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Mol Cell Biol. 2002 May; 22(10): 3281–3291.
PMCID: PMC133777

Saccharomyces cerevisiae Mer3 Is a DNA Helicase Involved in Meiotic Crossing Over

Abstract

Crossing over is regulated to occur at least once per each pair of homologous chromosomes during meiotic prophase to ensure proper segregation of chromosomes at the first meiotic division. In a mer3 deletion mutant of Saccharomyces cerevisiae, crossing over is decreased, and the distribution of the crossovers that occur is random. The predicted Mer3 protein contains seven motifs characteristic of the DExH box type of DNA/RNA helicases. The mer3G166D and the mer3K167A mutation, amino acid substitutions of conserved residues in a putative nucleotide-binding domain of the helicase motifs caused a defect in the transition of meiosis-specific double-strand breaks to later intermediates, decreased crossing over, and reduced crossover interference. The purified Mer3 protein was found to have DNA helicase activity. This helicase activity was reduced by the mer3GD mutation to <1% of the wild-type activity, even though binding of the mutant protein to single- and double-strand DNA was unaffected. The mer3KA mutation eliminated the ATPase activity of the wild-type protein. These results demonstrate that Mer3 is a DNA helicase that functions in meiotic crossing over.

During meiosis, two successive rounds of chromosome segregation take place following a single round of DNA replication. At the first meiotic division (meiosis I), pairs of homologous chromosomes (homologs) are synapsed and then segregated to opposite poles. In the prophase of meiosis I, DNA double-strand breaks (DSBs) are formed in a genetically programmed way, and two types of recombination events occur between homologs (3, 31, 45). Crossing over results in reciprocal exchanges of chromosome arms and serves to link the homologs to ensure proper segregation. The other type of recombination, gene conversion, is defined as nonreciprocal exchange of genetic information. To ensure that every pair of homologs sustains at least one crossover, the distribution of crossovers along and among chromosomes is regulated. The regulation of the distribution of crossovers along a chromosome is inferred from the observation of crossover interference, where multiple crossovers are less frequent than expected based on the frequency of single crossovers (38). The distribution of crossovers along and among chromosomes likely represents different manifestations of the same underlying regulation (10, 13, 29, 58). Despite the fact that crossing over is a key process required for faithful segregation of chromosomes during meiosis, the molecular mechanism of the process and its regulation remain elusive.

In Saccharomyces cerevisiae, there are a number of genes required for normal frequencies of crossing over that are not required for gene conversion, including ZIP1, ZIP2, ZIP3, MSH4, MSH5, MLH1, MLH3, and MER3 (1, 11, 25, 26, 41, 47, 57, 63). In addition to being required for normal frequencies of crossing over, ZIP1 and MER3 appear to be required to regulate the distribution of crossovers (41, 58). Meiosis-specific DSBs appear for a prolonged period of time in the absence of ZIP1 and MER3, suggesting that they have a role in the transition of DSBs to later intermediates (41, 54). Among these genes, the MER3 gene is of particular interest because the predicted Mer3 amino acid sequence contains the seven motifs characteristic of the DExH box type of DNA/RNA helicases (41). The Mer3 helicase motifs show significant homology to the DNA helicases encoded by yeast SGS1 and human BLM (the gene mutated in Bloom syndrome) (15, 18, 65). Previous studies have shown that such DNA helicases are involved in DNA recombination, replication, and cell cycle control (16, 19, 34, 64). These observations suggest that Mer3 is likely a DNA helicase that functions in meiotic recombination.

Here, we have characterized the phenotype of mer3 point mutants that have amino acid substitutions in a putative nucleotide-binding domain thought to be required for helicase activity and found that these mutants show a defect in the transition of DSBs to later intermediates, a decreased frequency of crossing over, impaired crossover interference, and a partial reduction in spore viability. These effects are similar to those caused by a mer3 deletion mutation, which in addition causes severe defects in progression through meiosis and in the production of viable spores. The Mer3 protein was purified and was found to have DNA helicase activity. The mutant Mer3GD protein was found to lack helicase activity, but it retained normal binding activity to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The mutant mer3KA proteins showed no detectable ATPase activity. These results indicate that the Mer3 helicase activity plays a critical role in crossover control during meiosis.

MATERIALS AND METHODS

Yeast strains, media, and genetic procedures.

Yeast strains used are listed in Table Table1.1. General genetic manipulations and preparation of media were performed according to the method of Sherman et al. (52). Transformation with plasmids and gene disruption and substitution constructs was performed by the lithium acetate procedure (27), and the correct genotype was verified by PCR analysis and DNA sequencing of the substitution site. To introduce mer3GD-URA3-mer3, mer3KA-URA3-mer3, and mer3-URA3-mer3-FLAG, relevant yeast haploid strains were transformed with DraI-digested pRDK4153, EcoRI-digested pRDK4154, and AflII-SalI-digested pRDK4155, respectively. To obtain URA3 popouts, uracil auxotrophs were selected by plating cells on SD plates supplemented with 5-fluoroorotic acid. Sporulation medium was prepared, and synchronous meiotic cultures were obtained as previously described (41), except that to obtain 23°C meiotic cultures, the temperature was shifted from 30°C to 23°C when the cells were transferred from YPA to sporulation medium.

