligate crossing over might be due, at least in part, to the fact that large chromosomes display more crossover interference than do small chromosomes (KABACK et al. 1999 ). By preventing excess exchanges on large chromosomes, interference might ensure that crossovers (generally presumed to be limited in number) are distributed such that every chromosome pair undergoes at least one exchange. Consistent with this hypothesis, mutations that reduce or eliminate interference randomize the distribution of crossovers among chromosomes such that chromosomes sometimes fail to cross over and therefore nondisjoin (SYM and ROEDER 1994 ; CHUA and ROEDER 1997 ).

We have found that a msh4 null mutation (like ndj1) significantly reduces crossover interference and results in SC formation that is delayed and often incomplete. Msh4 colocalizes with the Zip2 protein at sites of synapsis initiation and depends on both Zip2 and Zip1 for proper localization to chromosomes. Analysis of a msh4 zip1 double mutant indicates that Msh4 and Zip1 act in the same pathway of crossing over.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plasmids, disruptions, and strains:
Media were prepared and yeast manipulations were carried out using standard procedures (SHERMAN et al. 1986 ). All yeast transformants were verified by Southern blot analysis. Yeast strain genotypes are given in Table 1.

MSH4 gene disruptions were engineered as follows. pmsh485-2395 was derived from p6K (ROSS-MACDONALD and ROEDER 1994 ) by inserting linkers containing a NotI site into a BstBI site at nucleotide 80 and an SspI site at nucleotide 2393 within the MSH4 coding region. Appropriate NotI-BamHI and NotI-EcoRI fragments were ligated into pUC18 cut with EcoRI and BamHI to yield pmsh485-2395, in which nucleotides 85–2395 were replaced with GCGGCCGCAA. To create pmsh4ADE, pmsh485-2395 was digested with NotI; the ends were filled in with the Klenow fragment of DNA polymerase I and a 3.7-kb blunt-ended fragment containing the ADE2 gene was inserted. The msh4::ADE2 mutation was introduced into yeast by substitutive transformation (ROTHSTEIN 1991 ) using pmsh4ADE digested with NcoI and BamHI. p6H, containing msh4::LEU2, was derived from p5E (ROSS-MACDONALD and ROEDER 1994 ) by cutting with NdeI and circularizing the fragment containing MSH4 sequences, resulting in a plasmid in which part of a transposon (including LEU2) replaces nucleotides 168–2290 of the MSH4 coding region. The msh4::LEU2 disruption was introduced into yeast by transformation with p6H cut with NotI.

Strains producing tagged versions of the Msh4 protein were constructed as follows. In pU-Msh4-HA, a SacI-XhoI fragment containing the MSH4 gene marked with three copies of the HA tag at the 3' end was obtained from pJ8 (ROSS-MACDONALD and ROEDER 1994 ) and inserted between the XhoI and the SacI sites of pRS306 (SIKORSKI and HIETER 1989 ). The MSH4-HA gene was integrated (ROTHSTEIN 1991 ) at URA3 by transformation with pU-Msh4-HA targeted with StuI. pkan-Msh4C-3xHA was designed to allow simultaneous disruption of MSH4 and insertion of MSH4-HA. To make pkan-Msh4C-3xHA, p10H (ROSS-MACDONALD and ROEDER 1994 ) was cut with BstUI and EcoRI, and the 2.4-kb fragment containing the downstream portion of MSH4-HA was inserted into pFA6-Kan-MX4 (WACH et al. 1994 ) cut with EcoRI and EcoRV. The msh4::MSH4-HA-kan allele was introduced into yeast by transformation with pkan-Msh4C-3xHA cut with BsrGI to target integration at the MSH4 locus.

pU-Zip2C-GFP contains part of the ZIP2 gene tagged with green fluorescent protein (GFP; CHUA and ROEDER 1998 ). To generate pU-Zip2C-GFP, a 2.1-kb BamHI-SalI fragment containing the downstream portion of ZIP2::GFP (CHUA and ROEDER 1998 ) was inserted between the BamHI and SalI sites of pRS306 (SIKORSKI and HIETER 1989 ). The zip2::ZIP2-GFP-URA3 allele was introduced into yeast by integrating pU-Zip2C-GFP (targeted with EcoRI) at ZIP2. Other ZIP2 alleles were introduced as described (CHUA and ROEDER 1998 ).

pNDT80kan was constructed from pTP77 (TUNG et al. 2000 ). A 1.1-kb BglII-EcoRI fragment in NDT80 was replaced with a 1.4-kb BglII-EcoRI fragment from pFA6-Kan-MX4 (WACH et al. 1994 ) containing kan^r. NDT80 was disrupted by transformation with pNDT80kan targeted with XbaI and BamHI.

p11B, which contains SER1 disrupted by a transposon, was constructed as described (ROSS-MACDONALD et al. 1997 ). The ser1::mTn-3xHA/lacZ allele was introduced into yeast using p11B targeted with NotI. pR1723, containing his3-Nde, was derived from pRS303 (SIKORSKI and HIETER 1989 ) by first filling in the NdeI site in HIS3 and then inserting a SmaI-EagI fragment containing URA3 from YIp5 between the SmaI and EagI sites. The his3-Nde and ade2-Bgl alleles were introduced by two-step transplacement using pR1723 (targeted with NheI) and pR943 (ENGEBRECHT and ROEDER 1990 ), respectively. zip1::LEU2 and zip1::LYS2 alleles were introduced as described (SYM et al. 1993 ; SYM and ROEDER 1994 ).

Genetic analysis:
To calculate map distance, only four-spore-viable tetrads that did not show gene conversion of relevant markers were used. Crossover frequencies were compared as described (CHUA and ROEDER 1997 ).

For interference analysis, the number of nonparental ditypes (NPDs) expected in a particular interval was derived by applying the formula of PAPAZIAN 1952 , NPD = , and rounding the resulting figure to the nearest whole number. The ratio of NPDs observed to NPDs expected was then compared using the chi-square test as described (SYM and ROEDER 1994 ). To compare the NPD ratio calculated for the wild type with that calculated for the msh4 mutant, the chi-square coincidence test was applied to the values for observed and expected NPDs.

Cytology:
Strains isogenic with BR2495 were grown and sporulated as described (ROCKMILL et al. 1995 ). Strains isogenic or congenic with SK1 were prepared for meiosis by first growing to saturation in YPAD. Cells were then diluted 200-fold into YPA and grown at 30° for 15 hr, to an OD₆₀₀ of 0.9. Cells were then rinsed in 2% potassium acetate that was warmed to 30°, pelleted, and resuspended in warm 2% potassium acetate at a volume equal to their volume in YPA. Cells were shaken at 30°, and samples were harvested at various time points. Surface-spread meiotic nuclei were prepared as described (SYM et al. 1993 ), except that glass slides were not coated with plastic. Samples taken to assess timing of cell divisions were fixed and stained with 4',6-diamidino-2-phenylindole (DAPI) as described (ENGEBRECHT and ROEDER 1990 ).

Immunofluorescence on spread chromosomes was performed as described (SYM et al. 1993 ), with the following modifications. The solution for blocking and antibody dilutions was 3% bovine serum albumin, 1% teleostean gelatin (Sigma, St. Louis), and 0.02% thimerosal in phosphate-buffered saline. Rabbit anti-Zip1 antibodies (SYM et al. 1993 ) were diluted 200-fold and detected with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Rat anti-tubulin antibodies (KILMARTIN et al. 1982 ) were diluted 500-fold and detected with FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Hemagglutinin (HA)-tagged Msh4 was detected with mouse monoclonal antibody HA-11 (Covance) diluted 5000-fold followed by secondary antibodies conjugated to either Texas red or Cy3 (Jackson ImmunoResearch Laboratories). GFP-tagged Zip2 was detected with rabbit anti-GFP antibodies (CLONTECH, Palo Alto, CA) diluted 300-fold and secondary antibodies conjugated with Oregon Green 488 (Molecular Probes, Eugene, OR). Red1 protein was detected using rabbit anti-Red1 serum (SMITH and ROEDER 1997 ) diluted 100-fold followed by Oregon Green 488-conjugated secondary antibodies. All antibody binding was carried out at room temperature. Images were captured using a Nikon Eclipse E800 microscope and a Photometrics (Tucson, AZ) Sensys CCD camera.

