Coordination of the Initiation of Recombination and the Reductional Division in Meiosis in Saccharomyces cerevisiae

Corresponding author: Robert E. Malone, Department of Biological Sciences, University of Iowa, Iowa City, IA 52242., robert-malone{at}uiowa.edu (E-mail)

Communicating editor: M. LICHTEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Early exchange (EE) genes are required for the initiation of meiotic recombination in Saccharomyces cerevisiae. Cells with mutations in several EE genes undergo an earlier reductional division (MI), which suggests that the initiation of meiotic recombination is involved in determining proper timing of the division. The different effects of null mutations on the timing of reductional division allow EE genes to be assorted into three classes: mutations in RAD50 or REC102 that confer a very early reductional division; mutations in REC104 or REC114 that confer a division earlier than that of wild-type (WT) cells, but later than that of mutants of the first class; and mutations in MEI4 that do not significantly alter the timing of MI. The very early mutations are epistatic to mutations in the other two classes. We propose a model that accounts for the epistatic relationships and the communication between recombination initiation and the first division. Data in this article indicate that double-strand breaks (DSBs) are not the signal for the normal delay of reductional division; these experiments also confirm that MEI4 is required for the formation of meiotic DSBs. Finally, if a DSB is provided by the HO endonuclease, recombination can occur in the absence of MEI4 and REC104.

THE initiation of recombination in meiosis is formally different from the initiation of recombination in prokaryotes or in phages. In Escherichia coli bacterial conjugation, for example, the entering male chromosome is "preinitiated"; it contains a broken end (e.g., IPPEN-IHLER and MINKLEY 1986 ). Likewise, in phage the cutting of the cos site provides a double-strand break (DSB) that can serve to initiate recombination (THALER et al. 1987 ). In eukaryotes, however, broken DNA is carefully avoided during the mitotic cycle; indeed, broken DNA that is not repaired leads to a mitotic cell cycle arrest or lethality (GAME 1993 ; IVANOV and HABER 1997 ; KOGOMA 1997 ). Current evidence is consistent with the hypothesis that eukaryotic chromosomes entering meiosis are intact and strand breakage to initiate recombination must occur specifically in meiosis.

In the yeast Saccharomyces cerevisiae, ample data indicate that a key initiating event for meiotic recombination is the formation of many DNA DSBs in meiotic prophase I (e.g., CAO et al. 1990 ; SUN et al. 1991 ; GOLDWAY et al. 1993 ; WU and LICHTEN 1994 , WU and LICHTEN 1995 ; FAN et al. 1995 ; BULLARD et al. 1996 ). At least 11 genes, which have been called early exchange (EE) genes, are known to be required for the initiation of meiotic recombination (MAO-DRAAYER et al. 1996 ). Null mutations in most genes of the EE class completely abolish all forms of meiotic recombination; in these mutants, meiotic DSBs have not been detected [SPO11 and RAD50 (CAO et al. 1990 ); XRS2 (IVANOV et al. 1992 ); MRE11 (JOHZUKA and OGAWA 1995 ); MER2 (ROCKMILL et al. 1995 ); REC102, REC104, and REC114 (BULLARD et al. 1996 ); MRE2 (NAKAGAWA and OGAWA 1997 )]. In our strain background, DSBs also were not detectable in cells containing mutations in two genes demonstrated to code for components of the synaptonemal complex, HOP1 and RED1 (MAO-DRAAYER et al. 1996 ).

Surprisingly, the failure in S. cerevisiae to initiate meiotic recombination does not result in a block in the progression of cells through meiosis. Even though the probability of forming a viable spore in the absence of recombination is very low, most cells continue through meiosis and proceed through both the first and second divisions (PETES et al. 1991 ). The overall percentage of cells forming mature ascospores and asci is reduced in diploids containing an EE mutation [e.g., mei4 (MENEES et al. 1992 ); rec102 (BHARGAVA et al. 1992 ; COOL and MALONE 1992 ); rec104 (GALBRAITH and MALONE 1992 ); rec114 (PITTMAN et al. 1993 )]. The failure to stop meiosis when recombination does not initiate might suggest that there is no "communication" between the start of recombination and subsequent meiotic events.

We recently demonstrated in a set of isogenic strains that the initiation of meiotic recombination results in a normal delay of reductional division (GALBRAITH et al. 1997 ). We observed that cells containing mutations in several of the EE genes (REC102, REC104, and REC114) enter the first division approximately an hour earlier than wild-type (WT) cells. We found this intuitively pleasing because it would allow time for recombination to occur before homologs separate from each other in the first division. We argued that the initiation of recombination creates a signal that results in the delay of the reductional division. Interestingly, mutations in one EE gene (MEI4) confer a different phenotype. As has been previously reported (MENEES et al. 1992 ), mei4 mutants entered the first division at a time indistinguishable from the time of WT cells (GALBRAITH et al. 1997 ). This effect on the timing of the reductional division was one of the first differences in meiotic phenotypes conferred by mutations in the EE genes. These data motivated us to propose a model for the order of action of these EE genes and the relationship of recombination to meiosis (Figure 1; GALBRAITH et al. 1997 ). As predicted by this model, mutations in the REC104 gene were epistatic to mutations in the MEI4 gene (GALBRAITH et al. 1997 ).