TABLE 1.
 Yeast strains used in this study

Detection of meiosis-specific DNA DSBs.

DNA samples were prepared, and meiosis-specific DSBs in the his4::LEU2 region of chromosome III were detected by Southern blotting as essentially described before (41). DNA was digested with PstI, separated by electrophoresis on a 0.7% agarose gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA), and transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech). A 1.5-kb PstI-EcoRI fragment from pNKY291 (8) labeled with [α-32P]dATP by the random primer method was used as a probe for Southern hybridization. The signals were detected with a Fuji BAS2000 phosphorimager (Fuji) and were quantified with Image Gauge V3.3 (Fuji).

Plasmids.

The plasmids containing the mer3GD-URA3-mer3 (pRDK4153), mer3KA-URA3-mer3 (pRDK4154), and mer3-URA3-mer3-FLAG (pRDK4155) constructs were created by using the pRDK4151 vector. To create pRDK4151, a 2.5-kb EcoRI-ClaI fragment containing the MER3 N-terminal region was cloned between the EcoRI and ClaI sites of pTN149 (41), yielding the mer3-URA3-mer3. The mer3GD mutation was created by PCR-based mutagenesis (24) with the primers 5′-CCCTCGAGATGAAAACAAAGTTTGATCGCCTCGG, 5′-CCAACCGGATCAGACAAGACTGTATTATTTGAATTGGC, 5′-GCCAATTCAAATAATACAGTCTTGTCTGATCCGGTTGG, and 5′-CCAAGCTTCGGCATGCTTTTCAATTATTTCAATCAATTTGG (the altered nucleotide is underlined) and pRDK4157 template DNA (described below). The resulting 1.1-kb PCR product was digested with XhoI and HindIII and cloned between the XhoI and HindIII sites of pBluescriptII KS+ (Stratagene) to create pRDK4152. A 1.1-kb XhoI-SphI fragment which had the XhoI site filled in with Klenow fragment (New England Biolabs) was excised from pRDK4152 and cloned between the SnaBI and SphI sites of pRDK4151, yielding pRDK4153. The mer3KA mutation was created by site-directed mutagenesis as described by Kunkel (32), with the primer 5′-AAATAATACAGTGGCGGCTGATCCGGTTGG and a pBluescriptII SK+ (Stratagene)-based plasmid in which a 0.4-kb SpeI fragment containing the helicase motif I of MER3 was cloned at the SpeI site of the vector. The 0.4-kb SpeI fragment containing the mer3KA mutation was excised and substituted for the corresponding region of pRDK4151, to create pRDK4154. A 1.6-kb XbaI-SalI fragment containing the Flag sequence at the MER3 C terminus and SalI site introduced 173 bp downstream from the stop codon was cloned between the XbaI and SalI sites of pRDK4151, yielding pRDK4155. The Mer3 protein expression plasmid, pRDK4157, which contains the GAL10-MER3-FLAG gene, was created as follows. By using the reverse transcription-PCR (RT-PCR) product of MER3, which does not contain the MER3 intron (41), the MER3 cDNA was constructed. At the 5′ end of the MER3 cDNA, an XhoI site was introduced by PCR-based mutagenesis (CTCGAGATG; the XhoI site and the first codon are underlined and in boldface, respectively). At the 3′ end, MluI and HindIII sites were introduced by the same method (ACGCGTTAAGCTT; the restriction sites and the stop codon are underlined and in boldface, respectively). The sequence of the stop codon has also been changed from TGA. A DNA fragment constructed by annealing the oligonucleotides 5′-CGCGGGACGTACAAGGACGACGATGACAAGA and 5′-CGCGTCTTGTCATCGTCGTCCTTGTAGTCC was cloned into the MluI site, and the orientation resulting in the addition of TRDYKDDDDKTR (12 amino acids) containing the Flag epitope sequence at the Mer3 C terminus was confirmed by DNA sequencing. A putative URS1 element, which may repress the transcription in vegetatively growing yeast cells (17), present at position 109 to 117 (AGCCGCCAA) in the MER3 open reading frame (ORF), was changed by site-directed mutagenesis to AGTCGACAA, which did not change the Mer3 amino acid sequence. The resulting 3.6-kb XhoI-HindIII fragment containing MER3-FLAG with the mutated URS1 was cloned between the XhoI and HindIII sites of pRDK249 containing a GAL10 promoter, a 2 μm origin, and the URA3 marker (28), yielding pRDK4156. Then a 4.4-kb BamHI-HindIII fragment containing the GAL10-MER3-FLAG region from pRDK4156 was cloned between the BamHI and HindIII sites of YEplac112 containing a 2 μm origin and TRP1 (20), yielding pRDK4157. To express the mutant Mer3GD protein, a 1.1-kb XhoI-SphI fragment containing the mer3GD mutation was excised from pRDK4152 and cloned between the corresponding sites of pRDK4157 to obtain pRDK4158. To express the Mer3KA protein, a 1.0-kb SalI-SphI PCR fragment, which contains the mer3KA mutation, amplified from pRDK4154 was inserted between the corresponding sites of pRDK4152 to obtain pTN386, and then a 1.1-kb XhoI-SphI fragment from pTN386 was inserted between the corresponding sites of pRDK4157 to create the expression construct pTN483. All modified plasmids were sequenced to confirm that no additional mutations were introduced.