To quantify the colocalization of Msh4 and Zip2, the following procedure was used. First, image contrast and brightness were adjusted using Adobe Photoshop to compensate for the faintness of Msh4 foci in JN294. Msh4 and Zip2 foci that were judged to be brighter than background staining were copied separately onto transparent sheets in order to have a defined number of foci with distinct edges. The outlines of each spread were traced from the DAPI-stained DNA. Superposition of the sheets was used to count Msh4 and Zip2 foci that overlapped. To estimate the amount of overlap that would be found with random positioning, the copy of Zip2 foci was rotated 180° to randomize the position of the Zip2 foci with respect to the Msh4 foci. Any foci falling outside the area where the spread chromosomes overlapped were not included in the analysis. Within the overlapping region, the number of Msh4 and Zip2 foci and the number of overlapping foci were counted. Approximately 400 foci of each protein were scored for both JN294 and JN295.

Strain MY63, which has only untagged Msh4, was used to assess the background level of staining with antibodies against HA. Typically, spread nuclei from MY63 showed no foci.

To quantify Msh4 staining in wild-type and mutant spreads, Imagepoint IPLab Spectrum software was used to measure total intensity within each spread; background intensity was subtracted. At least 17 spreads for each strain were measured. The average values for the strains were found to be significantly different (P < 0.001) by a two-tailed t-test.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A msh4 mutant is defective in crossover interference:
Crossover interference can be detected as a deficit in double crossovers within a genetically marked interval, compared to the number expected based on the frequency of single crossovers. In yeast, four-strand double crossovers generate NPD tetrads. The frequency of NPDs expected in the absence of interference can be calculated from the observed frequency of tetratype (TT) tetrads due to single crossovers (PAPAZIAN 1952 ). The NPD ratio is the number of NPDs observed divided by the number expected (SNOW 1979 ). An NPD ratio <1.0 indicates that crossover interference is operating.

Tetrads from wild-type and msh4 strains were dissected and analyzed for recombination in seven genetic intervals representing three different chromosomes (Table 2). Map distances in msh4 strains were only 40–60% of those in wild type (Table 2), with the exception of the HOM3–TRP2 interval, which was unaffected (Table 2). In wild type, NPD ratios ranged from 0.13 to 0.34, indicating significant interference (Fig 1); the only exception was the HOM3–TRP2 interval, which did not display any interference in the strain background used for this analysis. Excluding the HOM3–TRP2 interval, NPD ratios in the msh4 mutant were consistently higher than those in wild type (Fig 1); this difference is statistically significant for the ADE2–SER1, CAN1–URA3, and URA3–HOM3 intervals (Table 2).

View larger version (44K): In this window In a new window Download PPT slide   Figure 1. The msh4 mutation reduces or eliminates interference. (A) Genetic map of the intervals tested. Gene order, the sizes of the intervals tested, and the overall size of each chromosome are indicated. (B) Interference values (NPD observed/NPD expected) were calculated for the seven intervals indicated. A ratio of 1.0 (dotted line) is indicative of no interference.

For the intervals examined on chromosomes V and XV, NPD ratios for the msh4 mutant were close to 1.0, as expected in the complete absence of interference (Fig 1). However, for the two intervals examined on chromosome III, NPD ratios were <1.0; the ratios for HIS4–CEN3 and CEN3–MAT were 0.67 and 0.38, respectively. For another test of interference on chromosome III, NPD ratios were calculated for the HIS4–MAT interval (which subsumes the HIS4–CEN3 and CEN3–MAT intervals; Fig 1). For this interval, the NPD ratio obtained in the msh4 mutant (0.68) was <1.0, but greater than the corresponding NPD ratio in wild type (0.36); both differences are statistically significant. Together, these data indicate that the msh4 mutation abolishes crossover interference in some intervals and significantly reduces interference in other intervals.

In theory, the observed increases in NPD ratios in the msh4 mutant could be due to an acquisition of chromatid interference instead of a loss of crossover interference. Chromatid interference would increase the number of four-strand double crossovers at the expense of two-strand and three-strand double crossovers. [There is no chromatid interference in wild-type yeast (MORTIMER and FOGEL 1974 ).] Chromatid interference would have to be quite strong to account for the excess of NPDs in the msh4 mutant; for the six intervals that show interference in the wild type, the NPD ratios in wild type average one-quarter those of the msh4 mutant. Thus, all or nearly all of the double crossovers occurring within these intervals in the mutant would have to be four-strand double crossovers. Such strong chromatid interference should be easily detected by examining tetrads with single crossovers in two adjacent intervals to determine the total number of chromatids involved. In both msh4 and wild type, double crossovers conformed to the 1:2:1 ratio of two-strand:three-strand:four-strand events expected in the absence of chromatid interference (Table 3). This analysis rules out the possibility that the increased NPD ratios in msh4 are due to chromatid interference and reinforces the conclusion that the Msh4 protein is required for crossover interference.

Msh4 and Zip1 act in the same recombination pathway:
To determine whether Msh4 and Zip1 operate in the same pathway, meiotic recombination and chromosome segregation were examined in the msh4 zip1 double mutant. The results of tetrad analysis indicate that map distances in the zip1 msh4 double mutant are similar to both single mutants; no statistically significant differences were observed between the double mutant and either single mutant strain for the six intervals examined (Table 4). Also, the frequency and pattern of spore viability in the double mutant were similar to those of the single mutants (Fig 2). In every case, tetrads containing 4, 2, or 0 viable spores were present in excess, compared to tetrads containing 3 or 1 viable spores; this pattern is indicative of meiosis I nondisjunction events (SYM and ROEDER 1994 ). The frequency of chromosome III nondisjunction was estimated from the frequency of two-spore-viable tetrads in which both spores are disomic for chromosome III (and therefore mating incompetent due to heterozygosity at the MAT locus). Such tetrads represented 5.2% of total tetrads in the zip1 strain, 4.9% in the msh4 strain, and 4.4% in the zip1 msh4 strain. Thus, by all three criteria—map distances, spore viability, and chromosome III nondisjunction—Msh4 and Zip1 act in the same pathway.

View larger version (31K): In this window In a new window Download PPT slide   Figure 2. Spore viability in msh4 and zip1 mutant strains. The distribution of four-, three-, two-, one-, and zero-spore-viable tetrads is shown for wild-type (RM97), msh4 (RM99), zip1 (RM98), and msh4 zip1 (RM100) strains. The number of tetrads dissected was 144 for wild type, 528 for msh4, 600 for zip1, and 648 for msh4 zip1.

Chromosome synapsis is delayed and often incomplete in the msh4 mutant:
Although pachytene nuclei have been observed in the msh4 mutant (ROSS-MACDONALD and ROEDER 1994 ), the extent and timing of SC formation were not quantitated. To test the possibility that the msh4 mutation reduces the efficiency of chromosome synapsis, SC formation was examined in msh4 and wild-type cells by staining spread meiotic chromosomes with anti-Zip1 antibodies. Spread nuclei were simultaneously stained with anti-tubulin antibodies to assess spindle formation. The DNA-binding dye DAPI was used to visualize chromosomes. Nuclei were classified according to the extent of Zip1 localization and the presence or absence of a meiosis I spindle as described (Fig 3, A–D; SMITH and ROEDER 1997 ).

