Mnd1p: An evolutionarily conserved protein required for meiotic recombination

Abstract

We used a functional genomics approach to identify a gene required for meiotic recombination, YGL183c or MND1. MND1 was spliced in meiotic cells, extending the annotated YGL183c ORF N terminus by 45 aa. Saccharomyces cerevisiae mnd1–1 mutants, in which the majority of the MND1 coding sequence was removed, arrested before the first meiotic division with a phenotype reminiscent of dmc1 mutants. Physical and genetic analysis showed that these cells initiated recombination, but did not form heteroduplex DNA or double Holliday junctions, suggesting that Mnd1p is involved in strand invasion. Orthologs of MND1 were identified in protists, several yeasts, plants, and mammals, suggesting that its function has been conserved throughout evolution.

Sexually reproducing organisms have a specialized developmental pathway for gametogenesis in which diploid cells undergo meiosis to produce haploid germ cells. Before the first meiotic division, while cells contain replicated pairs of homologs, recombination occurs. The process of recombination serves at least three purposes: (i) to provide an opportunity for damage to homologs to be repaired, (ii) to generate diversity, and (iii) to facilitate proper chromosome segregation.

Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs) made by the Spo11p endonuclease (1, 2). The 5′ ends of these breaks are then resected (3) in a process involving RAD50/MRE11. Several genes, including RAD51 and DMC1, promote invasion of the homologous chromosome by a 3′ single-stranded DNA end. After DNA synthesis, second-end capture, and ligation, double Holliday junctions or joint molecules (JMs) that contain heteroduplex DNA (hDNA) are formed (4, 5). For each JM, resolution is required for the completion of recombination and proper chromosome separation at the first meiotic division. In the original DSB repair model, crossover (CO) and noncrossover (NCO) recombinants are proposed to be alternatively resolved products of a single intermediate JM species (6). However, recent kinetic evidence suggests that CO and NCO products can be produced from different intermediates (7).

Using a functional genomics approach, we identified a meiosis-specific protein in S. cerevisiae that is required for recombination. The gene encoding this activity is YGL183c, or MND1 [Meiotic Nuclear Divisions (8)]. Sequence of the cDNA corresponding to MND1 revealed that the meiotic transcript was spliced, yielding a coding sequence 45 aa longer than the annotated YGL183c ORF. mnd1–1 mutants in which base pairs 136–661 of correct MND1 gene sequence had been deleted arrested before the first meiotic division. These cells initiated recombination but did not form hDNA or JMs. Our results suggest that Mnd1p is required for stable hDNA formation. We identified orthologs of MND1 in protists, several yeasts, Arabidopsis thaliana, Mus musculus, and Homo sapiens, suggesting that the function of Mnd1p has been conserved throughout two billion years of evolution.

Materials and Methods

Strains.

The plasmid for replacement of base pairs 136–661 of YGL183c with the gene for kanamycin resistance was obtained from the Eurofan I deletion project at the Institute for Microbiology at Johann Wolfgang Goethe-University Frankfurt, Frankfurt. This mutation is referred to as mnd1–1. KBY80 (leu2∷hisG/leu2∷hisG ho∷LYS2 lys2/lys2, ura3/ura3) was the wild-type (wt) diploid SK1 strain used for assaying nuclear division. JG702 and JG802 are isogenic with KBY80 with mnd1–1∷kan^r/mnd1–1∷kan^r and dmc1∷LEU2/dmc1∷LEU2, respectively. JG721, JG710, and JG709 are all isogenic to JG702 with spo11∷URA3/spo11∷URA3,spo13∷hisG/spo13∷hisG, and rad17∷hisG-URA3/rad17∷hisG-URA3, respectively. For measuring heteroallele recombination in the SK1 background, the wt diploid strain JG751 was used (arg4-Nsp/arg4-Bgl his4-B/his4-X leu2/leu2 trp1/trp1 ura3/ura3) (9). JG750 is isogenic to JG751 with mnd1–1∷kan^r/mnd1–1∷kan^r. For the physical analysis of DSBs at HIS4 in S288c, the diploid wt strain QFY105 was used (trp1/TRP1 arg4/ARG4 tyr7/TYR7 ade6/ade6 ura3/ura3 LEU2/leu2 HIS4/his4-IR9 rad50S-URA3/rad50S-URA3) (10). JG166 is isogenic to QFY105 except for the HIS4 gene (HIS4/his4-AAG) (11) and mnd1–1∷kan^r/mnd1–1∷kan^r. For the physical analysis of hDNA at HIS4 in S288c, the diploid wt strain DNY86 was used (trp1/TRP1 arg4/ARG4 tyr7/TYR7 ade6/ade6 ura3/ura3 LEU2/leu2 his4-IR15/his4-IR16) (12). JG190 is isogenic to DNY86 with mnd1–1∷kan^r/mnd1–1∷kan^r. For measuring heteroallele recombination in S288c, the wt diploid strain used was MS1. This strain is isogenic with DNY86 with the exception of the HIS4 locus (his4–713/his4-Sal) (12). JG197 is isogenic with mnd1–1∷kan^r/mnd1–1∷kan^r. For the analysis of DSBs, COs, and JMs in SK1, we used MJL2442 (7). JG774, JG820, and JG819 are all isogenic to MJL2442 with mnd1–1∷kan^r/mnd1–1∷kan^r, dmc1∷LEU2/dmc1∷ARG4, or both, respectively.