View larger version (9K): In this window In a new window Download PPT slide   Figure 1. Original model for the involvement of EE genes in the timing of reductional division (from Figure 6 of GALBRAITH et al. 1997 ). The initiation of recombination generates a transient signal that causes a delay of the first division. Some EE genes, including REC102, REC104, and REC114 are required to form the transient signal. Mutations in these genes result in an earlier reductional division. The MEI4 gene is an example of an EE gene that acts after the formation of the signal. mei4 mutations do not significantly affect the timing of reductional division (MENEES et al. 1992 ; GALBRAITH et al. 1997 ).

The experiments in this article address several questions raised by previous work that demonstrated a relationship between the initiation of meiotic recombination and the timing of the first meiotic division (GALBRAITH et al. 1997 ). All the EE genes examined in that article were meiosis-specific; that is, MEI4, REC102, REC104, and REC114 are expressed only in meiosis (COOL and MALONE 1992 ; GALBRAITH and MALONE 1992 ; MENEES et al. 1992 ; PITTMAN et al. 1993 , respectively). We wondered whether mutations in the classic EE gene RAD50, which is required for mitotic recombination repair as well as for the initiation of meiotic recombination (MALONE 1983 ; ALANI et al. 1990 ; CAO et al. 1990 ; JOHZUKA and OGAWA 1995 ), also would result in an earlier reductional division. Although an elegant study of the timing of meiotic events has been published for the special allele rad50S (ALANI et al. 1990 ), we could not find published data about the timing of the first division in a null rad50 mutation.

The data in our initial work also suggested that rec102 cells might enter the first division even earlier than rec104 cells (GALBRAITH et al. 1997 ). In this article we ask whether that is true, and if there are in fact two classes of EE mutants that are "early" and "earlier" with respect to the timing of the first division. The previous results indicated that rec104 mutations were epistatic to mei4 mutations, consistent with REC104 acting before MEI4. We ask whether other mutations are epistatic to mei4, as predicted by the model. Finally, the model shown in Figure 1 proposes that the creation of DSBs is not the signal that results in the delay of the first division. We test that in two ways. First, we ask whether DSBs occur in the mei4 mutant. If breaks occur, then DSBs would remain a candidate for the signal between recombination and the first division. Second, we ask whether a specific DSB induced during meiotic prophase I in an EE mutant with an early reductional division can restore normal timing. The data in this article allow us to keep the general model for communication between the initiation of recombination and the first meiotic division but cause us to revise it to account for the new observations (see DISCUSSION).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plasmids:
To obtain the rec102-2::LYS2 allele, a 2.7-kb EcoRI-KpnI fragment from pCM208 (COOL and MALONE 1992 ) containing REC102 was cloned into pRS426 (CHRISTIANSON et al. 1992 ) to generate pJK8. The 1.2-kb BspEI-BbuI fragment of pJK8 containing REC102 was then removed and replaced with the 4.8-kb EcoRI-PstI fragment containing the LYS2 gene from the YDp-K plasmid (BERBEN et al. 1991 ) to obtain pJK27. The deletion starts at bp -605 and ends at bp +609 of REC102.

pL32 and pJH10 were gifts from J. E. Haber (Brandeis University). pL32 contains the pSPO13-HO fusion (MALKOVA et al. 1996 ). To obtain a selectable marker for yeast transformation, the primers G418a (5'-CCCCGGTACCCAGCTGAAGCTTCGTACGC-3') and G418b (5'-CCCCGGTACCGCATAGGCCACTAGTGGATTTG-3'), both of which contained a KpnI restriction site at their ends, were used to amplify by PCR the P_TEF-kan^r-T_TEF fragment from pUG6 (GULDENER et al. 1996 ). All oligonucleotide primers used in this study were synthesized at Integrated DNA Technologies (Coralville, IA). The amplified fragment was directly cloned into the unique KpnI site at the 5' end of the remaining LYS2 coding region in pL32 to obtain pRM283, which contains the lys2::kan^r::pSPO13-HO construct. pJH10 contains the MAT-inc allele, which is a single base pair substitution C:G T:A at the Y/Z border that inhibits the HO endonuclease digestion (WEIFFENBACH et al. 1983 ). To acquire a selectable marker for integrating the MAT-inc allele into our strain, a 6.4-kb EcoRI-EcoRI fragment of pJH10 containing MAT-inc was first cloned into pRS306 (SIKORSKI and HIETER 1989 ) in which the unique BamHI site is disrupted (pRM280). Primers TRP5a (5'-GGAAAGAACTGGATcCTCTAGACC) and TRP5b (5'-GTAACAATTGGATCcTATACGGTG-3') were used to amplify by PCR a fragment containing the TRP5 gene (-276 to +2556) from yeast genomic DNA; the fragment was cloned downstream of MAT-inc to generate pRM285. (c refers to a single base substitution to generate a BamHI site.)