View larger version (37K): In this window In a new window Download PPT slide   Figure 3. Chromosome synapsis and spindle formation in a msh4 mutant. msh4 (RM51) and wild-type (RM53) strains were introduced into sporulation medium at time 0; chromosomes were spread at the indicated times. Synapsis was assessed by staining with anti-Zip1 antibodies, and spindles were detected by simultaneous staining with anti-tubulin antibodies. At least 100 nuclei were scored for each strain at each time point. (A–D) Time course of synapsis and spindle formation. Nuclei were divided into several categories to reflect the different stages in meiotic cell cycle progression: no Zip1 staining (not shown), (A) spotty Zip1 staining, (B) partly continuous Zip1 staining (in which a mixture of spots and linear stretches of Zip1 are seen, but Zip1 does not extend along the entirety of the chromosomes), (C) pachytene (in which Zip1 is fully continuous along all chromosomes, except in the nucleolar region), and (D) spindles (indicative of meiotic chromosome segregation). The kinetics of appearance and disappearance of nuclei in the last four categories are shown. (E) Polycomplexes. For each class of Zip1 and tubulin staining, the fraction of spreads containing a polycomplex was assessed. Results shown represent pooled data from the 5.5- and 6.5-hr time points in the data set of Fig 3A–D. (F) Chromosome synapsis during ndt80 arrest. Zip1 and tubulin staining was quantified in an ndt80 strain (JN305) at 9.5 hr and a msh4 ndt80 strain (JN304) at 11.5 hr.

Nuclei with partly continuous Zip1 staining (indicative of incomplete synapsis) appeared at the same time in the mutant as in wild type, but these nuclei were longer lived and accumulated to a higher level in msh4 (Fig 3B). By 4.5 hr, wild-type cells were at the peak of pachytene, while msh4 cells displayed very few pachytene nuclei (Fig 3C). In this and other time courses (not shown), the maximum number of pachytene nuclei was reduced approximately twofold, and the time point at which maximal synapsis was observed was often delayed compared to wild type (Fig 3C). The appearance of nuclei containing meiosis I spindles was consistently delayed in msh4 (Fig 3D).

Polycomplexes are structured aggregates of SC components, including Zip1. Polycomplexes are observed in only a minority of meiotic prophase nuclei from wild type, but they are almost always present in strains overproducing Zip1 and in most mutants defective in SC formation (LOIDL et al. 1994 ; SYM and ROEDER 1995 ). Polycomplexes can be detected as regions of intense Zip1 staining with no DNA staining. In the strain background used for this analysis, a polycomplex was observed in only a fraction of wild-type nuclei (Fig 3E). In the msh4 mutant, however, nuclei with polycomplexes were far more frequent; late in meiotic prophase, nearly all nuclei with fully or partially synapsed chromosomes contained a polycomplex (Fig 3E).

The reduced number of pachytene nuclei observed in the msh4 mutant can be explained in two ways: not all msh4 cells go through pachytene, or each msh4 cell spends an abnormally short time in pachytene. A mutation that induces pachytene arrest, ndt80 (XU et al. 1995 ), was used to distinguish these possibilities. As expected, the ndt80 strain displayed pachytene arrest, with 92% of cells in pachytene (Fig 3F). In contrast, the msh4 ndt80 strain arrested with only 24% pachytene nuclei; 72% of the nuclei displayed partly linear Zip1 staining (Fig 3F). This result suggests that many msh4 cells fail to fully synapse their chromosomes, even when cells are held in midmeiotic prophase.

Although previous results suggested the msh4 mutant is not delayed in meiotic nuclear division (ROSS-MACDONALD and ROEDER 1994 ), the defect in meiotic prophase prompted a careful time course analysis of nuclear division in msh4 and wild-type strains. DAPI was used to visualize nuclei, and the number of mono-, bi-, and tetra-nucleate cells was monitored over time (Fig 4). In the msh4 mutant, the production of both bi-nucleate cells (products of the meiosis I division) and tetra-nucleate cells (products of the meiosis II division) was delayed by 3 hr. Thus, both meiotic divisions are delayed in the msh4 mutant, as expected from the delay in meiotic prophase progression.

View larger version (20K): In this window In a new window Download PPT slide   Figure 4. Time course of meiotic nuclear divisions. Wild-type (RM53) and msh4 (RM51) strains were induced to undergo meiosis. At the indicated times, cells were fixed with formaldehyde and subsequently stained with DAPI. Cells were scored as mono-, bi-, or tetra-nucleate; at least 200 cells were counted for each sample.

Timing and pattern of Msh4 localization relative to Zip1 and Zip2:
The Msh4 protein has been shown to localize to 60 foci on meiotic chromosomes (ROSS-MACDONALD and ROEDER 1994 ). The localization of Msh4 relative to Zip1 was examined in cells (strain JN202) producing Msh4 protein tagged with the hemagglutinin epitope (Msh4-HA). This analysis was carried out in a BR2495 strain background, in which meiosis proceeds more slowly than in the SK1 strains used in the genetic experiments. Spread meiotic nuclei were prepared at 14.5 hr in meiosis; because meiosis is not completely synchronous, nuclei at all stages of meiotic prophase are present at this time. Nuclei were classified as to the extent of synapsis (as in Fig 3) and then examined for Msh4 foci. None (0/20) of the spreads without Zip1 staining showed any Msh4 foci. Of the spreads with spotty Zip1 staining (indicative of the initiation of synapsis), 72% (38/53) had Msh4 foci; the number of Msh4 foci appeared to be higher in nuclei in which Zip1 spots were more intensely staining. Together, these observations suggest that Msh4 localizes to chromosomes shortly after Zip1 assembly initiates. 100% (50/50) of nuclei with partly continuous Zip1 staining (i.e., partially synapsed chromosomes) displayed Msh4 foci. In addition, 100% (120/120) of nuclei with fully synapsed chromosomes had Msh4 foci on chromosomes, demonstrating that Msh4 foci are present throughout pachytene.

Msh4 foci show extensive overlap with Zip3 foci (AGARWAL and ROEDER 2000 ), predicting that Msh4 also colocalizes with Zip2 (since Zip2 and Zip3 colocalize). The extent of overlap between Zip2 and Msh4 was assessed in cells producing Msh4-HA and Zip2 tagged with green fluorescent protein (Zip2-GFP). A typical spread nucleus at pachytene is shown in Fig 5A&NDASH;C. On average, 80% of Msh4 foci overlapped a Zip2 focus, and 66% of Zip2 foci overlapped a Msh4 focus. Random positioning would cause only 32% of Msh4 foci and 26% of Zip2 foci to overlap (see MATERIALS AND METHODS). Therefore, most, but not all, Msh4 and Zip2 foci have the same subnuclear location. Zip2 foci that lack Msh4 were more frequent than Msh4 foci lacking Zip2. This difference could be explained if Zip2 localization normally precedes Msh4 localization; in this case, some of the Zip2 foci observed in spreads could be those to which Msh4 has not yet localized.

View larger version (63K): In this window In a new window Download PPT slide   Figure 5. Localization of Msh4 protein. (A–F) Colocalization of Msh4 and Zip2. HA-tagged Msh4 and GFP-tagged Zip2 were visualized by immunofluorescence. Both tagged proteins are fully functional, as assessed by homozygous tagged strains having wild-type spore viability (J. E. NOVAK, unpublished data; CHUA and ROEDER 1997 ). Msh4 is shown in red (A, D); Zip2 in green (B, E). The images are fused in C and F to show the extent of colocalization. A–C show a wild-type nucleus (JN295); D–F show a zip1 nucleus (JN294). (G–I) Localization of Msh4 in wild type and meiotic mutants. Images show Msh4 in red and DNA (visualized by DAPI) in blue. (G) wild type (JN202); (H) zip2 mutant (JN220); (I) zip1 mutant (JN232). In D, the brightness of Msh4 staining has been artificially increased to permit accurate quantitation of colocalization with Zip2. Bars, 2 µm.

Proper localization of the Msh4 protein depends on Zip1 and Zip2:
To understand better the relationship between Msh4 and the Zip proteins, Msh4 localization was examined in zip1 and zip2 strains, and Zip2 localization was assessed in a msh4 strain. Wild-type and mutant strains were tested at 15 hr, roughly the time of maximum pachytene nuclei and Msh4 localization in the BR2495 strain background used for this analysis. In this strain background, both zip1 and zip2 arrest in prophase of meiosis I (SYM et al. 1993 ; CHUA and ROEDER 1998 ); therefore, the mutants were also tested at 20 hr. To compare cells at a similar stage of meiosis, only spreads with extensive Red1 staining on chromosomes were chosen for analysis. Red1 is a component of the cores of meiotic chromosomes; Red1 localization is maximal at pachytene (SMITH and ROEDER 1997 ).