Sporulation Conditions.

For S288c strains, cultures grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) overnight at 30°C were diluted into SPS (0.5% yeast extract, 1.0% peptone, 0.17% yeast nitrogen base, 1.0% KOAc, 0.5% ammonium sulfate, 0.05 M potassium phthalate) to OD₆₀₀ = 0.2 and shaken for 18–20 h at 30°C. Cells were collected by centrifugation, washed once with 1% KOAc, and then resuspended in the same volume of 1% KOAc as the SPS culture. Cultures were shaken at room temperature and aliquots were removed at the indicated times. For SK1 strains, YPD overnight cultures were diluted into YPA (1% KOAc, 2% peptone, 1% yeast extract) to OD₆₀₀ = 0.2 and shaken for 14–16 h at 30°C. Cells were collected by centrifugation, washed once with SPM (1% KOAc, 0.02% raffinose), and then resuspended in the same volume of SPM as the YPA culture. Cultures were shaken at 30°C and aliquots were removed at the indicated times. For assaying nuclear division, formaldehyde was added to 4.5% for at least 1 h before proceeding with the staining protocol. For return-to-growth experiments, cells were diluted and plated on synthetic dextrose (SD) complete medium to monitor cell viability or on SD medium lacking histidine or arginine to detect recombination. For preparation of RNA and DNA, cells were collected by centrifugation, frozen in liquid nitrogen, and stored at −80°C until processed further.

Assaying for Nuclear Division.

After incubation in formaldehyde, cells were washed twice in PBS, resuspended in 1 μg/ml 4′,6-diamindine-2-phenylindole (DAPI) in PBS, and incubated for 12 min in the dark. Cells were washed twice with PBS, resuspended in PBS, and imaged with an Olympus BX60 fluorescence microscope.

DNA Preparation.

For one-dimensional (1D) gels, genomic DNA was prepared essentially as described (12), with the addition of a phenol:chloroform extraction before the final ethanol precipitation. For two-dimensional (2D) gels, genomic DNA was prepared exactly as described (13).

RNA and cDNA Preparation.

Poly(A)⁺ RNA from KBY80 at 6 h in sporulation was prepared as described (14). Reverse transcription was carried out with 400 ng of poly(A)⁺ RNA and primer 1 (5′-ACCACATTTTTCCACCGAA). The resulting cDNA was used as a template in a PCR containing primer 1 and primer 2 (5′-CCACCGTTATTCTTTGCGAT).

Southern Blotting.

Standard Southern blotting protocols were used (15). Visualization and quantitation of bands was accomplished with a PhosphorImager and imagequant software. The probe for the Southern blot found in Fig. 4 B and C was a PCR fragment from HIS4 (SGD coordinates 66663–68236 for chromosome III). The probe used in Fig. 5B was a PCR fragment from ARG4 (Saccharomyces Genome Database coordinates 140119–141267 for chromosome VIII). These DNA fragments were radiolabeled by using standard random hexamer labeling with exo-free Klenow and α-³²P-dCTP (15).

Figure 4.