Strains:
Genotypes of yeast strains are listed in Table 1. All strains for kinetic studies are isogenic and are the derivatives of the homothallic diploid K65-3D. All Rec^- mutations (with the exception of rad50S) are null mutations that delete all or almost all of the coding region. Strains built by transformation were confirmed by both Southern analysis and genetic tests. Strains built by crossing were confirmed by PCR and genetic tests. Wild-type (K65-3D), rec104 (K65-104-4A), and mei4 (K65-m4) strains are decribed in GALBRAITH et al. 1997 . One-step gene replacement (ROTHSTEIN 1991 ) was performed to obtain rad50 and rec104 rad50 strains. The null rad50 mutation was the rad50::URA3 allele from pRM62 (MALONE et al. 1990 ). Also, the 5.7-kb BglI-NsiI fragment from pJK27 was used to make strains containing rec102-2::LYS2 by one-step gene replacement (ROTHSTEIN 1991 ).

Two sequential one-step gene replacements were performed to obtain JK8-7. First, the 7.6-kb PvuII-XhoI fragment from pRM285 was transformed into K65-3D to obtain the MATa/MAT-inc::TRP5 strain. This strain was then transformed with a 5.1-kb lys2::kan^r::pSPO13-HO PCR fragment made using the PCR primers Ho#1 [5'-CACCGCATCATCCAAGGATAG-3'] and Ho#2 [5'-GTAACGATGAAGCTGAGGAG-3']. Transformants were selected on YPD plus Geneticin plates (100 µg/ml Geneticin; Bethesda Research Laboratories, Gaithersburg, MD; GULDENER et al. 1996 ). Two sequential one-step gene replacements were also performed to generate JK8-5 and JK8-8.

RM96-15A, a closely related congenic strain containing the rad50S allele (BULLARD et al. 1996 ), was crossed with K65-Het104mei4, a K65-3D derivative heterozygous for both mei4 and rec104 (GALBRAITH et al. 1997 ). The resulting diploid was dissected to obtain JK8-11-5B (MATa mei4::URA3 rad50S::URA3), JK8-11-5C (MAT mei4::URA3 rad50S::URA3), JK8-11-2B (MATa rad50S::URA3), and JK8-11-1D (MAT rad50S::URA3). JK8-11-5B was crossed with JK8-11-5C to get JK8-12; and JK8-11-2B was crossed with JK8-11-1D to get JK8-13. JK8-12 was used for measuring meiotic DSBs in mei4 cells, and JK8-13 was used as the MEI4 control.

Media, growth, and sporulation conditions:
Media, growth, and sporulation conditions have been described previously (GALBRAITH et al. 1997 ). For each experiment, all cultures were grown in the same medium and were treated identically. At least one culture of a WT strain (K65-3D) and one culture of a rec104 strain (K65-104-4A) were included as a normal and an early timing control. We note, as previously observed (GALBRAITH et al. 1997 ), that the exact kinetics of sporulation can vary slightly from one experiment to another, but the relative timing does not.

Staining nuclei with 4',6-diamidino-2-phenylindole and classifying cells:
Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and cells were examined with a fluorescent microscope as described previously (GALBRAITH et al. 1997 ). For time points 2 hr, at least 400 cells were counted; for all subsequent time points at least 1000 cells were counted. Mononucleate cells include those that have not entered meiosis and those that have just begun the meiotic process but have not yet undergone a division. Binucleate cells consist of cells that have undergone the first meiotic division. We define tetranucleate cells as cells having three or four nuclei (i.e., cells have undergone both the first and the second meiotic divisions). Not all tetranucleate cells go on to form mature asci, especially in EE mutants. Thus, in EE mutants, the final percentage of sporulation is always less than the percentage of tetranucleate cells.

DNA analysis:
The physical examination of DSB DNA was described in BULLARD et al. 1996 . To detect DSBs at the MAT locus, DNA was digested with EcoRI, subjected to electrophoresis, transferred to Hy-Bond N (Amersham, Arlington Heights, IL), and probed with the 1.1-kb HindIII-BamHI fragment isolated from pRM285. In every experiment using the pSPO13-HO construct, the presence of DSBs at the MAT locus was verified by Southern analysis. To directly compare the appearance of the HO-induced DSB with a natural meiotic DSB, the filter used for measuring the HO DSBs in the JK8-7 (Rec⁺) diploid was reprobed with the 122-bp EcoRI-HindIII fragment isolated from pRM9 (MALONE et al. 1994 ) to detect DSBs at the HIS2 hotspot (BULLARD et al. 1996 ). To examine DSBs at the THR4 hotspot (WU and LICHTEN 1995 ), DNA was digested with BglII and probed with a 0.9-kb fragment upstream of the THR4 gene. Primers THf1 (5'-TGCCCGATGATAAGGTCTCC-3') and THr1 (5'-ATGTCACTCGTTCTTGCAGC-3') were used to obtain the probe by PCR.

All imaging and quantification analysis was done using a Molecular Dynamics (Sunnyvale, CA) PhosphorImager Model 445SI as per instructions from the manufacturer.