At 15 hr, Msh4 foci were detectable by immunofluorescence in zip1 and zip2 nuclei, but they were consistently much fainter than those in wild-type nuclei (Fig 5, G–I). Msh4 foci in the zip2 strain tended to be slightly fainter than those in zip1. In a typical experiment, the intensity of Msh4 staining (measured by total intensity in CCD camera images) averaged 200,000 for wild-type nuclei, 70,000 for the zip1 mutant, and 53,000 for the zip2 mutant. At 20 hr, each mutant displayed slightly brighter Msh4 staining than at 15 hr, but still less intense compared to the wild type at 15 hr (not shown). Thus, Msh4 localization is partly dependent on both Zip1 and Zip2. In contrast, Zip2 foci appear normal in pattern and intensity in the msh4 mutant (data not shown), indicating that Zip2 does not depend on Msh4 for its localization to chromosomes.

The abnormal localization of Msh4 in zip1 cells may be caused by a simple reduction in the amount of Msh4 localized to chromosomes or it may reflect a more profound disruption of the normal pattern of localization. To address this issue, a zip1 mutant was examined to determine whether Msh4 still colocalizes extensively with Zip2, which localizes normally in the absence of Zip1 (CHUA and ROEDER 1998 ). Fig 5D&NDASH;F, shows the localization of Msh4 and Zip2 in a typical zip1 mutant nucleus. On average, 54% of Msh4 foci overlapped a Zip2 focus, and 48% of Zip2 foci overlapped a Msh4 focus. These values are substantially less than the corresponding 80% and 66% overlap observed in the wild type, though still higher than the estimate for random overlap (27% of Msh4 and 24% of Zip2 foci). In a zip1 mutant, Zip2 protein localizes to axial associations (CHUA and ROEDER 1998 ), which are connections between aligned homologous chromosomes seen in zip1 mutants (SYM et al. 1993 ). Msh4 did not localize to the majority of axial associations visible in a zip1 mutant (data not shown). Thus, in the absence of Zip1, Msh4 localization is abnormal with respect to Zip2.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A msh4 mutation impairs chromosome synapsis:
Careful analysis of the timing and extent of SC formation in the msh4 mutant has revealed a defect in chromosome synapsis. SC formation is delayed compared to wild type, and full synapsis is achieved in less than half of all nuclei. A polycomplex is formed in nearly all cells, including those in which chromosomes synapse fully, consistent with a decrease in Zip1 localization to chromosomes. The defect/delay in SC formation is accompanied by a delay in both spindle formation and nuclear division. It is likely that the msh4 delay in meiosis reflects the operation of checkpoint triggered by the defect in SC formation or perhaps by an associated defect in the resolution of recombination intermediates (BAILIS and ROEDER 2000 ).

Msh4 is found largely at the same sites on chromosomes as the Zip2 protein. Zip2 is required for Zip1 localization to chromosomes, and Zip2 foci correspond to the sites at which Zip1 begins to polymerize on chromosomes (CHUA and ROEDER 1998 ). The localization of Msh4 to sites of synapsis initiation can account for the observed defect/delay in SC formation in the msh4 mutant. Although Msh4 seems to localize to chromosomes slightly after Zip1, it may play a role in stabilizing the association of Zip1 with chromatin, or it may help organize chromatin-associated Zip1 into SC. Alternatively, msh4 may have a defect in synapsis extension, rather than synapsis initiation. We favor this interpretation for two reasons. First, nuclei with partially synapsed chromosomes appear at the same time in the mutant as they do in wild type, but nuclei of this type persist longer and accumulate to a higher level in the mutant than they do in wild type (Fig 3B). Second, msh4 nuclei often display chromosomes with linear stretches of SC that fail to extend along the full length of the chromosome. This contrasts with the situation observed in the zip3 mutant (defective in synapsis initiation) in which some chromosomes synapse fully while others fail to exhibit any Zip1 staining (AGARWAL and ROEDER 2000 ). Msh4 might affect Zip1 polymerization at a distance by promoting conversion of Zip1 to a conformation that more efficiently polymerizes along chromosomes.

Homologs of the MSH4 and MSH5 genes have been identified in both humans and mice (PAQUIS-FLUCKLINGER et al. 1997 ; HER and DOGGETT 1998 ; WINAND et al. 1998 ; BOCKER et al. 1999 ; DE VRIES et al. 1999 ; EDELMANN et al. 1999 ; KNEITZ et al. 2000 ). The human MSH4 and MSH5 proteins, like the yeast proteins, have been shown to form a complex (WINAND et al. 1998 ; BOCKER et al. 1999 ). Similar to yeast Msh4, the mouse Msh4 protein localizes to discrete foci on meiotic chromosomes during the zygotene and pachytene stages of meiotic prophase (KNEITZ et al. 2000 ). Analysis of Msh4 and Msh5 knockout mice has demonstrated an important role for the proteins in chromosome synapsis (DE VRIES et al. 1999 ; EDELMANN et al. 1999 ; KNEITZ et al. 2000 ). Knockout mice are sterile, and nuclei with fully synapsed chromosomes are never observed. There is a limited amount of SC formation, but this occurs in only a fraction of nuclei and often involves nonhomologous chromosomes. These observations suggest that chromosome synapsis in mice is more dependent on the Msh4-Msh5 complex than is synapsis in yeast.

Msh4 functions in crossing over:
Several observations indicate that Msh4-Msh5-Zip3-Zip2 foci correspond to the sites of meiotic recombination events. First, the formation of these foci appears to require the initiation of meiotic recombination (CHUA and ROEDER 1998 ; AGARWAL and ROEDER 2000 ; our unpublished observations). Second, in a mutant (rad50S) in which meiotic recombination is blocked at an early stage, the Zip2 and Zip3 proteins colocalize with the Mre11 protein; furthermore, Zip3 co-immunoprecipitates with Mre11 from meiotic cell extracts (CHUA and ROEDER 1998 ; AGARWAL and ROEDER 2000 ). Mre11 interacts with Rad50 and Xrs2 to form a complex that is involved in the formation and processing of meiotic double-strand breaks (the initiators of meiotic recombination events; USUI et al. 1998 ). Third, the formation of axial associations (to which Zip2 localizes) requires two strand-exchange proteins, Dmc1 and Rad51 (ROCKMILL et al. 1995 ). Finally, Zip3 interacts physically with Rad51 and Rad57, a cofactor to Rad51-mediated strand exchange (AGARWAL and ROEDER 2000 ).

Other observations indicate that Msh4-Msh5-Zip3-Zip2 foci correspond specifically to the sites of reciprocal recombination events (i.e., crossovers). Null mutations in the genes encoding all four of the known components of these foci reduce crossing over two- to threefold without affecting gene conversion. Furthermore, Msh4-Msh5-Zip3-Zip2 foci, like crossovers, display interference; these complexes are nonrandomly positioned along chromosomes such that two foci rarely occur close together (JENNIFER FUNG and G. S. ROEDER, unpublished data).

The Msh4 protein may be directly involved in crossing over. Homology to MutS proteins suggests that Msh4 has DNA-binding activity. MutS proteins bind duplex DNA, with especially high affinity for DNA containing mismatched base pairs (MODRICH 1987 ; KOLODNER and MARSISCHKY 1999 ). However, at least one of the yeast MutS proteins, Msh2, also binds strongly to Holliday junctions and promotes their resolution in vitro by purified Holliday junction-cleaving enzymes (ALANI et al. 1997 ; MARSISCHKY et al. 1999 ). It is tempting to speculate that Msh4 (and/or its partner, Msh5) binds to Holliday junctions and promotes their resolution in favor of crossing over.