Physical analysis of HIS4 hotspot, S288c. (A) Genomic DNA was isolated from QFY105 (rad50S) and JG166 (rad50S mnd1–1) after 0 and 24 h in sporulation medium. DNA was digested with BglII, electrophoresed on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized to a ³²P-labeled DNA fragment indicated as probe. The 3.0-kb fragment is the parental band and the 1.9-kb band corresponds to the DSB. (B) Genomic DNA was isolated from DNY86 (wt) and JG190 (mnd1–1) at the indicated times after transfer to sporulation medium. A 30-bp palindrome sequence containing a PstI site (inserted at the SalI site of his4) was present on one homolog of chromosome III and a 32-bp palindrome sequence containing a BamHI site (inserted at the SalI site of his4) was present on the other homolog of chromosome III. DNA was digested with PstI, PvuII (P), and BamHI. The 1.3- and 1.1-kb bands are the parental fragments and the 2.4-kb band corresponds to hDNA. (C) JG197 (mnd1–1, squares) and MS1 (wt, diamonds) were put into sporulation media at time 0 h and aliquots were removed and plated on SD-complete or SD-histidine at indicated times. Time in hours is depicted on the x axis and the frequency of HIS+ prototrophs is shown according to the log scale on the y axis.

Figure 5.

Physical analysis of chromosome III, SK1. (A) Map of chromosome III. One homolog had a URA3-ARG4 casette inserted into the leu2 gene and the other homolog had the same cassette, plus a 36-bp palindrome at the EcoRI site in arg4 (his4∷arg4-pal), inserted into the his4 gene. HIS4 and LEU2 were offset by 16.7 kb. (B) Genomic DNA was isolated from a sporulating culture of a wt strain (MJL2442) or mnd1–1 strain (JG774) at the hours indicated. DNA was digested with XhoI, electrophoresed on a 0.5% agarose gel, transferred to a nylon membrane, and probed with a ³²P-labeled DNA sequence derived from ARG4. The parental fragments are 12.4 and 12.7 kb, the DSB fragments are 3.7 and 2.4 kb, and the CO fragments are 5.2 and 19.8 kb. (C) Genomic DNA was isolated from the same sporulating cultures depicted in B at 5 h. This DNA was digested with XmnI and analyzed by native/native 2D electrophoresis. DNA will migrate according to molecular weight in the first dimension and both molecular weight and shape in the second dimension. The 4- and 5-kb parental fragments migrate at their expected position, but JMs are retarded in the second dimension and migrate above the arc of linear fragments (shown in the interpretive panel). (D) DSBs at the ARG4-URA3 hotspot in SK1. Genomic DNA was isolated from a sporulating culture of a wt strain (MJL2442), mnd1–1 strain (JG774), dmc1 strain (JG820), and mnd1–1dmc1 strain (JG819) at the hours indicated. Analysis was carried out as in B.

2D Native/Native Gels.

Methods used were exactly as described (7).

Results

Identification of Genes Involved in Meiotic Recombination.

A transcriptional profile of the meiotic process reveals four main categories of induction: (i) metabolic genes induced by nitrogen starvation, (ii) early (I and II) genes that include genes involved in synapsis of homologous chromosomes and recombination, (iii) middle genes, which are involved in processes such as the mechanics of meiotic division and spore morphogenesis, and (iv) late genes, which are mostly involved in spore wall formation (14). Of the 32 genes in the early I cluster, more than half have been shown to have sporulation defects. To identify genes involved in meiotic recombination, we constructed deletions of 12 genes in the early I cluster that had uncharacterized phenotypes in meiosis and analyzed the ability of these mutants to form tetrads (Table 1, which is published as supporting information on the PNAS web site, www.pnas.org). Of these 12, YOR177c and YGL183c were the only genes essential for tetrad formation. YOR177c has since been named MPC54 and is a meiosis-specific component of the spindle pole body (16). YGL183c has been named MND1; mutants have been shown to arrest before the first meiotic division (ref. 8, see Fig. 2A).

Figure 2.

Nuclear division. Cells were collected at 11 h postintroduction into sporulation media, stained with DAPI, and photographed. (A) mnd1–1; (B) mnd1–1spo11; (C) mnd1–1spo13; and (D) mnd 1–1rad17. (Magnifications: ×100.)

MND1 Does Not Play a Role in Mitotic DNA Metabolism.