Measurement of commitment to meiotic recombination at the MAT locus:
Commitment to meiotic recombination was measured by a return-to-growth assay (RESNICK et al. 1986 ). Cells were removed from sporulation medium at various time points and plated on rich medium (YPD) to allow mitotic growth. At least 200 colonies from two independent cultures from each time point were tested to determine the frequency of -mating cells.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

rad50 mutants have an earlier reductional division than rec104 mutants:
Our earlier work indicated that some EE genes (REC102, REC104, and REC114 vs. MEI4) can be distinguished because null mutations confer different effects on the timing of the reductional division (GALBRAITH et al. 1997 ). Of the known EE genes, RAD50 is perhaps the most heavily studied (e.g., MALONE 1983 ; ALANI et al. 1990 ; CAO et al. 1990 ; PADMORE et al. 1991 ; RAYMOND and KLECKNER 1993 ; JOHZUKA and OGAWA 1995 ). To understand the role of RAD50 (an EE gene that is not meiosis-specific) in the relationship between recombination and the first division, we examined the effect of a rad50 null mutation on the timing of the first division. In each experiment (Figure 2A and Figure B) one culture of a rad50 diploid, a rec104 diploid (early timing control), and a WT diploid (normal timing control) were examined. rad50 cells undergo reductional division ~2 hr earlier than WT cells and ~1 hr earlier than the rec104 mutant. This becomes clearer in an expanded view of early time points (Figure 2C). To verify this new rad50 phenotype, we simultaneously examined the kinetics of the first division in four additional independent rad50 cultures (with WT and rec104 controls). All four independent rad50 cultures initiated the first division about an hour earlier than the rec104 mutant (data not shown). We conclude that a rad50 null mutant initiates reductional division even earlier than a rec104 mutant. The two different phenotypes allowed us to examine epistatic interactions between the rad50 and rec104 mutations. The timing in the rad50 rec104 double mutant is indistinguishable from that in the rad50 single mutant (Figure 2A and Figure B). From these observations we conclude that rad50 is epistatic to rec104.

View larger version (28K): In this window In a new window Download PPT slide   Figure 2. Timing of the first and second division in rad50 diploids. (A and B) Percentage of binucleate cells vs. time in two independent experiments. Isogenic WT (K65-3D; ), rec104 (K65-104-4A; ), rad50 (JN10-2-15B; ), and rec104 rad50 (JN10-2-54D; x) diploids were sporulated. Binucleate cells represent cells that have undergone the first meiotic division. Final sporulation for the WT, rec104, rad50, and rec104 rad50 diploids averaged 74, 26, 31, and 31% mature asci, respectively. (C) An expanded view of the early times showing the standard deviations, from the two experiments in A and B. (D) Percentage of tetranucleate cells vs. time in sporulation. Tetranucleate cells represent cells having gone through both meiotic divisions. Data were averaged from the two independent experiments with error bars indicating the standard deviations.

In previous work, EE mutants exhibiting a more rapid reductional division still proceeded through the second division at the normal time (GALBRAITH et al. 1997 ). Figure 2D shows that (like rec102, rec104, and rec114) a rad50 mutation does not alter the timing of the initiation of the second division. This is consistent with the idea that the timing of the second division is, to some extent, independent of the timing of the first division (GALBRAITH et al. 1997 ).

rec102 mutants, like rad50, have an even earlier reductional division than rec104 mutants:
The discovery that the rad50 mutants have an even earlier reductional division than rec104 mutants reminded us of previous work, which hinted that rec102 mutants might also confer this phenotype (GALBRAITH et al. 1997 ). To determine if rec102 mutants truly go through the reductional division more rapidly than rec104 mutants, we examined rec102 and rec104 again in detail (Figure 3). In both experiments, rec102 cells (JK5-1-5D) began the first division about 1/2 to 1 hr earlier than isogenic rec104 cells (K65-104-4A). The kinetics of the reductional division in the rec102 rec104 double mutant (JK5-1-1B) are almost identical to those in the rec102 single mutant, which leads to the conclusion that rec102, like rad50, is epistatic to rec104 (Figure 3).

View larger version (26K): In this window In a new window Download PPT slide   Figure 3. (A and B) Percentage of binucleate cells vs. time in sporulation. A rec102 diploid has an even earlier reductional division than a rec104 diploid. Wild-type (K65-3D; ), rec104 (K65-104-4A; ), rec102 (JK5-1-5D; ), and rec102 rec104 (JK5-1-1B; x) diploids were sporulated. Samples were taken and stained as described in MATERIALS AND METHODS. A and B represent two independent experiments. Final sporulation results (24 hr) for the WT, rec104, rec102, and rec102 rec104 diploids averaged 82, 37, 35, and 36% mature asci, respectively.

rec102 is epistatic to mei4:
Our previous data (GALBRAITH et al. 1997 ) indicated that the EE genes REC104 and MEI4 could be placed into different groups because mutations in the two genes conferred different phenotypes with respect to the timing of reductional division: mei4 cells undergo the first meiotic division at the normal time in contrast to the earlier division in rec104 mutants. The rec104 mutation is epistatic to mei4, which suggests a model for the order of action of these genes (Figure 1). In that article we predicted that all EE mutants conferring an earlier reductional division should be epistatic to mei4. We tested this prediction by comparing the timing of the reductional division in the rec102 mei4 double mutant with that in each single mutant (Figure 4). As we found previously, mei4 cells (K65-m4) begin reductional division at a time indistinguishable from WT cells. The timing of the reductional division in the rec102 mei4 double mutant (JK5-1-1A) is early and is indistinguishable from the timing in the rec102 single mutant (JK5-1-5D). This result indicates that rec102 is epistatic to mei4, consistent with the hypothesis that EE genes act in a linearly dependent pathway (see DISCUSSION).