The number of Msh4-Msh5-Zip3-Zip2 foci observed at pachytene (55) is significantly less than the average number of crossovers that occur in a meiotic cell (90). One explanation for this discrepancy is that the foci are transient and asynchronous, such that not all foci are detected at any given point in time. (Note, however, that in ndt80 cells arrested at pachytene, the number of Zip2 foci is still significantly <90.) If Msh4 does normally act at all crossover sites, then loss of Msh4 might have the result that recombination intermediates that would normally generate crossovers are randomly resolved such that only 50% result in crossing over. An alternative possibility is that only about half of all crossovers require Msh4 (and associated proteins) and serve as sites of Msh4 localization; the remaining crossovers occur by a Msh4-independent mechanism (ROSS-MACDONALD and ROEDER 1994 ; NAKAGAWA et al. 1999 ; ZALEVSKY et al. 1999 ). In this case, in the absence of Msh4, all recombination intermediates that normally serve as Msh4 substrates would be resolved without crossing over.

If there is more than one pathway of crossing over, then these pathways might require different gene products. However, all observations to date suggest that all of the gene products that specifically promote meiotic crossing over act in the same pathway. Epistasis tests have shown that Msh5 and Mlh1 (and presumably Mlh3) act in the same pathway as Msh4 (HOLLINGSWORTH et al. 1995 ; HUNTER and BORTS 1997 ). It has been proposed that a Msh4-Msh5 dimer interacts with the Mlh1-Mlh3 dimer to form a tetramer analogous to other MutS-MutL complexes observed in eukaryotic cells (KOLODNER and MARSISCHKY 1999 ; NAKAGAWA et al. 1999 ). Exo1 and probably Mer3 also act in the same pathway as Msh4 (KHAZANEHDARI and BORTS 2000 ; KIRKPATRICK et al. 2000 ; TSUBOUCHI and OGAWA 2000 ; our unpublished observations). Here, we have shown that Msh4 and Zip1 are in the same epistasis group; Zip3 and Zip2 presumably also act in the Msh4-dependent pathway, since these proteins colocalize with Msh4 and Msh5. Thus, if there is a second crossover pathway, gene products that act exclusively in this pathway have yet to be identified.

The role of the Zip1 protein in crossing over has been controversial (SYM et al. 1993 ; SYM and ROEDER 1994 ; STORLAZZI et al. 1996 ; CHUA and ROEDER 1997 ; ROEDER 1997 ). Does this SC building block also participate directly in recombination or does the defect in crossing over in the zip1 mutant result indirectly from an alteration in meiotic chromosome structure? Our analysis of Msh4 localization provides a plausible explanation for the role of Zip1 in crossing over. Since Zip1 is required for localization of Msh4 in wild-type amounts and to the correct locations, the zip1 defect in crossing over might be a consequence of a failure of Msh4 function.

Msh4 plays a role in crossover interference:
Our data indicate that the Msh4 protein plays an important role in crossover distribution. In the absence of Msh4, crossover interference is abolished in some intervals and reduced in others. Below, we consider several possible models for how Msh4 might mediate interference. Note that these models are not mutually exclusive.

One possibility is that msh4 affects interference by impairing SC formation. As described in the Introduction, a number of observations suggest that the interference signal is transmitted along the SC. The finding that a msh4 mutation impairs both synapsis and interference provides additional support for this hypothesis. The incomplete and delayed SC formation observed in the msh4 mutant could limit transmission of the interference signal along chromosomes. The simplest version of this model is that zippering up of the SC serves as the mechanistic basis for signal transmission (EGEL 1978 ; MAGUIRE 1988 ). Synapsis initiates at sites designated to become crossovers, and SC zippering extends outward (probably in both directions) from each site. Subsequent crossovers (or commitments to crossing over) are inhibited in nearby chromosomal regions that have already synapsed.

The view that SC zippering serves as the mechanistic basis for transmission of the interference signal is a specific version of a more general class of models in which a crossover initiates a structural change that prevents subsequent crossing over and is transmitted along the chromosome in a time-dependent manner. In the model proposed by KING and MORTIMER 1990 , a crossover (or commitment to crossing over) promotes polymerization of a recombination inhibitor along the chromosome. In the model of KABACK et al. 1999 , crossing over triggers a conformational chain reaction in which a chromosomal component undergoes an allosteric change that both blocks recombination and causes a neighboring component to undergo the same change in conformation. Yet another possibility is that transmission of the interference signal involves changes in chromatin structure; Kleckner and colleagues have suggested that this might occur by a crossover-induced relief of physical stress (KLECKNER 1996 ; STORLAZZI et al. 1996 ). By any of these models, if the proposed structural change in the chromosome must occur within the context of SC, then the msh4 defect in interference might still be attributed to the defect in synapsis. Alternatively, Msh4 might be involved in initiating (rather than transmitting) the structural change that travels along the chromosome. Msh4 could play such a role regardless of whether signal transmission involves SC zippering, polymerization of a recombination inhibitor, conformational change in a chromosomal protein, or release of tension.

Another possible explanation for the effect of msh4 on interference assumes the operation of two distinct pathways of crossing over. According to this model, crossovers in one pathway require Msh4 and display interference, whereas crossovers in the other pathway are independent of Msh4 and do not exhibit interference. The msh4 mutation would thus eliminate interference by inactivating the first pathway. This model accounts neatly for the observation that the HOM3–TRP2 interval shows no reduction in crossing over in msh4 and no interference in wild type; presumably, all of the crossovers in this interval are of the Msh4-independent variety. In this context, it is interesting to note that mutation of the Caenorhabditis elegans homolog of MSH4 or MSH5 completely abolishes crossing over (ZALEVSKY et al. 1999 ; KELLY et al. 2000 ), indicating that all crossovers occur by a Msh4-dependent mechanism in this organism. Furthermore, interference in worms (and many other eukaryotes) is much stronger than it is in yeast. This difference in the strength of interference across species can be economically explained by proposing that Msh4-dependent crossovers (which display interference) are diluted by Msh4-independent crossovers (that lack interference) in budding yeast but not in worms (or most eukaryotes).

A very different explanation for crossover interference is suggested by the counting model of Stahl and colleagues (FOSS et al. 1993 ; FOSS and STAHL 1995 ). According to this model, adjacent crossovers must be separated by a specific number of noncrossover recombination events (two in the case of yeast; FOSS et al. 1993 ). A test of this model indicated that data from budding yeast cannot be adequately explained by such a counting mechanism (FOSS and STAHL 1995 ). However, if only a subset of crossovers exhibits interference (as proposed above), then the counting model could still apply. Counting might be due to the clustering of multiple recombination intermediates (three in yeast) to produce a single late recombination nodule, with only one intermediate in each such nodule being resolved in favor of crossing over (STAHL 1993 ). In the context of the counting model, Msh4 might be required for clustering, to promote crossing over within a nodule, or to designate which of the intermediates within a cluster is chosen to be resolved in favor of crossing over.

Summary:
Msh4, a protein known to be involved in meiotic crossing over, is also required for crossover interference. Furthermore, Msh4 localizes to sites of synapsis initiation and promotes SC formation. Msh4 therefore adds to the growing list of proteins that promote both interference and synapsis, providing support for the hypothesis that the SC is required for interference.

	FOOTNOTES

¹ Current address: Bristol-Myers Squibb, Applied Genomics, P.O. Box 5400, Princeton, NJ 08543-5400.

	ACKNOWLEDGMENTS

We are grateful to Christopher Sarnowski for developing programs used for genetic analysis; to Jennifer Fung, Laurent Maloisel, Beth Rockmill, and Hideo Tsubouchi for comments on the manuscript; and to Franklin Stahl, Penelope Chua, and Pedro San-Segundo for comments on a previous version of the manuscript. This work was supported by National Institutes of Health (NIH) grant GM28904 to G.S.R., by the Howard Hughes Medical Institute, and NIH National Research Service Award GM17654 to J.E.N.