Purification of Mer3 protein.

The pRDK4157 plasmid was introduced into the protease-deficient yeast strain RKY1293 by transformation. An initial culture was made by growing cells overnight at 30°C in minimal medium lacking tryptophan and supplemented with 3% (vol/vol) glycerol and 2% (wt/vol) lactic acid. This culture was used to inoculate 1 liter of the same medium, except that lactic acid was present at a concentration of 0.02% (wt/vol) instead of 2% (wt/vol). When the culture reached an optical density at 600 nm of 1 to 1.3, the GAL10 promoter was induced by the addition of galactose to 2% (wt/vol), and cell growth was continued for 8 h at 30°C. All subsequent operations were performed at 4°C unless otherwise indicated, and the buffers used for all steps up to the elution of the Mer3 protein from the Flag-affinity gel were supplemented with a mixture of protease inhibitors (phenylmethylsulfonyl fluoride and benzamidine at 1 mM each; pepstatin A, aprotinin, and leupeptin at 1 μg/ml each).

Cells from 4 liters of culture were harvested by centrifugation (11,000 × g for 5 min), washed with cold water, and suspended in 100 ml of buffer P (50 mM NaPO4 [pH 8.0], 200 mM NaCl, 5 mM EDTA, 1 mM β-mercaptoethanol). After standing on ice for 30 min, the cell suspension was centrifuged as described above and resuspended in 0.2 ml of buffer P per g of cells. The cells were lysed by the addition of an equal volume of acid-washed glass beads (425 to 600 μm in diameter; Sigma) followed by blending in 1.5-ml-tubes for four periods of 2 min each with a Bead-beater (Biospec Products). The cell extract was separated from the glass beads by centrifugation, and the beads were washed with 0.7 ml of buffer P per 1.5-ml tube. The cell extract and washes were combined and centrifuged at 15,000 × g for 30 min, and the supernatant was mixed with 1 ml of anti-Flag M2 affinity gel slurry (Sigma) in two 15-ml tubes and incubated for 1.5 h with rocking. The gel was collected by centrifugation (500 × g, 3 min) and washed twice with 20 ml of buffer T (20 mM Tris HCl [pH 7.4], 200 mM NaCl, 5 mM EDTA, 10% [vol/vol] glycerol, 1 mM dithiothreitol [DTT]). The gel was then transferred to a 1.5-ml tube and washed with 1 ml of buffer T four times. The bound protein was eluted by incubation of the Flag gel in 1 ml of buffer T containing 0.2 mg of Flag peptide per ml (Sigma) for 1.5 h with rocking. The elution step was repeated twice. The Flag elution fraction was clarified by centrifugation (15,000 × g for 10 min) and loaded at 0.5 ml/min onto a MonoQ HR5/5 column (Pharmacia) equilibrated in buffer T. After washing the column with 5 ml of buffer T, the bound protein was eluted with a 10-ml linear gradient from 0.2 to 1 M NaCl in buffer T. The peak of the Mer3 protein, which eluted at ~280 mM NaCl, was directly applied at 1 ml/min onto a 1-ml HiTrap heparin column (Pharmacia) equilibrated with buffer T containing 300 mM NaCl. After washing with 5 ml of buffer T containing 300 mM NaCl, the protein was eluted with a 5-ml linear gradient of 0.3 to 1 M NaCl in buffer T. The Mer3 protein eluted at ~440 mM was dialyzed for 1 h against 100 volumes of buffer S (20 mM Tris HCl [pH 6.9], 250 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT) and then loaded at 0.5 ml/min onto a MonoS HR5/5 column (Pharmacia) equilibrated in buffer S. After washing the column with 5 ml of buffer S, the bound protein was eluted with a 5-ml linear gradient from 0.5 to 1 M NaCl in buffer S. The Mer3 protein eluted at ~440 mM NaCl and was dialyzed twice against 100 volumes of storage buffer (10 mM Tris HCl [pH 7.6], 400 mM NaCl, 50% glycerol, 1 mM β-mercaptoethanol) and stored at −80°C. Fractions containing Mer3 protein were identified by SDS-PAGE, and the protein concentration was determined by the Bradford method (7) with the Bio-Rad protein assay. The resulting protein was >95% pure as assessed by SDS-PAGE followed by staining the gels with Coomassie blue. The mutant Mer3GD and Mer3KA proteins were purified exactly as described for the wild-type Mer3 protein.

DNA substrates.