View larger version (22K): In this window In a new window Download PPT slide   Figure 4. A rec102 mutation is epistatic to a mei4 mutation. Isogenic WT (K65-3D; ), mei4 (K65-m4; ), rec102 (JK5-1-5D; ), and rec102mei4 (JK5-1-1A; x) diploids were sporulated. Values shown are the means plus standard deviations of two independent cultures. Final sporulation results (24 hr) for the WT, rec102, mei4, and rec102 mei4 diploids averaged 82, 37, 38, and 36% mature asci, respectively.

MEI4 is required for the formation of meiosis-specific DSBs:
Null mutations in the EE genes tested thus far [SPO11 and RAD50 (CAO et al. 1990 ); XRS2 (IVANOV et al. 1992 ); MRE11 (JOHZUKA and OGAWA 1995 ); MER2 (ROCKMILL et al. 1995 ); REC102 REC104, and REC114 (BULLARD et al. 1996 ); MRE2 (NAKAGAWA and OGAWA 1997 )] all prevent meiotic DSBs. The published phenotypes of mei4 mutants strongly suggest that a mei4 mutation should prevent the formation of meiotic DSBs (MENEES et al. 1992 ; NAG et al. 1995 ); however, this has not been reported. The JK8-12 diploid is homozygous for mei4::URA3 in a rad50S background. rad50S allows meiotic DSBs to accumulate (ALANI et al. 1990 ; CAO et al. 1990 ). No meiotic DSBs can be detected at the THR4 hotspot (GOLDWAY et al. 1993 ; WU and LICHTEN 1994 ) in mei4 mutant cells, whereas meiotic DSBs can be detected 3 hr after cells enter sporulation in MEI4 control (Figure 5). Similar results were found at the HIS2 hotspot (data not shown). This confirms that MEI4 is required for the formation of meiosis-specific DSBs, and is consistent with our previous proposal that DSBs are not the signal for coordinating meiotic recombination and the reductional division (GALBRAITH et al. 1997 ; and see DISCUSSION).

View larger version (46K): In this window In a new window Download PPT slide   Figure 5. MEI4 is required for the formation of meiosis-specific DSBs. JK8-12 (mei4::URA3 rad50S::URA3) and JK8-13 (rad50S::URA3) cells were sporulated and DNA was prepared from samples taken at various time points. Southern analysis was done by digestion of genomic yeast DNA with BglII and probing with the 0.9-kb fragment upstream of THR4 (see MATERIALS AND METHODS). This generates a 10.5-kb parental band and a 3.8-kb major meiotic DSB band.

The DSB induced by pSPO13-HO can induce recombination at the MAT locus in mei4 and rec104 mutants:
The original model (Figure 1; GALBRAITH et al. 1997 ) proposed that the initiation of recombination creates a signal that normally delays the reductional division. Because a mei4 diploid does not form meiotic DSBs, but enters the reductional division at a time indistinguishable from that of WT cells, DSBs do not appear to be the signal. However, although DSBs are not necessary for the normal delay, the presence of DSBs during prophase I might be recognized, communicate to the first division that recombination has started, and delay the first division. To test this possibility we introduced a DSB into EE mutant cells that normally have no breaks. MALKOVA et al. 1996 showed that the fusion of the HO endonuclease gene to the SPO13 promoter can induce a DSB at the MAT locus during meiotic prophase I and that the kinetics of break formation by HO are indistinguishable to the kinetics of DSB formation at the THR4 hotspot. We therefore used the pSPO13-HO construct to introduce a DSB in rec104 and mei4 mutants in early meiosis.

Southern blot analysis was performed to examine the appearance of the pSPO13-HO-induced DSBs at the MAT locus (Figure 6). Quantitative analysis confirms that the pSPO13-HO construct induces DSBs at the MAT locus in our strain background during the early stages of meiosis. Comparison with DSBs at the HIS2 hotspot (BULLARD et al. 1996 ) indicates that the HO DSBs appear at the same time as naturally occurring meiotic DSBs (Figure 6C). This result also demonstrates that DSBs created by HO can occur normally in an EE mutant [as found by MALKOVA et al. 1996 in their strain background].

View larger version (40K): In this window In a new window Download PPT slide   Figure 6. Timing of HO-induced DSB formation at the MAT locus. (A) Structure of the MAT loci. After EcoRI digestion, the 1.5-kb HindIII-BamHI probe (thick line) detects two parental bands (6.3 kb for MATa and 6.0 kb for MAT-inc-TRP5) and a 3.8-kb DSB band at the MATa locus caused by HO. The open boxes refer to the MAT loci (ASTELL et al. 1981 ), and the black box refers to the 2.8-kb TRP5 gene. The asterisk indicates the -inc mutation at the junction of Y and Z region, which prevents digestion by HO. B, BamHI; R, EcoRI. (B–E) Appearance of DSBs at the MAT locus. Two independent cultures of a WT [+pSPO13-HO] diploid (JK8-7; ), a rec104 [+pSPO13-HO] diploid (JK8-8; ), and a mei4 [+pSPO13-HO] diploid (JK8-5; ) were sporulated. (Although only one culture is shown in B and C, both cultures were examined by Southern analysis and used to quantitate the results shown in D and E.) Numbers above the lanes in B and C refer to the number of hours the cells were in sporulation. The parental and the DSB bands are indicated. (C) The appearance of DSBs at the HIS2 hotspot (BULLARD et al. 1996 ). The same filter used in B for measuring HO-induced DSB in JK8-7 was reprobed with the 122-bp EcoRI-HindIII fragment upstream of the HIS2 gene (see MATERIALS AND METHODS). The parental and the DSB bands are indicated. (D) The fraction of total DNA in the DSB band was quantified as described in MATERIALS AND METHODS and BULLARD et al. 1996 . (E) Commitment to gene conversion at the MAT locus induced by pSPO13-HO. The two cultures of WT [+pSPO13-HO] (JK8-7; ), rec104 [+pSPO13-HO] (JK8-8; ), and mei4 [+pSPO13-HO] (JK8-5; ) diploids were examined by return-to-growth experiments for induction of recombination at the MAT locus (see MATERIALS AND METHODS). At least 200 cells were examined for recombination at each time point for each culture to determine the percentage of -mating cells resulting from a gene conversion event at the MAT locus.