Manuscript received February 9, 2001; Accepted for publication April 24, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AGARWAL, S. and G. S. ROEDER, 2000  Zip3 provides a link between recombination enzymes and synaptonemal complex proteins. Cell 102:245-255[Medline].

ALANI, E., S. LEE, M. F. KANE, J. GRIFFITH, and R. D. KOLODNER, 1997  Saccharomyces cerevisiae MSH2, a mispaired base recognition protein, also recognizes Holliday junctions in DNA. J. Mol. Biol. 265:289-301[Medline].

BAHLER, J., T. WYLER, J. LOIDL, and J. KOHLI, 1993  Unusual nuclear structures in meiotic prophase of fission yeast: a cytological analysis. J. Cell Biol. 121:241-256[Abstract/Free Full Text].

BAILIS, J. M. and G. S. ROEDER, 2000  The pachytene checkpoint. Trends Genet. 16:395-403[Medline].

BOCKER, T., A. BARUSEVICIUS, T. SNOWDEN, D. RASIO, and S. GUERRETTE et al., 1999  hMSH5: a human MutS homologue that forms a novel heterodimer with hMSH4 and is expressed during spermatogenesis. Cancer Res. 59:816-822[Abstract/Free Full Text].

CHU, S., J. DERISI, M. EISEN, J. MULHOLLAND, and D. BOTSTEIN et al., 1998  The transcriptional program of sporulation in budding yeast. Science 282:699-703[Abstract/Free Full Text].

CHUA, P. R. and G. S. ROEDER, 1997  Tam1, a telomere-associated meiotic protein, functions in chromosome synapsis and crossover interference. Genes Dev. 11:1786-1800[Abstract/Free Full Text].

CHUA, P. R. and G. S. ROEDER, 1998  Zip2, a meiosis-specific protein required for the initiation of chromosome synapsis. Cell 93:349-359[Medline].

CONRAD, M. N., A. M. DOMINGUEZ, and M. E. DRESSER, 1997  Ndj1, a meiotic telomere protein required for normal chromosome synapsis and segregation in yeast. Science 276:1252-1255[Abstract/Free Full Text].

DE VRIES, S. S., E. B. BAART, M. DEKKER, A. SIEZEN, and D. G. DE ROOIJ et al., 1999  Mouse MutS-like protein Msh5 is required for proper chromosome synapsis in male and female meiosis. Genes Dev. 13:523-531[Abstract/Free Full Text].

DONG, H. and G. S. ROEDER, 2000  Organization of the yeast Zip1 protein within the central region of the synaptonemal complex. J. Cell Biol. 148:417-426[Abstract/Free Full Text].

EDELMANN, W., P. E. COHEN, B. KNEITZ, N. WINAND, and M. LIA et al., 1999  Mammalian MutS homologue 5 is required for chromosome pairing in meiosis. Nat. Genet. 21:123-127[Medline].

EGEL, R., 1978  Synaptonemal complex and crossing-over: structural support or interference? Heredity 41:233-237[Medline].

EGEL-MITANI, M., L. W. OLSON, and R. EGEL, 1982  Meiosis in Aspergillus nidulans: another example for lacking synaptonemal complexes in the absence of crossover interference. Hereditas 97:179-187[Medline].

ENGEBRECHT, J. and G. S. ROEDER, 1990  MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis. Mol. Cell. Biol. 10:2379-2389[Abstract/Free Full Text].

FIORENTINI, P., K. N. HUANG, D. X. TISHKOFF, R. D. KOLODNER, and L. S. SYMINGTON, 1997  Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro. Mol. Cell. Biol. 17:2764-2773[Abstract].

FOSS, E., R. LANDE, F. W. STAHL, and C. M. STEINBERG, 1993  Chiasma interference as a function of genetic distance. Genetics 133:681-691[Abstract].

FOSS, E. J. and F. W. STAHL, 1995  A test of a counting model for chiasma interference. Genetics 139:1201-1209[Abstract].

HAVEKES, F. W., J. H. DE JONG, C. HEYTING, and M. S. RAMANNA, 1994  Synapsis and chiasma formation in four meiotic mutants of tomato (Lycopersicon esculentum). Chromosome Res. 2:315-325[Medline].

HER, C. and N. A. DOGGETT, 1998  Cloning, structural characterization, and chromosomal localization of the human orthologue of Saccharomyces cerevisiae MSH5 gene. Genomics 52:50-61[Medline].

HOLLINGSWORTH, N. M., L. PONTE, and C. HALSEY, 1995  MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair. Genes Dev. 9:1728-1739[Abstract/Free Full Text].

HUANG, K. N. and L. S. SYMINGTON, 1993  A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology. Mol. Cell. Biol. 13:3125-3134[Abstract/Free Full Text].

HUNTER, N. and R. H. BORTS, 1997  Mlh1 is unique among mismatch repair proteins in its ability to promote crossing over during meiosis. Genes Dev. 11:1573-1582[Abstract/Free Full Text].

KABACK, D. B., D. BARBER, J. MAHON, J. LAMB, and J. YOU, 1999  Chromosome size-dependent control of meiotic reciprocal recombination in Saccharomyces cerevisiae: the role of crossover interference. Genetics 152:1475-1486[Abstract/Free Full Text].

KANE, S. M. and R. ROTH, 1974  Carbohydrate metabolism during ascospore development in yeast. J. Bacteriol. 118:8-14[Abstract/Free Full Text].

KELLY, K. O., A. F. DERNBURG, G. M. STANFIELD, and A. M. VILLENEUVE, 2000  Caenorhabditis elegans msh-5 is required for both normal and radiation-induced meiotic crossing over but not for completion of meiosis. Genetics 156:617-630[Abstract/Free Full Text].

KHAZANEHDARI, K. A. and R. H. BORTS, 2000  EXO1 and MSH4 differentially affect crossing-over and segregation. Chromosoma 109:94-102[Medline].

KILMARTIN, J. V., B. WRIGHT, and C. MILSTEIN, 1982  Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J. Cell Biol. 93:576-582[Abstract/Free Full Text].

KING, J. S. and R. K. MORTIMER, 1990  A polymerization model of chiasma interference and corresponding computer simulation. Genetics 126:1127-1138[Abstract].

KIRKPATRICK, D. T., J. R. FERGUSON, T. D. PETES, and L. S. SYMINGTON, 2000  Decreased meiotic intergenic recombination and increased meiosis I nondisjunction in exo1 mutants of Saccharomyces cerevisiae.. Genetics 156:1549-1557[Abstract/Free Full Text].

KLECKNER, N., 1996  Meiosis: how could it work? Proc. Natl. Acad. Sci. USA 93:8167-8174[Abstract/Free Full Text].

KNEITZ, B., P. E. COHEN, E. AVDIEVICH, L. ZHU, and M. F. KANE et al., 2000  MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice. Genes Dev. 14:1085-1097[Abstract/Free Full Text].

KOLODNER, R. D. and G. T. MARSISCHKY, 1999  Eukaryotic DNA mismatch repair. Curr. Opin. Genet. Dev. 9:89-96[Medline].

LOIDL, J., F. KLEIN, and H. SCHERTHAN, 1994  Homologous pairing is reduced but not abolished in asynaptic mutants of yeast. J. Cell Biol. 125:1191-1200[Abstract/Free Full Text].

MAGUIRE, M. P., 1988  Crossover site determination and interference. J. Theor. Biol. 134:565-570[Medline].

MARSISCHKY, G. T., S. LEE, J. GRIFFITH, and R. D. KOLODNER, 1999  Saccharomyces cerevisiae MSH2/6 complex interacts with Holliday junctions and facilitates their cleavage by phage resolution enzymes. J. Biol. Chem. 274:7200-7206[Abstract/Free Full Text].

MODRICH, P., 1987  DNA mismatch correction. Annu. Rev. Biochem. 56:435-466[Medline].

MOENS, P. B., 1969  Genetic and cytological effects of three desynaptic genes in the tomato. Can. J. Genet. Cytol. 11:857-869.