Oligonucleotides (high-performance liquid chromatography [HPLC] purified) were purchased from CyberSyn, Inc. For DNA-binding assays, the 50-nucleotide (nt) oligonucleotide T70 (5′-TCGATAGTCTCTAGACAGCATGTCCTAGCAAGCCAGAATTCGGCAGCGTC) was first 5′ end labeled with [γ-32P]ATP (NEN Life Science Products) by using T4 polynucleotide kinase (New England Biolabs). The labeled oligonucleotide T70 was mixed with a one- to twofold molar of the 50-nt oligonucleotide T74 that is the complementary strand of T70 in annealing buffer (10 mM Tris HCl [pH 7.6], 1 mM EDTA, 0.5 M NaCl), the mixture heated to 94°C for 5 min, and then cooled to 20°C over a 2-h period. The annealed DNAs were separated by PAGE on a 10% polyacrylamide gel run in TBE (90 mM Tris borate [pH 7.6], 2 mM EDTA). The band containing annealed DNAs was excised, and the DNA was eluted by soaking the gel slice in elution buffer (0.5 M NaCl, 0.6 M Na acetate, 1 mM EDTA [pH 8.0]) overnight at 4°C followed by extraction with phenol-chloroform and precipitation with ethanol. The DNA was then dissolved in buffer containing 10 mM Tris HCl (pH 7.6) and 250 mM NaCl. To prepare an ssDNA substrate, the 5′-end-labeled oligonucleotide T70 was purified as described above. For DNA helicase assays, HPLC-purified oligonucleotide M13-50 (5′-CATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC; complementary to nt 6230 to 6279 of M13mp18 circular ssDNA) was 5′ end labeled and purified with a QIAquick nucleotide removal kit (Qiagen). The labeled DNA was mixed with an equal molar amount of M13mp18 circular ssDNA in annealing buffer (10 mM Tris HCl [pH 7.6], 1 mM EDTA, 100 mM NaCl), and the mixture was heated to 95°C for 5 min and then incubated at 65°C for 20 min, followed by incubation at 23°C for 20 min. The annealed DNA was purified by gel filtration through a 0.12-cm2 by 25-cm Bio-Gel A 5m (Bio-Rad) column equilibrated and run in the annealing buffer. The concentration of purified DNAs was determined with a Beckman DU 640B spectrophotometer.

Gel mobility shift assays.

Indicated amounts of the Mer3 protein and 1 nM DNA (concentrations are in terms of moles of molecules) were incubated in 20-μl volumes of DNA binding buffer (20 mM Tris HCl [pH 7.6], 50 mM NaCl, 2 mM DTT, 100 μg of bovine serum albumin [BSA] per ml). Prior to assembling the reaction, the ssDNA substrate was incubated at 100°C for 3 min to disrupt secondary structure. After incubation for 30 min on ice, 5 μl of loading buffer A (40 mM Tris HCl [pH 8.0], 25% glycerol, 400 μg of BSA per ml) was added to the binding reaction mixture. The DNA-protein complexes were analyzed by electrophoresis through nondenaturing 5% polyacrylamide gels (19:1) run in low-ionic-strength buffer (6.7 mM Tris HCl [pH 8.0], 3.3 mM Na Acetate, 2 mM EDTA), and the DNA-containing bands were visualized with a phosphorimager (445 SI; Molecular Dynamics).

ATPase assays.

Reaction mixtures containing 20 mM Tris HCl (pH 7.6), 50 mM NaCl, 5 mM MgCl2, 2 mM DTT, 100 μg of BSA per ml, 0.02 Ci of [γ-32P]ATP per ml (ICN Biomedical, Inc.), 1.5 μg of poly(dA) (Amersham Pharmacia Biotech) or M13mp18 RFI (Life Technologies Inc.) per ml, and 0.5 mM ATP (Amersham Pharmacia Biotech) were preincubated at 30°C for 5 min, and the reaction was initiated by the addition of 4 nM Mer3 protein. At the indicated times after the addition of Mer3, 5-μl portions of the reaction mixtures were removed and terminated by the addition of 2 μl of 0.2 M EDTA. One microliter of each reaction mixture was spotted onto polyethyleneimine cellulose thin-layer chromatography plates (Merck). The plates were developed in a mixture of 1 M formic acid and 0.5 M LiCl and dried, and the amounts of 32Pi and [γ-32P]ATP in the reaction mixture were determined with a phosphorimager (445 SI; Molecular Dynamics).

DNA helicase assays.

The indicated amounts of protein and DNA (concentrations are in terms of moles of nucleotides) were incubated in 20-μl volumes containing DNA helicase buffer (20 mM Tris HCl [pH 7.6], 50 mM NaCl, 2 mM DTT, 100 μg of BSA per ml, 2 mM MgCl2, 2 mM ATP). All reaction mixtures were preincubated at 30°C for 5 min, started by the addition of the Mer3 protein, and incubated for the times indicated in the individual experiments. Reactions were stopped by the addition of 5 μl of stop buffer (50 mM Tris HCl [pH 7.6], 50 mM EDTA, 2.5% SDS) and 0.5 μl of 25 mg of proteinase K per ml, followed by incubation at 37°C for 10 min. The DNA products were then analyzed by electrophoresis through nondenaturing 8% polyacrylamide gels run in TBE. The gels were dried, and the radiolabeled DNA was visualized with a phosphorimager. For time course experiments, a single reaction mixture (100 μl) was prepared and incubated, 10-μl aliquots were removed at each time point, and 2.5 μl of stop buffer and 0.25 μl of proteinase K at 25 mg/ml were added to each 10-μl aliquot prior to analysis.

RESULTS

Amino acid substitutions in the nucleotide-binding domain of Mer3 cause defects in meiotic progression, DSB transitions, and viable spore formation.