MALKOVA et al. 1996 showed that the DSB generated by pSPO13-HO can induce meiotic recombination at the MAT locus in rad50 cells, which are normally completely deficient in meiotic recombination. We asked if the HO-induced DSB could also induce recombination in rec104 or mei4 mutants. The HO DSBs could result in conversion of the MATa allele to the MAT-inc allele, generating MAT-inc/MAT-inc diploids, which can be measured by mating tests (see MATERIALS AND METHODS). The pSPO13-HO DSBs induce recombination at the MAT locus in both rec104 and mei4 diploids with a frequency and kinetics indistinguishable from a WT strain (Figure 6E). This result is consistent with the proposal that REC104 and MEI4 are not absolutely required for recombination after the formation of DSBs (see DISCUSSION).

The HO-induced DSB at the MAT locus has no effect on the timing of meiotic divisions in mei4 and rec104 mutants:
As a control, we first tested the effect of the HO DSB on the timing of the reductional division in mei4 cells. A comparison of the mei4 mutant with and without the pSPO13-HO construct is shown in Figure 7. The timing of the first division is indistinguishable between the mei4 diploid and the mei4 diploid containing pSPO13-HO. Both strains initiate reductional division at about the same time as WT cells.

View larger version (27K): In this window In a new window Download PPT slide   Figure 7. The effect of the HO-induced DSB at the MAT locus on the timing of the meiotic divisions in mei4 and rec104 diploids. (A) Percentage of binucleate cells for two cultures of mei4 [+pSPO13-HO] (JK8-5; ), WT (K65-3D; ), and mei4 (K65-m4; ) diploids sporulated at the same time. (B) Percentage of tetranucleate cells vs. time in sporulation. Same symbols as in A. (C) Percentage of binucleate cells for two cultures of rec104 [+pSPO13-HO] (JK8-8; ), rec104 (K65-104-4A; ), as well as a culture of WT (K65-3D; ); diploids all sporulated at the same time. (D) Percentage of tetranucleate cells vs. time in sporulation. Same symbols as in C.

Unlike mei4 cells, rec104 cells start reductional division ~1 hr earlier than WT cells (Figure 2, Figure 3, and GALBRAITH et al. 1997 ). We therefore tested whether a DSB induced by pSPO13-HO could restore normal timing of the reductional division in a rec104 mutant. The timing of the reductional division in rec104 cells containing pSPO13-HO is identical to that in rec104 cells (Figure 7). From this experiment we conclude that the pSPO13-HO-induced DSB is not sufficient to restore normal timing of the first division in rec104 cells.

We also examined the effect of the artificial HO DSB on the timing of the second meiotic division in mei4 and rec104 cells. The data show that there is no alteration of timing in mei4 or rec104 diploids (Figure 7B and Figure D), as we would have predicted from earlier observations and the model.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Epistatic interactions among the EE gene mutants:
Our previous work (GALBRAITH et al. 1997 ) revealed one of the first phenotypic differences conferred by null EE mutations. Mutations in REC102, REC104, and REC114 confer an earlier reductional division than WT cells. In contrast, a mei4 mutant undergoes the first division with timing indistinguishable from that of WT cells (Figure 4 and GALBRAITH et al. 1997 ). The work presented here adds RAD50 to the group of EE genes required for normal timing of the reductional division. In addition, we observe a new phenotype in the rad50 and rec102 mutants: an even earlier reductional division than rec104 mutants. The difference in timing allowed us to test epistatic interactions among the EE mutations. We have demonstrated that (1) rec104 is epistatic to mei4 (GALBRAITH et al. 1997 ), (2) rec102 is epistatic to mei4 (Figure 4), (3) rad50 is epistatic to rec104 (Figure 2), and (4) rec102 is epistatic to rec104 (Figure 3). These results are consistent with the hypothesis that EE genes, at least with respect to the timing of the first division, function in a linearly dependent pathway (see below).