MORTIMER, R., and S. FOGEL, 1974 Genetical interference and gene conversion, pp. 263–275 in Mechanisms in Recombination, edited by R. GRELL. Plenum Press, New York.

NAKAGAWA, T. and H. OGAWA, 1999  The Saccharomyces cerevisiae MER3 gene, encoding a novel helicase-like protein, is required for crossover control in meiosis. EMBO J. 18:5714-5723[Medline].

NAKAGAWA, T., A. DATTA, and R. D. KOLODNER, 1999  Multiple functions of MutS- and MutL-related heterocomplexes. Proc. Natl. Acad. Sci. USA 96:14186-14188[Free Full Text].

OLSON, L. W. and F. K. ZIMMERMANN, 1978  Meiotic recombination and synaptonemal complexes in Saccharomyces cerevisiae.. Mol. Gen. Genet. 166:151-159[Medline].

PAPAZIAN, H. P., 1952  The analysis of tetrad data. Genetics 37:175-188[Free Full Text].

PAQUIS-FLUCKLINGER, V., S. SANTUCCI-DARMANIN, R. PAUL, A. SAUNIERES, and C. TURC-CAREL et al., 1997  Cloning and expression analysis of a meiosis-specific MutS homolog: the human MSH4 gene. Genomics 44:188-194[Medline].

POCHART, P., D. WOLTERING, and N. HOLLINGSWORTH, 1997  Conserved properties between functionally distinct MutS homologs in yeast. J. Biol. Chem. 272:30345-30349[Abstract/Free Full Text].

ROCKMILL, B., M. SYM, H. SCHERTHAN, and G. S. ROEDER, 1995  Roles for two RecA homologs in promoting meiotic chromosome synapsis. Genes Dev. 9:2684-2695[Abstract/Free Full Text].

ROEDER, G. S., 1997  Meiotic chromosomes: it takes two to tango. Genes Dev. 11:2600-2621[Free Full Text].

ROSS-MACDONALD, P. and G. S. ROEDER, 1994  Mutation of a meiosis-specific MutS homolog decreases crossing over but not mismatch correction. Cell 79:1069-1080[Medline].

ROSS-MACDONALD, P., A. SHEEHAN, G. S. ROEDER, and M. SNYDER, 1997  A multipurpose transposon system for analyzing protein production, localization, and function in Saccharomyces cerevisiae.. Proc. Natl. Acad. Sci. USA 94:190-195[Abstract/Free Full Text].

ROTHSTEIN, R., 1991  Targeting, disruption, replacement and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 194:281-301[Medline].

SHERMAN, F., G. R. FINK and J. B. HICKS, 1986 Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SIKORSKI, R. and P. HIETER, 1989  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.. Genetics 122:19-27[Abstract/Free Full Text].

SMITH, A. V. and G. S. ROEDER, 1997  The yeast Red1 protein localizes to the cores of meiotic chromosomes. J. Cell Biol. 136:957-967[Abstract/Free Full Text].

SNOW, R., 1979  Maximum likelihood estimation of linkage and interference from tetrad data. Genetics 92:231-245[Abstract/Free Full Text].

STAHL, F. W., 1993 Genetic recombination: thinking about it in phage and fungi, pp. 1–9 in The Chromosome, edited by J. S. HESLOP-HARRISON and R. B. FLAVELL. BIOS Scientific Publishers, London.

STORLAZZI, A., L. XU, A. SCHWACHA, and N. KLECKNER, 1996  Synaptonemal complex (SC) component Zip1 plays a role in meiotic recombination independent of SC polymerization along the chromosomes. Proc. Natl. Acad. Sci. USA 93:9043-9048[Abstract/Free Full Text].

STRICKLAND, W. N., 1958  An analysis of interference in Aspergillus nidulans.. Proc. R. Soc. Lond. Ser. B 149:82-102[Medline].

SYM, M. and G. S. ROEDER, 1994  Crossover interference is abolished in the absence of a synaptonemal complex protein. Cell 79:283-292[Medline].

SYM, M. and G. S. ROEDER, 1995  Zip1-induced changes in synaptonemal complex structure and polycomplex assembly. J. Cell Biol. 128:455-466[Abstract/Free Full Text].

SYM, M., J. ENGEBRECHT, and G. S. ROEDER, 1993  ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis. Cell 72:365-378[Medline].

TISHKOFF, D. X., A. L. BOERGER, P. BERTRAND, N. FILOSI, and G. M. GAIDA et al., 1997  Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2. Proc. Natl. Acad. Sci. USA 94:7487-7492[Abstract/Free Full Text].

TSUBOUCHI, H. and H. OGAWA, 2000  Exo1 roles for repair of DNA double-strand breaks and meiotic crossing over in Saccharomyces cerevisiae.. Mol. Biol. Cell 11:2221-2233[Abstract/Free Full Text].

TUNG, K.-S. and G. S. ROEDER, 1998  Meiotic chromosome morphology and behavior in zip1 mutants of Saccharomyces cerevisiae.. Genetics 149:817-832[Abstract/Free Full Text].

TUNG, K.-S., E.-J. E. HONG, and G. S. ROEDER, 2000  The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80. Proc. Natl. Acad. Sci. USA 97:12187-12192[Abstract/Free Full Text].

USUI, T., T. OHTA, H. OSHIUMI, J.-I. TOMIZAWA, and H. OGAWA et al., 1998  Complex formation and functional versatility of Mre11 of budding yeast in recombination. Cell 95:705-716[Medline].

WACH, A., A. BRACHAT, R. POHLMANN, and P. PHILIPPSEN, 1994  New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae.. Yeast 10:1793-1808[Medline].

WANG, T.-F., N. KLECKNER, and N. HUNTER, 1999  Functional specificity of MutL homologs in yeast: evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction. Proc. Natl. Acad. Sci. USA 96:13914-13919[Abstract/Free Full Text].

WINAND, N. J., J. A. PANZER, and R. D. KOLODNER, 1998  Cloning and characterization of the human and Caenorhabditis elegans homologs of the Saccharomyces cerevisiae MSH5 gene. Genomics 53:69-80[Medline].

XU, L., M. AJIMURA, R. PADMORE, C. KLEIN, and N. KLECKNER, 1995  NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae.. Mol. Cell. Biol. 15:6572-6581[Abstract].

ZALEVSKY, J., A. J. MACQUEEN, J. B. DUFFY, K. J. KEMPHUES, and A. M. VILLENEUVE, 1999  Crossing over during Caenorhabditis elegans meiosis requires a conserved MutS-based pathway that is partially dispensable in budding yeast. Genetics 153:1271-1283[Abstract/Free Full Text].

This article has been cited by other articles:

				T. Tsubouchi, A. J. MacQueen, and G. S. Roeder Initiation of meiotic chromosome synapsis at centromeres in budding yeast Genes & Dev., November 15, 2008; 22(22): 3217 - 3226. [Abstract] [Full Text] [PDF]

				T. J. Getz, S. A. Banse, L. S. Young, A. V. Banse, J. Swanson, G. M. Wang, B. L. Browne, H. M. Foss, and F. W. Stahl Reduced Mismatch Repair of Heteroduplexes Reveals "Non"-interfering Crossing Over in Wild-Type Saccharomyces cerevisiae Genetics, March 1, 2008; 178(3): 1251 - 1269. [Abstract] [Full Text] [PDF]

				T. Snowden, K.-S. Shim, C. Schmutte, S. Acharya, and R. Fishel hMSH4-hMSH5 Adenosine Nucleotide Processing and Interactions with Homologous Recombination Machinery J. Biol. Chem., January 4, 2008; 283(1): 145 - 154. [Abstract] [Full Text] [PDF]

				N. Mitra and G. S. Roeder A Novel Nonnull ZIP1 Allele Triggers Meiotic Arrest With Synapsed Chromosomes in Saccharomyces cerevisiae Genetics, June 1, 2007; 176(2): 773 - 787. [Abstract] [Full Text] [PDF]