The predicted Mer3 amino acid sequence contains seven motifs characteristic of DNA/RNA helicases (21). Motif I, which is also called a P-loop or Walker motif A, constitutes a nucleotide-binding domain in many proteins containing this motif (22, 62). To determine whether the Mer3 motif I plays an important role in meiosis, conserved amino acids in motif I were altered by site-directed mutagenesis, and the mutations were introduced into the MER3 genomic locus (Fig. (Fig.1A,1A, mer3G166D and mer3K167A). Such mutations are known to eliminate ATP hydrolysis by the Walker A type of ATPases (23, 55). Interestingly, it has been observed that a mer3 deletion causes more severe defects in meiotic nuclear divisions and in the transition of DSBs to later intermediates at 30°C than at to 23°C, although a similar reduction of crossovers is observed at both temperatures (41; see below). Isogenic diploid strains containing the mutations were synchronously sporulated at 30 and 23°C, and meiotic nuclear divisions were monitored by DAPI (4′,6′-diamidino-2-phenylindole) staining (Fig. (Fig.1B,1B, 30°C; and C, 23°C). As was seen before (41), a deletion of MER3 resulted in approximately 10% of the wild-type level of the meiotic divisions at 30°C, while at 23°C, the mer3 deletion delayed the appearance of the meiotic divisions, but eventually reached the wild-type level. In the mer3GD and mer3KA mutants, ~3- and ~4-h delays in the appearance of meiotic divisions were observed at 30 and 23°C, respectively, although eventually wild-type levels of meiotic divisions occurred, consistent with a similar meiotic progression defect at both temperatures. The delay in the induction of meiotic divisions observed in mer3GD and mer3KA mutants prompted us to test whether these mutations effect meiosis-specific DSBs. Chromosomal DNA was prepared from synchronously sporulating cultures and DSBs formed in the his4::LEU2 hot spot (Fig. (Fig.1D)1D) were detected by Southern blotting (see Materials and Methods). Figure Figure1E1E shows the Southern blot demonstrating the formation of DSBs at 30°C. In wild-type cells, DSBs appeared at 5 h after transfer of the cells into sporulation medium and gradually disappeared thereafter. In mer3GD and mer3KA cells, the DSBs appeared at 5 h, were present through the 9-h time point, and then disappeared. The amount of DSBs formed at site I was measured (Fig. (Fig.1F,1F, 30°C; and G, 23°C). These data show that in the mer3GD and mer3KA mutants, the DSBs were present for a longer period of time and accumulated to higher levels than were seen in the wild-type control, indicating that the transition of DSBs to later intermediates is impaired in the mer3GD and the mer3KA mutants. However, the defect in the DSB transition in the mer3GD and the mer3KA mutants was not as severe as that previously seen for the mer3Δ mutant (41).

FIG. 1.
Effect of the mer3GD and mer3KA amino acid substitutions on meiotic nuclear divisions and meiosis-specific DSB formation. The isogenic SK1 strains TNY821 (wild type), TNY822 (mer3Δ), TNY823 (mer3GD), and TNY824 (mer3KA) were used. (A) Positions ...

A high incidence of nondisjunction of homologs at meiosis I and a decrease in spore viability have been observed in the mer3 deletion mutant (41). Tetrads of the mer3GD and mer3KA mutants formed at 30 and 23°C were dissected, and the spore viability and the proportion of tetrad types (4-, 3-, 2-, 1-, and 0-sv for four-spore-viable, three-spore-viable, etc.) were determined (Table (Table2).2). At 30°C, the mer3GD and the mer3KA mutant had decreased spore viability compared to the wild-type strain (P < 10−10). The proportion of 4-sv tetrads was decreased, and the proportions of 2- and 0-sv tetrads were increased in the mer3GD and mer3KA mutants compared to the wild-type control. These results are consistent with the idea that nondisjunction of homologs at meiosis I is elevated in the mer3GD and mer3KA mutants. Essentially similar phenotypes were observed in the mer3 point mutants at 30 and 23°C, although the spore viability was further decreased at 23°C. In comparison to the mer3GD and the mer3KA mutants, the mer3Δ mutant showed both more pronounced decreases in the proportion of 4-sv tetrads and more pronounced increases in the proportion of 2- and 0-sv tetrads. While the mer3Δ mutant has a more severe phenotype, both the mer3GD and the mer3KA mutations do cause significant defects in progression through meiosis, in the DSB transition, and in the formation of viable spores. A similar level and time course of Mer3 expression were observed in wild-type, mer3GD, and mer3KA cells during meiosis by Western blotting with anti-Mer3 antibodies (data not shown), indicating that the mer3GD and mer3KA mutant phenotypes were due to impairment of some enzymatic activity of Mer3 rather than a defect in Mer3 expression or stability.

TABLE 2.
 Proportion of tetrad types in mer3 mutant strains

The mer3G166D and mer3K167A mutations decrease the frequency of crossing over and impair crossover interference.