MND1 was disrupted in the Eurofan 1 project and analyzed for defects in DNA metabolism and meiosis. Specifically, the mutant strain was assayed for DNA damage response, UV sensitivity, gamma ray sensitivity, mitotic mutation rate in the CAN1 gene, mitotic recombination between direct and inverted repeats, hydroxyurea sensitivity, and ability to sporulate. In all cases but sporulation, the mnd1–1 mutation had no effect (http://mips.gsf.de/proj/eurofan/eurofan_2.html). Transcription of this gene is not induced under any of the 300 growth conditions that have been examined in yeast except for sporulation (http://www.transcriptome.ens.fr/ymgv), suggesting that this gene is only involved in processes that occur during sporulation.

MND1 Was Spliced During Meiosis.

The predicted ORF for MND1 can be extended at the 5′ end by homology to orthologs of MND1 in other organisms (17). When 132 bp upstream of the annotated YGL183c ORF are translated, the resulting peptide sequence is 36% identical to the N terminus of the orthologous human protein. This region does not contain any stop codons and is in-frame with the rest of the gene, yet it does not contain a Met start codon, as confirmed by sequencing genomic DNA. However, sequence of the predominant form of the cDNA in meiotic cells (Fig. 1A) revealed a spliced transcript in which an 83-bp intron extends the N terminus of the annotated YGL183c ORF by 45 aa. The MND1 intron contains a 5′ consensus splice site (GUAUGU), a 3′ consensus splice site (CAG), and a noncanonical branch point (cACUAAC) (Fig. 1A).

Figure 1.

Mnd1 was spliced in meiotic cells. (A) PCR amplification from genomic DNA and cDNA reveals a spliced transcript in meiotic cells. U indicates unspliced, and S indicates spliced. In the diagram at the right, the 5′ and 3′ splice and branchpoint sequences and PCR primers are indicated. The white box shows the extension of the reading frame for YGL183c in the 5′ direction. The gray bars correspond to the region for which an alignment is shown in B. (B) clustalx was used to align N termini of predicted orthologous Mnd1 genes from S. cerevisiae, K. lactis, S. pombe, H. sapiens, P. falciparum, and A. thaliana.

Orthologs of MND1 Exist in Several Fungi, Protists, Plants, and Animals.

We found orthologs of MND1 in the hemiascomycetous yeasts Saccharomyces byanus (79% identity), Saccharomyces kluyveri (82% identity), Kluyveromyces thermotolerans (37% identity), Kluyveromyces lactis (31% identity), Debaryomyces hansenii (43% identity), in Schizosaccharomyces pombe (21% identity), in the plant A. thaliana (14% identity), in the protists Encephalitozoon cuniculi (21% identity), Giardia lamblia (22% identity), and Plasmodium falciparum (15% identity), and in the mammals mouse (23% identity) and human (24% identity). Notably, we were unable to identify orthologs of MND1 in Caenorhabditis elegans or Drosophila melanogaster. The alignment among the N termini of proteins (Fig. 1B) revealed a very highly conserved region that does not have a significant match to any known functional domain. Amino acids 118–139 encode a putative nuclear localization signal (18).

MND1 Functions After DSB Formation.

mnd1–1 mutants in the SK1 strain background arrested before the first meiotic division (Fig. 2A). To further dissect the role of MND1, we explored the phenotype of an mnd1–1 mutant in combination with null mutations in (i) SPO11, which makes DSBs, (ii) SPO13, which causes a single MII-like division (19), and (iii) RAD17, a checkpoint gene that detects unresolved recombination intermediates (20). Each of these double mutants was sporulated and DNA was stained with DAPI. In the mnd1–1spo11 mutant, tetrads were formed with wt efficiency (Fig. 2B), as they are in a spo11 mutant, but the spores were not viable (0/40 spores from 10 dissected tetrads grew), presumably caused by the massive chromosome segregation defects observed in spores in spo11 mutants (21). Deletion of SPO13 results in viable dyads when combined with a mutation that blocks the initiation of recombination (e.g., a SPO11 deletion). In mnd1–1spo13, the cells arrested with one DAPI-staining body (Fig. 2C). This result indicates that MND1 operates after DSB formation. These results are consistent with a previous report that shows that in a spo11spo13 background an mnd1–1 mutant can form some viable spores in dyads (8). Finally, in the mnd1–1 rad17 mutant, meiotic divisions occurred and four DAPI-staining bodies were produced (Fig. 2D), but mature spores were not formed. This finding indicates that the arrest in an mnd1–1 strain was caused by the RAD17-mediated checkpoint that detects unresolved recombination intermediates.