In addition to the timing difference, another phenotypic difference among EE mutants was reported by OHTA et al. 1998 . Mutations in four EE genes (MRE11, RAD50, XRS2, and MRE2) alter the micrococcal nuclease (MNase) sensitivity at a meiotic DSB site. The mre2 and mre11 mutants (type 1) confer a reduction in MNase sensitivity relative to WT cells, whereas the MNase sensitivity in rad50 and xrs2 mutants (type 2) reaches a higher level than the WT level. MNase sensitivity in the mre11 rad50 double mutant is the same as that in the mre11 single mutant, indicating that mre11 (type 1) is epistatic to rad50 (type 2) with respect to MNase sensitivity (OHTA et al. 1998 ). If the recombination pathway affects both chromatin structure (MNase sensitivity) and the timing of the first division in the same way, then we would predict that mre11 should have a very early first division (i.e., it should be upstream of rad50).

Different phenotypes conferred by EE mutations are examined in this article and in the work of OHTA et al. 1998 . RAD50 was the only gene examined in both studies. A null mutation in RAD50 confers a very early first division and an increase in MNase sensitivity at a meiotic DSB site. It is possible that progressive alterations in chromatin structure are the signals for the timing of the reductional division. If this united view is true, one would also predict that rec102 mutants should alter chromatin like rad50 and that a rec104 or rec114 mutation might have different effects on chromatin structure than a rad50 mutation. Finally, in this scheme, the meiotic chromatin of mei4 mutants should appear like that from WT cells.

A revised model for communication between initiation of recombination and reductional division:
GALBRAITH et al. 1997 proposed a model to explain the delay in reductional division caused by initiation of recombination. In this article we present data showing that rec102 and rad50 mutations confer a new phenotype, an even earlier first division than a rec104 mutation. Furthermore, mutations in both REC102 and RAD50 are epistatic to a mutation in REC104. To account for this information, a revised model is presented in Figure 8. We propose that the EE genes may be sorted into three groups. The first group includes RAD50 and REC102. Mutations in these genes result in a very early reductional division, ~2 hr earlier than division in WT cells. We propose that RAD50 and REC102 normally function to generate an initiation signal with partial ability to delay the first division. The second group would include REC104 and REC114. Mutations in these genes cause the reductional division to occur ~1 hr earlier than it occurs in WT cells but ~1 hr later than in the rec102 or rad50 mutant. We propose that these genes would be required to alter the putative negative signal to the fully functional stage. As previously indicated (GALBRAITH et al. 1997 ), the signal must cause only a transient delay because WT cells do, of course, proceed through meiotic divisions. The third group of EE genes would include MEI4. Mutations in this last group of EE genes do not affect timing of the first division. We suppose that Mei4p acts after the formation of the signal. The fact that the EE^- mutants fall into three classes suggests that the process that recognizes recombination initiation and leads to a delay in the first division is complex and that different steps in initiation may generate separate signals.

View larger version (10K): In this window In a new window Download PPT slide   Figure 8. A model for the interaction between initiation of recombination and reductional division. EE genes can be classified into three groups. Genes in the first group (e.g., RAD50) can generate an initial signal, "S," which has partial activity to delay the reductional division. The genes in the second group (e.g., REC104) result in the modification of S to "S*". S* is the fully functional signal responsible for the normal 2-hr transient delay of the reductional division. The genes in the third group like MEI4 are not involved in the generation of the signal. Mutations in the first group of genes (e.g., RAD50) eliminate the initial signal and therefore result in a very early reductional division. Mutations in the second group of genes (e.g., REC104) cannot generate the modifier, "M", so no S* is made. However, the initial signal S is still made, thus leading a partial delay of the division. These mutants have a reductional division that is earlier than that of WT cells, but later than that of mutants in the first group. Mutations in the third group of genes (e.g., MEI4) do not affect formation of the transient signal, and hence the initiation of reductional division is delayed as it should be and occurs at the normal time.

Our data indicate that recombination initiation is not needed for the first division (consistent with all published reports) and also that, in its absence, the first division can begin up to 2 hr earlier. In fact, in rec102 or rad50 cells, the first division begins at a time when recombination would normally be starting. This indicates that the first division apparatus is ready at the time of recombination, but is normally prevented from acting when recombination initiates. PADMORE et al. 1991 found (in the SK1 background) that the first division normally occurs ~15 min after the completion of recombination. The transient 2-hr delay of the first division caused by the initiation of recombination makes sense because it would allow time for recombination to proceed before attempting to segregate chromosomes.

Meiotic DSBs do not serve as the transient signal for the delay of the first division:
Both the original model (Figure 1; GALBRAITH et al. 1997 ) and the revised model in Figure 8 predict that meiotic DSBs are not the transient signal that normally delays the first meiotic division. This prediction is consistent with the phenotypes conferred by the rad50S alleles, which allow the accumulation of meiotic DSBs, but do not delay the first division or arrest cells through meiosis, although the second division is delayed in rad50S strains (ALANI et al. 1990 ; CAO et al. 1990 ). This work shows that MEI4 is required for the formation of meiotic DSBs. This result excludes meiotic recombination DSBs as a candidate for the transient signal, because MEI4 is required for generating them, and yet mei4 cells undergo reductional division with timing indistinguishable from that of WT cells.

Though meiotic DSBs cannot be the normal signal for delay of the first division, we wondered if a DSB occurring early in meiosis (or the recombination associated with it) could serve as a signal to delay the first division. It has been demonstrated in yeast that a single DSB can cause G2 arrest during mitosis, and one unrepaired DSB even on a dispensable plasmid can lead to lethality (BENNETT et al. 1993 ; FAIRHEAD and DUJON 1993 ; GAME 1993 ). Because mitotic cells appear to recognize a single DSB, the pSPO13-HO-induced break during meiotic prophase I might be recognized and restore normal timing in rec104 cells. Such breaks should, however, have no effect on a mei4 mutant.