To determine whether the mer3GD or the mer3KA mutations effect crossing over, the frequency of reciprocal exchanges in the CAN1-URA3, URA3-HOM3, and HOM3-TRP2 intervals on chromosome V (Table (Table1)1) were examined by tetrad analysis. At 30°C, the frequency of crossing over in the CAN1-URA3 and URA3-HOM3 intervals, but not in the HOM3-TRP2 interval, was decreased by both the mer3GD and mer3KA mutations to approximately 60% of the wild-type level (P < 10−10) (Table (Table3).3). Similar but slightly more severe phenotypes were observed at 23°C. These crossover defects were essentially the same as previously observed for the mer3Δ mutant (41). The effect of either of these two point mutations on crossover interference was analyzed in two ways. First, the frequency of nonparental ditype tetrads (NPDs), which is indicative of double-crossovers in an interval, was compared to the frequency expected in the absence of crossover interference, where crossovers would occur randomly (Table (Table4).4). At 30°C, in the wild-type strain, the frequency of NPDs observed in the CAN1-URA3 and URA3-HOM3 intervals was less than the expected frequency (P < 10−5), showing that there was crossover interference. In the case of the mer3GD and mer3KA mutants, there was almost no difference between NPDs observed and NPDs expected (0.03 < P < 0.12), indicating that these two mer3 mutations impair crossover interference. Second, to confirm that the mer3KA and mer3GD mutations impair crossover interference, the distribution of zero-, one- and double-crossover events in the CAN1-URA3 and URA3-HOM3 intervals was examined (Table (Table5).5). In the wild-type strain, the distribution of crossovers observed was different from the distribution predicted in the absence of crossover interference according to a Poisson distribution (P < 10−13). In the mer3GD and mer3KA mutants, the distribution of crossovers observed was close to a Poisson distribution (10−2 > P > 10−4). In addition, the distribution in these two mer3 mutants was completely different from the wild-type distribution (P < 10−22). Interestingly, in the HOM3-TRP2 interval, crossover interference was not observed in the wild-type strain, and the mer3GD and mer3KA mutations did not cause a significant reduction of crossing over in this interval. Similar defects in crossover interference were observed in the mer3GD and mer3KA mutants at 23°C (Tables (Tables44 and and5).5). Previous studies with the mer3Δ mutant showed a somewhat stronger defect in crossover interference than that observed here with the mer3GD or the mer3KA mutant (41).

TABLE 3.
 Frequency of crossing over in mer3 mutant strains
TABLE 4.
 Analysis of NPD events
TABLE 5.
 Distribution of zero-, one-, and double-crossover events in mer3 mutant strains

Purification of the Mer3 protein.

The genetic analyses described above suggest that the putative nucleotide-binding domain of the Mer3 helicase motifs has an important role in crossing over. To aid in the purification of the Mer3 protein, a Flag epitope was introduced at the C terminus of Mer3 (Fig. (Fig.2A).2A). To determine if the MER3-FLAG gene encoded a functional protein, the strain RKY4227 was constructed by introducing the MER3-FLAG gene at the MER3 chromosome locus under control of the native promoter. At 30°C, RKY4227 showed a spore viability of 97% (193/200, viable/total spores), similar to that seen in wild-type cells (Table (Table2),2), indicating that the tagged Mer3 protein was fully functional. The Mer3 -FLAG protein was then overexpressed from a multicopy plasmid under control of the GAL10 promoter in vegetatively growing yeast cells by the addition of galactose to the medium. The Mer3-FLAG protein was purified to >95% homogeneity by affinity chromatography on an anti-Flag antibody matrix followed by conventional chromatography through MonoQ, heparin, and MonoS columns (Fig. (Fig.2B).2B). The purified protein had a molecular mass of ~140 kDa determined by SDS-PAGE (6.5% polyacrylamide), which is consistent with the predicted molecular weight of 136,574 for the Mer3-FLAG protein. Western blot analysis with anti-Flag monoclonal antibody confirmed that this protein species was the Mer3-FLAG protein (Fig. (Fig.2C).2C). The mutant Mer3GD and Mer3KA proteins were also purified by this procedure, resulting in protein preparations of purity similar to that of the wild-type Mer3 protein (data not shown).

FIG. 2.
Purification of the Mer3 protein. (A) Schematic structure of the Mer3 protein. The helicase domain and Flag tag and their respective amino acid coordinates are illustrated as solid and hatched boxes, respectively. (B) SDS-PAGE (6.5% polyacrylamide) analysis ...

DNA-binding properties and ATPase activity of the Mer3, Mer3GD, and Mer3KA proteins.

The ability of the Mer3 protein to bind to 50-nt ssDNA and to 50-bp dsDNA with blunt ends was characterized by gel mobility shift assays. The Mer3 protein was able to form stable complexes with both ss- and dsDNA, although there was a slight preference for ssDNA at a high concentration of Mer3 (Fig. (Fig.3A3A and B). Denaturing the dsDNA prior to the addition of the Mer3 protein resulted in increased formation of protein-DNA complexes, confirming the slightly higher preference of Mer3 for ssDNA (Fig. 3E and F). For each substrate, approximately 50% of the DNA was converted to protein-DNA complexes at a ratio of four Mer3 molecules per one DNA molecule. The mutant Mer3GD protein had the same DNA-binding properties as the wild-type Mer3 protein (Fig. 3C and D).

FIG. 3.
DNA binding and ATPase activities of the Mer3 protein. Binding of Mer3 protein to oligonucleotide substrates was detected by gel mobility shift assays as described in Materials and Methods. Amounts of the Mer3 or Mer3GD protein are indicated. (A) Binding ...