Because spore formation was defective in the mnd1–1 strain, we measured recombination by the return-to-growth assay, which allows for the recovery of diploid cells that have initiated meiotic recombination between heteroalleles by DSBs but are unable to repair them at later stages. Cells were withdrawn from sporulation medium and transferred to nutrient-rich medium to assess commitment to meiotic recombination (22). In general, heteroallelic recombination reflects NCO recombination, or gene conversion. However, heteroallelic recombination in a return-to-growth experiment may not require wt end resection and/or heteroduplex formation because a dmc1 strain behaves like wt in this assay despite having hyperresected DSBs (23) and no detectable JMs (24). The average frequency of ARG+ prototrophs at 7 h in the wt SK1 strain was 6.5 × 10⁻² and 1.1 × 10⁻² in an mnd1–1 strain (Fig. 3). Because Mnd1 is not expressed during mitotic growth, the difference at time 0 h is presumably stochastic. When the difference in the baseline is adjusted, the induction of recombination is similar; the mnd1–1 strain induced recombination an average of 49-fold and the wt strain induced recombination an average of 74-fold at the 7 h time point (see Fig. 3 Inset). Furthermore, the kinetics of recombination induction appeared to be similar; commitment to meiotic recombination began at about 3 h after transfer to sporulation media and leveled off by 6 h in both wt and mnd1–1 strains. Similar results were observed at the his4 locus (data not shown). These data suggest that MND1 is not required for the induction of the high levels of recombination characteristic of meiosis.

Figure 3.

Heteroallele recombination at ARG4 in SK1. JG750 (mnd1–1) and JG751 (wt) were put into sporulation media at time 0 h, and aliquots were removed and plated on SD-complete or SD-arginine at indicated times. Data points were the average measurement from two independent isolates.

MND1 Was Required for Stable hDNA Formation.

Initiation of recombination may be directly monitored by examining DSBs. The HIS4 promoter sequence contains a hotspot for DSB formation (Fig. 4A). DSBs at the HIS4 locus in S288c-derived strains have been characterized in detail (10–12, 25). In a rad50S strain, the ends of DSBs are not processed, facilitating visualization by Southern blot (26). We analyzed the levels of HIS4 DSBs formed in a rad50S strain in an otherwise wt strain background (QFY105) and an mnd1–1 strain (JG166, Fig. 4A). At 24 h, the levels of DSBs in these strains were comparable; rad50S had 1.7% DSB and rad50S mnd1–1 had 2.5% DSB. This result indicates that the level of DSBs was not diminished by mutation of MND1. When we measured DSBs in a RAD50 strain background (where DSBs are processed), we observed no difference in the levels of DSBs in wt and mnd1–1 strains (data not shown).

Nag and Petes (12) developed a physical assay to detect hDNA at the HIS4 locus during meiotic recombination. The strain used in this assay, DNY86, contains one chromosome with a BamHI site inserted into the SalI site of the HIS4 gene and a second chromosome with a PstI site inserted into the same SalI site in HIS4. hDNA will contain one DNA strand with a BamHI site and one strand with a PstI site and therefore will not be cut with either enzyme. hDNA first occurs post-DSB, when 3′ single-stranded DNA from the break site invades the homologous chromosome. This recombination intermediate, referred to as a single-end invasion, is relatively unstable, but can be observed if DNA is crosslinked with psoralen before extraction from cells (24). hDNA will persist in the cell in both NCO and CO recombination intermediates and products. Nag and Petes (12) demonstrated that hDNA cannot be detected in a rad50 strain (which cannot make DSBs), but can be detected in a rad52 strain, which is defective in a later stage of recombination. The hDNA detected by this assay is presumably derived from both CO and NCO recombination intermediates and products.

We used this physical assay to determine whether stable hDNA formation occurred in mnd1–1 mutants. The 2.4-kb band diagnostic of hDNA in wt was not formed in the isogenic mnd1–1 strain (Fig. 4B). We always observed a background band at 2.4 kb that was meiosis independent. Although hDNA was not detected at the his4 locus in the mnd1–1 strain, heteroallelic recombination at this same his4 locus in a return-to-growth assay was reduced only 2-fold in an mnd1–1 strain and did not show any delay in recombination induction relative to the isogenic wt strain (Fig. 4C). Even though DSBs were plentiful in an mnd1–1 strain, and could be used efficiently for heteroallele recombination in a return-to-growth experiment, this strain was not able to form stable hDNA.