				E. de Boer, P. Stam, A. J. J. Dietrich, A. Pastink, and C. Heyting Two levels of interference in mouse meiotic recombination PNAS, June 20, 2006; 103(25): 9607 - 9612. [Abstract] [Full Text] [PDF]

				H. Wang and C. Hoog Structural damage to meiotic chromosomes impairs DNA recombination and checkpoint control in mammalian oocytes J. Cell Biol., May 22, 2006; 173(4): 485 - 495. [Abstract] [Full Text] [PDF]

				A. J. Bartrand, D. Iyasu, S. M. Marinco, and G. S. Brush Evidence of Meiotic Crossover Control in Saccharomyces cerevisiae Through Mec1-Mediated Phosphorylation of Replication Protein A Genetics, January 1, 2006; 172(1): 27 - 39. [Abstract] [Full Text] [PDF]

				N. K. Kolas, A. Svetlanov, M. L. Lenzi, F. P. Macaluso, S. M. Lipkin, R. M. Liskay, J. Greally, W. Edelmann, and P. E. Cohen Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I J. Cell Biol., November 7, 2005; 171(3): 447 - 458. [Abstract] [Full Text] [PDF]

				M. Oliver-Bonet, P.J. Turek, F. Sun, E. Ko, and R.H. Martin Temporal progression of recombination in human males Mol. Hum. Reprod., July 1, 2005; 11(7): 517 - 522. [Abstract] [Full Text] [PDF]

				F. A.T. de Vries, E. de Boer, M. van den Bosch, W. M. Baarends, M. Ooms, L. Yuan, J.-G. Liu, A. A. van Zeeland, C. Heyting, and A. Pastink Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation Genes & Dev., June 1, 2005; 19(11): 1376 - 1389. [Abstract] [Full Text] [PDF]

				S. Y. Lam, S. R. Horn, S. J. Radford, E. A. Housworth, F. W. Stahl, and G. P. Copenhaver Crossover Interference on Nucleolus Organizing Region-Bearing Chromosomes in Arabidopsis Genetics, June 1, 2005; 170(2): 807 - 812. [Abstract] [Full Text] [PDF]

				T. L. Peoples-Holst and S. M. Burgess Multiple branches of the meiotic recombination pathway contribute independently to homolog pairing and stable juxtaposition during meiosis in budding yeast Genes & Dev., April 1, 2005; 19(7): 863 - 874. [Abstract] [Full Text] [PDF]

				J. L. Argueso, J. Wanat, Z. Gemici, and E. Alani Competing Crossover Pathways Act During Meiosis in Saccharomyces cerevisiae Genetics, December 1, 2004; 168(4): 1805 - 1816. [Abstract] [Full Text] [PDF]

				S. Neyton, F. Lespinasse, P. B. Moens, R. Paul, P. Gaudray, V. Paquis-Flucklinger, and S. Santucci-Darmanin Association between MSH4 (MutS homologue 4) and the DNA strand-exchange RAD51 and DMC1 proteins during mammalian meiosis Mol. Hum. Reprod., December 1, 2004; 10(12): 917 - 924. [Abstract] [Full Text] [PDF]

				J. Loidl and H. Scherthan Organization and pairing of meiotic chromosomes in the ciliate Tetrahymena thermophila J. Cell Sci., November 15, 2004; 117(24): 5791 - 5801. [Abstract] [Full Text] [PDF]

				E. Sanchez-Moran, G. H. Jones, F. C. H. Franklin, and J. L. Santos A Puromycin-Sensitive Aminopeptidase Is Essential for Meiosis in Arabidopsis thaliana PLANT CELL, November 1, 2004; 16(11): 2895 - 2909. [Abstract] [Full Text] [PDF]

				J. D. Higgins, S. J. Armstrong, F. C. H. Franklin, and G. H. Jones The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis Genes & Dev., October 15, 2004; 18(20): 2557 - 2570. [Abstract] [Full Text] [PDF]

				H. Bhuiyan and K. Schmekel Meiotic Chromosome Synapsis in Yeast Can Occur Without Spo11-Induced DNA Double-Strand Breaks Genetics, October 1, 2004; 168(2): 775 - 783. [Abstract] [Full Text] [PDF]

				F. W. Stahl, H. M. Foss, L. S. Young, R. H. Borts, M. F. F. Abdullah, and G. P. Copenhaver Does Crossover Interference Count in Saccharomyces cerevisiae? Genetics, September 1, 2004; 168(1): 35 - 48. [Abstract] [Full Text] [PDF]

				L. Maloisel, J. Bhargava, and G. S. Roeder A Role for DNA Polymerase {delta} in Gene Conversion and Crossing Over During Meiosis in Saccharomyces cerevisiae Genetics, July 1, 2004; 167(3): 1133 - 1142. [Abstract] [Full Text] [PDF]

				K. A. Borkovich, L. A. Alex, O. Yarden, M. Freitag, G. E. Turner, N. D. Read, S. Seiler, D. Bell-Pedersen, J. Paietta, N. Plesofsky, et al. Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism Microbiol. Mol. Biol. Rev., March 1, 2004; 68(1): 1 - 108. [Abstract] [Full Text] [PDF]

				N. M. Hollingsworth and S. J. Brill The Mus81 solution to resolution: generating meiotic crossovers without Holliday junctions Genes & Dev., January 15, 2004; 18(2): 117 - 125. [Full Text] [PDF]

				T. de los Santos, N. Hunter, C. Lee, B. Larkin, J. Loidl, and N. M. Hollingsworth The Mus81/Mms4 Endonuclease Acts Independently of Double-Holliday Junction Resolution to Promote a Distinct Subset of Crossovers During Meiosis in Budding Yeast Genetics, May 1, 2003; 164(1): 81 - 94. [Abstract] [Full Text] [PDF]

				M. Shinohara, K. Sakai, A. Shinohara, and D. K. Bishop Crossover Interference in Saccharomyces cerevisiae Requires a TID1/RDH54- and DMC1-Dependent Pathway Genetics, April 1, 2003; 163(4): 1273 - 1286. [Abstract] [Full Text] [PDF]

				J. L. Argueso, A. W. Kijas, S. Sarin, J. Heck, M. Waase, and E. Alani Systematic Mutagenesis of the Saccharomyces cerevisiae MLH1 Gene Reveals Distinct Roles for Mlh1p in Meiotic Crossing Over and in Vegetative and Meiotic Mismatch Repair Mol. Cell. Biol., February 1, 2003; 23(3): 873 - 886. [Abstract] [Full Text]

				E. R. Hoffmann, P. V. Shcherbakova, T. A. Kunkel, and R. H. Borts MLH1 Mutations Differentially Affect Meiotic Functions in Saccharomyces cerevisiae Genetics, February 1, 2003; 163(2): 515 - 526. [Abstract] [Full Text] [PDF]

				A. J. MacQueen, M. P. Colaiacovo, K. McDonald, and A. M. Villeneuve Synapsis-dependent and -independent mechanisms stabilize homolog pairing during meiotic prophase in C. elegans Genes & Dev., September 15, 2002; 16(18): 2428 - 2442. [Abstract] [Full Text] [PDF]

				T. Nakagawa and R. D. Kolodner The MER3 DNA Helicase Catalyzes the Unwinding of Holliday Junctions J. Biol. Chem., July 26, 2002; 277(31): 28019 - 28024. [Abstract] [Full Text] [PDF]

				S. Santucci-Darmanin, S. Neyton, F. Lespinasse, A. Saunieres, P. Gaudray, and V. Paquis-Flucklinger The DNA mismatch-repair MLH3 protein interacts with MSH4 in meiotic cells, supporting a role for this MutL homolog in mammalian meiotic recombination Hum. Mol. Genet., July 15, 2002; 11(15): 1697 - 1706. [Abstract] [Full Text] [PDF]

				H. Tsubouchi and G. S. Roeder The Mnd1 Protein Forms a Complex with Hop2 To Promote Homologous Chromosome Pairing and Meiotic Double-Strand Break Repair Mol. Cell. Biol., May 1, 2002; 22(9): 3078 - 3088. [Abstract] [Full Text] [PDF]