ATPase activity of the Mer3 protein was examined in the presence of poly(dA) or M13mp18 replication form I (M13 RF) and in the absence of DNA by thin-layer chromatography (Fig. (Fig.3G).3G). Addition of poly(dA) or M13 RF significantly increased the amount of ATP hydrolyzed by Mer3 in the absence of DNA, indicating that ATPase activity of Mer3 is highly stimulated by either ss- or dsDNA. The Mer3GD protein had almost no significant dsDNA-stimulated ATPase activity and had significantly reduced (threefold reduced versus wild type) ssDNA-stimulated ATPase activity, while the Mer3KA protein had no detectable ATPase activity in the presence or absence of DNA (40) (Fig. 3H and I).

The Mer3 protein has DNA helicase activity.

The Mer3 protein was tested for DNA helicase activity by using a DNA substrate containing a 32P-labeled 50-nt fragment annealed to M13mp18 circular ssDNA (Fig. (Fig.4A).4A). Displacement of the labeled fragment by the addition of the Mer3 protein was observed as increasing amounts of Mer3 were added to the assay (Fig. (Fig.4A,4A, lanes 3 to 5). When Mg2+ or ATP was omitted from the reaction, no activity was observed (Fig. (Fig.4A,4A, lanes 9 and 10). In studies published elsewhere, we have shown that the Mer3 helicase can displace 100- and 631-nt fragments in a reaction that is almost entirely dependent on the ssDNA binding protein RPA and have shown that Mer3 translocates along ssDNA in the 3′-to-5′ direction (40). The mutant Mer3GD protein displaced only a small amount of the labeled fragment (Fig. (Fig.4A,4A, lanes 6 to 8), indicating that the ability to bind to ss- and dsDNA was insufficient for helicase activity. The amounts of the fragment displaced in a similar reaction mixture containing different concentrations of Mer3 were measured (Fig. (Fig.4B).4B). At approximately 2 nM Mer3 protein, which corresponds to about seven Mer3 molecules per DNA substrate, strand displacement reached a plateau. Figure Figure4C4C shows a time course of the strand displacement reaction. When the Mer3 protein was present, over 50% of the labeled fragments were displaced within 5 min, and the reaction was essentially complete within 10 min. In contrast, the initial velocity of the strand displacement in the presence of the Mer3GD protein was less than 1% of the wild-type level. These results show that the Mer3 protein has DNA helicase activity and that this activity was highly impaired by the mer3GD mutation. The Mer3KA protein was not tested for helicase activity, since the Mer3 helicase activity is completely dependent on ATP hydrolysis, and hence the Mer3KA protein would be unlikely to have helicase activity, since it is completely defective for ATPase activity.

FIG. 4.
DNA helicase activity of the Mer3 protein. (A) Displacement of a 32P-labeled 50-nt fragment annealed to M13mp18 circular ssDNA was measured as described in Materials and Methods. Reaction mixtures contained 1 μM (in nucleotides) DNA substrate ...

DISCUSSION

Meiotic crossing over is regulated to take place at least once between each pair of homologs to ensure proper segregation of chromosomes at meiosis I. Meiotic crossing over is a highly regulated process involving a number of features, including programmed induction of recombination proteins (9, 61) and recombination initiation structures (i.e., DNA DSBs) (56), double Holliday junctions (4, 12, 50), and specific enzyme systems required for crossing over and for prevention of excessive crossovers, called interference (31, 39, 45). Although a number of gene products have been found to be specifically required for crossing over, the exact function of these gene products and the molecular mechanism of crossover control have remained elusive. The S. cerevisiae meiosis-specific gene MER3, which has been suggested to encode a DNA helicase on the basis of its amino acid sequence, is an important component of the crossover control system (41). A mer3 deletion mutation decreases sporulation, and among the cells that proceed through meiosis, there are decreased crossing over and an absence of crossover interference, leading to a high incidence of nondisjunction of homologs at meiosis I.

In the present study were constructed two site-directed mutations, mer3GD and mer3KA, which cause amino acid substitutions of conserved residues in a putative nucleotide-binding domain of the conserved helicase motifs of Mer3. After a delay in induction of meiotic nuclear divisions, the mer3GD and mer3KA mutant diploid cells sporulated normally, but showed a defect in the DSB transition, nonrandom spore death, decreased frequencies of crossing over, and a nearly randomized distribution of crossovers. The Mer3 protein was purified and shown to bind to both ss- and dsDNA, have DNA-stimulated ATPase activity, and have DNA helicase activity. In studies published elsewhere, we have shown that the Mer3 helicase can displace 100- and 631-nt fragments in a reaction that is almost entirely dependent on the ssDNA binding protein RPA and have shown that Mer3 translocates along ssDNA in the 3′-to-5′ direction (40). The mutant Mer3GD protein was almost entirely defective (<1% wild-type activity) for the DNA helicase activity, but it retained the DNA-binding properties of the wild-type protein. The mutant Mer3KA protein was completely defective for ATPase activity and is unlikely to have helicase activity, since DNA unwinding by Mer3 was dependent on ATP hydrolysis and Mg2+. These genetic and biochemical results indicate that the Mer3 protein functions as a DNA helicase in crossover control and suggest that Mer3 protein may also play a second role that depends on its DNA-binding ability, but not its DNA helicase or ATPase activity.