In the Southern blot shown in Fig. 4B, the mnd1–1 strain contained a band at 2.0 kb. This band most likely reflects DSBs that have 5′ ends that have been resected past the PstI or BamHI restriction enzyme sites (which have been inserted at the SalI site, approximately 630 bp away from the DSB site); the 3′ single-stranded DNA is therefore resistant to digestion. This distance is consistent with what has been observed genetically, which is that heteroduplexes at HIS4 often span a distance of 1.8 kb (25). Thus, in mnd1–1 strains, 5′ end resection has occurred, and long single-stranded 3′ tails are present but unable to stably invade the homologous chromosome to form hDNA.

MND1 Was Required to Complete CO Recombination.

We explored the status of recombination intermediates in an mnd1–1 strain at a synthetically created hotspot in the SK1 strain background by using the procedures of Allers and Lichten (7). In brief, the LEU2 locus on one chromosome III homolog has a cassette inserted into it that contains URA3 and ARG4. The other homolog has a similar cassette inserted into the HIS4 locus (Fig. 5A). These markers make it possible to monitor DSBs, COs, and JMs. Fig. 5B shows a Southern blot of DNA from a meiotic time series of a wt strain (MJL2442) and an mnd1–1 strain (JG774) digested with XhoI. The parental fragments were 12.4 and 12.7 kb, DSBs were 3.7 and 2.4 kb, and CO products were 19.8 and 5.2 kb. DSBs appeared at 3 h after transfer to sporulation medium in both strains in the time course shown, and by 2 h in an independent time course (data not shown). However, the mnd1–1 strain had ≈2-fold more DSBs at the 3, 4, 5, 6, and 7 h time points. We could detect the 5.2-kb CO product (the 19.8-kb CO product did not transfer well presumably because of its large size) in the wt strain by 5 h in the time course shown, and by 4 h in an independent time course. The CO product was undetectable in the mnd1–1 strain in both time courses, even at 7 and 8 h. Thus, MND1 was required to complete CO recombination.

MND1 Was Required for JM Formation.

We further investigated the defect in the mnd1–1 strain by assessing JM formation. JMs are considered to be the hDNA-containing precursors of all CO recombinants and some or all NCO recombinants (5, 7). JMs can be detected by 2D native/native gels in which the first dimension separates DNA based on mass and the second dimension separates DNA based on mass and shape. The bulk of genomic DNA migrates in an arc pattern, but DNA with deviations in shape, such as JMs, migrates off the arc (7). We prepared genomic DNA from the aforementioned two time courses from wt and mnd1–1 by using a protocol that preserves JMs (13). JMs could be detected in wt between 3.5 and 6 h (5 h, Fig. 5C) but were absent at 4, 5, 6, and 8 h in mnd1–1 (5 h, Fig. 5C).

hDNA and JM formation may be impaired for at least two reasons in an mnd1–1 strain: (i) the 5′ ends are not resected or (ii) strand invasion itself is impaired. A comparison between DSBs in mnd1–1, dmc1, mnd1–1dmc1, and wt strains revealed elongated smears in the case of the dmc1 and mnd1–1dmc1 strains, a short smear in the case of wt, and an intermediate smear in the case of mnd1–1 (Fig. 5D). This result suggests that 5′ ends are resected and strand invasion is impaired in an mnd1–1 strain. Furthermore, in an mnd1–1dmc1 strain, the DSBs showed the same resection phenotype as in a dmc1 strain, indicating that these two genes are epistatic for end resection.

Discussion

We have taken a functional genomics approach to identifying new genes involved in meiotic recombination. We analyzed the meiotic phenotype of null mutations in uncharacterized genes with expression profiles similar to genes known to be involved in meiotic recombination. In this way, we identified MND1. Like many genes in this expression cluster (see Table 1), MND1 is spliced during meiosis. Mutation of this gene caused an arrest before the first meiotic division. The cells arrested because of the RAD17-mediated checkpoint, which detects unresolved recombination intermediates. MND1 is not required for the initiation of meiotic recombination, because DSB formation is at or above wt levels at four different DSB sites. Recombination rates between heteroalleles of arg4 and his4 in return-to-growth experiments indicate that induction of recombination is nearly normal in an mnd1–1 strain. However, neither hDNA nor JMs can be detected, indicating that these recombination intermediates are not present or are highly unstable. COs, a product of JMs, are undetectable. The DSB ends in an mnd1–1 strain are slightly hyperresected compared with a wt strain, but less resected than ends in a dmc1 strain. Based on this analysis, Mnd1p appears to be required for 3′ single-stranded ends to stably invade the homologous chromosome.