The data presented here demonstrate that Mer3 is a DNA helicase that can catalyze the displacement of a 50-nt fragment paired with a circular ssDNA; other studies have more extensively characterized the Mer3 helicase and demonstrated it can displace much longer fragments (631 nt) paired with a circular ssDNA, particularly in the presence of RPA (40). Similar strand displacement reactions have been shown to be carried out by comparable amounts of other helicases thought to function in recombination, such as E. coli RuvAB and RecQ, yeast Sgs1 and Srs2, and human BLM (5, 30, 35, 46, 59, 60). In S. cerevisiae, there are a number of genes encoding predicted DNA helicases that have been shown to be involved in DNA recombination, including SGS1, SRS2, RAD54, TID1/RDH54, and MER3 (14, 33, 41, 49, 53, 64, 65). Of these proteins, Sgs1, Srs2, and Mer3 have been shown to be helicases (35; this study), whereas, thus far, Rad54 and Tid1/Rdh54 have only been demonstrated to have an unwinding activity that changes the topology of dsDNA (37, 43, 44). Each of these proteins appears to function in a different type of recombination or at a different step in recombination. Among these proteins, the Mer3 protein is unique in that it is expressed only in meiosis and is required for meiotic progression, DSB transitions, crossing over, and crossover interference. There are several steps during recombination at which a DNA helicase could act, including unwinding of dsDNA during initial strand invasion, strand exchange at early steps of recombination, and branch migration of Holliday junctions later during recombination (42, 48, 51). The observation that mer3 mutants exhibit similar defects in crossing over, as seen in msh4 or msh5 deletion mutants, suggests that Mer3 could function in branch migration and/or resolution of Holliday junctions, as has been suggested for Msh4 and Msh5 (25, 39, 47). In contrast, the observations that a subset of DSBs produced in meiosis appear for a prolonged period of time (mer3GD, mer3KA, and 23°C mer3Δ) or persist (30°C mer3Δ) and that there is either a delay (mer3GD, mer3KA, and 23°C mer3Δ) or arrest (30°C mer3Δ) of meiotic progression in mer3 mutants suggest that Mer3 could function at an early step of the recombination pathway that promotes crossing over. We favor the idea that Mer3 functions at an early step in recombination and is required for DSB transitions. Correct transition of DSBs to later intermediates would be required for crossing over and could be a focus for crossover control. This would explain why DSBs accumulate in mer3 mutants and why there are defects in both crossing over and crossover control. We cannot exclude that Mer3 also acts late in recombination and is required for maturation of a late recombination intermediate that, in the absence of Mer3, is unstable and dissociates into DSB intermediates. Clearly a more detailed analysis of Mer3 helicase activity, its substrate specificity, and relationships with other recombination proteins will be required to address the question of at which steps of recombination the Mer3 helicase acts.

An interesting feature of the data presented here is the difference in phenotype caused by the mer3GD and mer3KA mutations compared to the mer3Δ mutation. Both the mer3GD and mer3KA mutations cause a defect in the DSB transition, decrease crossing over, impair crossover interference, and delay progression of meiosis, but they do not reduce the proportion of cells that ultimately complete meiosis. In contrast, the mer3Δ mutation confers more severe defects in meiotic progression and in the formation of viable spores (Fig. (Fig.11 and Table Table2).2). As described in the section above, a mer3Δ mutant shows a temperature-sensitive phenotype. This could be due to the fact that temperature affects the stability of the recombination intermediate (e.g., DNA strand exchange products; DNA-protein complex) or even a higher-order protein complex that can be formed in the absence of Mer3. Nonetheless, the mer3GD and mer3KA mutations confer recombination defects at both temperatures, suggesting that the helicase and ATPase activities are crucial functions of Mer3. The Mer3GD protein might have a level of residual DNA helicase activity that is sufficient for meiotic progression and for viable spore formation, although this seems unlikely in the case of the Mer3KA protein. Thus, it is possible that the Mer3 protein has another function besides DNA unwinding. Since the Mer3 and Mer3GD proteins have the same ability to bind DNA, this DNA binding activity by itself may be required for meiotic progression. Thus, it is possible that the absence of Mer3 bound to DNA may trigger the meiotic checkpoint that results in meiotic arrest prior to meiosis I (2, 6, 36, 66). Alternatively, it is possible that Mer3 bound to DNA may stabilize recombination intermediates and/or recruit other factors, even in the absence of helicase activity. Similarly, Mer3 protein could stabilize a critical higher-order protein complex, a function that might not require helicase activity. If this is the case, then it is possible that it is the inability to properly assemble such structures or protein complexes or process recombination intermediates formed in this context that triggers meiotic arrest. It is also formally possible that the helicase and ATPase activities are not required for recombination at all and that the mer3GD and mer3KA mutations cause partial defects in some other function of Mer3, although we consider this unlikely.

Acknowledgments

We are grateful to Hernan Flores-Rozas, Jason Kahana, and Albrecht Clement for advice on protein purification and John Weger and Jill Green for DNA sequencing. We also thank Neelam Amin, Ruchira Das Gupta, Martin Hess, Patrick Lau, Kyungjae Myung, and Hernan Flores-Rozas for comments on the manuscript.

This work was supported by National Institutes of Health grant GM26017 to R.D.K. T.N. was supported by the Japan Society for the promotion of Science and Human Frontier Science Program.

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