Recombination between heteroalleles in the return-to-growth assay reflects recombination events initiated during meiosis that are then resolved during vegetative growth. Our results indicate that either (i) a DSB is enough to stimulate heteroallele recombination and the remainder of the recombination event can be completed by using vegetative proteins or (ii) strand invasion is normally required for elevated recombination levels but a vegetative protein can substitute for Mnd1p.

Dmc1p and Rad51p are both yeast orthologs of Escherichia coli RecA protein. However, these proteins appear to have distinct roles in the cell. Dmc1p shows meiosis-specific expression and the null strain displays an arrest before meiosis I (23), whereas a rad51 strain has both mitotic and meiotic defects, and the meiotic defect is not as severe (27). Similar to mnd1–1, the meiosis I block in a dmc1 strain can be bypassed by mutation of the RAD17-mediated checkpoint (20). A dmc1 strain does not contain any single-end invasions (24). Based on these observations and others, both Hunter and Kleckner (24) and Shinohara et al. (28) have proposed that Dmc1p and Rad51p are asymmetrically located on the 3′ single-stranded tails of DSBs, and that the Dmc1p-associated end is the end that initiates strand invasion. However, eukaryotic Dmc1p is poor at promoting extensive strand exchange as measured in vitro (29, 30). It has thus been proposed that other proteins may be required for this reaction to occur efficiently in vivo. Based on the similarity in phenotype between mnd1–1 and dmc1 mutants, we propose that the defect in an mnd1–1 strain is at a similar step in recombination, and furthermore, that these two proteins may act in concert in vivo.

Our results support a model in which Mnd1p operates downstream of Dmc1p binding to DNA (Fig. 6). DSBs in a dmc1 strain are hyperresected, presumably because Dmc1p normally protects DNA ends from degradation by cellular nucleases. In an mnd1–1 strain, the ends appear more protected from degradation, suggesting that Dmc1p, or a protein complex containing Dmc1p, is bound. The dmc1mnd1–1 strain has the same phenotype as the dmc1 strain, indicating that the defect in end protection in the double mutant strain is identical to the defect in the dmc1 strain. In addition, Dmc1p foci are present by cytology in an mnd1–1 strain (T. Holzen and D. Bishop, personal communication). Based on these observations, we disfavor the possibility that Mnd1p is required for Dmc1p to efficiently assemble on single-stranded DNA in vivo, but rather propose that Mnd1p operates downstream of Dmc1p binding to DNA. Mnd1p may be a cofactor for strand invasion.

Figure 6.

Dmc1p is depicted as an oval, Mnd1p is depicted as a circle. Mnd1p may be present in multiple copies; only one is shown for illustration. Human Dmc1p forms octameric rings that can stack on DNA (36, 37); only one octamer is shown for illustration. Mnd1p could potentially be involved in one or more of the following: (i) disassembly of Dmc1p from single-stranded DNA, (ii) distortion of the unbroken homolog to facilitate invasion by the Dmc1p-associated single-stranded DNA, or (iii) efficient strand invasion/assimilation activity along with Dmc1p.

Observing physical recombination intermediates in eukaryotes other than yeast has been difficult. Thus, the only means we have to compare recombination mechanisms across evolution is comparative genomics. With the identification of SPO11, MND1, and DMC1 orthologs in yeasts, mouse, human, and plants, the likelihood of evolutionarily conserved recombination machinery for these organisms grows stronger. We and others have been unable to identify an ortholog of either MND1 or DMC1 in D. melanogaster or C. elegans. Synaptonemal complex formation also differs for these organisms; Spo11p is not required for synapsis in flies and worms (31, 32), but is required in mouse and yeast cells (33–35). These differences raise the issue as to whether flies and worms have evolved recombination mechanisms that differ substantially from those of yeast, mouse, and human.

Abbreviations

hDNA

heteroduplex DNA

JM

joint molecule

CO

crossover

NCO

noncrossover

DSB

double-strand break

wt

wild type

SD

synthetic dextrose

DAPI

4′,6-diamindine-2-phenylindole

1D

one-dimensional

2D

two-dimensional