To address this question, we used a pSPO13-HO construct to induce a DSB at the MAT locus during meiotic prophase I in rec104 and mei4 cells that normally have no DSBs. The percentage of DSBs created by the HO endonuclease reaches 30% of the total DNA by 12 hr. If one assumes that only one of the four chromatids is cut per cell, then we conclude that all the cells have a break by then (4 x 30% 100%). Previous results (KOLODKIN et al. 1986 ; MALKOVA et al. 1996 ), however, suggest that it is more likely that both chromatids are often cut; in this case ~60% of the cells have the break (2 x 30%). Because an average of 5–15% of the cells in our strains do not enter meiosis, we conclude that ~70% of the cells passing through meiosis have the HO breaks. There is a degree of asynchrony in the population; not all cells move through meiosis at the same time and hence the HO break and the normal meiotic breaks occur over a period of several hours. We note that the kinetics of the induction of HO breaks in this experiment are the same as the kinetics of real recombination DSBs in the same WT cells. Further, the kinetics of the HO break in the Rec^- mutants are the same as in the WT Rec⁺ cells. Because at least 60% of the cells contain the HO break, and because they follow the same kinetics as normal recombination breaks, we argue it would be possible to see the MI delay if it could be caused by the HO break.

The HO break did not affect the timing of the first division in mei4 cells. More interestingly, the HO break did not restore normal timing of the first division in a strain (rec104) with an earlier first division. This result is consistent with the hypothesis that DSBs are not the transient signal arising from the initiation of recombination and indicates that the presence of one DSB during meiosis prophase I is not sufficient to institute the normal delay of reductional division. An alternative possibility, but one we think unlikely, is that a single meiotic DSB would be capable of delaying MI in a rec104 strain, but that the HO break is not recognized by the delay system. This might result from the fact that the HO break can occur outside the context of normal recombination DSBs. For example, the HO break does not require the gene products of the meiosis-specific EE genes (e.g., REC104, MEI4, MER2). Finally, we note that the HO-induced break appears (Figure 6) to persist over a somewhat longer period than the DSBs at THR4 or HIS2. One interpretation is that the HO breaks are not processed with the same efficiency as normal meiotic recombination DSBs. An alternative interpretation is that the HO endonuclease is present for a longer time than the meiotic recombination initiation complex.

The transient signal proposed in Figure 8 could be a protein(s) or a post-translational modification of a protein(s). Alternatively, it could be an altered DNA or chromosomal structure (e.g., see earlier discussion about meiotic chromatin), although it cannot be meiotic DSBs. Studies from different groups have shown that chromatin becomes more accessible at recombination hotspots before the formation of DSBs (e.g., OHTA et al. 1994 , OHTA et al. 1998 ; WU and LICHTEN 1994 , WU and LICHTEN 1995 ; FAN and PETES 1996 ). Using the technique of fluorescence in situ hybridization (FISH), WEINER and KLECKNER 1994 demonstrated that homologous chromosomes associate with each other after premeiotic DNA synthesis prior to recombination. Consistent with the idea that homologs associate before meiotic DSB formation, examination of the HIS2 hotspot indicates that a "hot" chromosome with increased DSBs can stimulate DSBs and recombination in trans on a "cold" homolog (BULLARD et al. 1996 ). These authors argued that the best explanation was association or interaction of homologs prior to DSB formation. XU and KLECKNER 1995 found that a 32-bp BamHI linker at the synthetic HIS4::LEU2 hotspot also appeared to act in trans and argued that homologs must interact prior to the formation of DSBs. Though different quantitative results were obtained at the HIS2 hotspot and the HIS4::LEU2 hotspot, both observations agree with the proposal that homologs interact before meiotic DSBs are formed. This early "interacting" process between homologous chromosomes could be the basis of the transient signal to delay the first meiotic division.

Adding a DSB can induce gene conversion in a mei4 or rec104 mutant:
MALKOVA et al. 1996 demonstrated that the pSPO13-HO-induced DSB can cause meiotic recombination in null rad50 cells, which are normally deficient in meiotic recombination. The data in this article show that this construct can also induce gene conversion to the same level as that of the WT control in the other two EE mutants, rec104 and mei4. Formally all of these results suggest that RAD50, MEI4, and REC104 are only required before DSB formation. However, the break created by HO is different than the normal meiotic DSB. It is also possible that some genes required to make meiotic DSBs are needed after the breaks are formed. Certain recessive alleles of RAD50 [rad50S alleles (ALANI et al. 1990 ; CAO et al. 1990 )] or MRE11 [mre11S (NAIRZ and KLEIN 1997 ); mre11-58 (TSUBOUCHI and OGAWA 1998 )] cause a failure of DSB processing, allow the accumulation of DSBs, and confer the phenotype of a late exchange mutation. Thus, for the moment, we hesitate to completely exclude MEI4 and REC104 from all post-DSB meiotic roles.

Meiosis may be thought of as a well-controlled intracellular developmental process. High levels of meiotic recombination and the subsequent reductional division are two of the unique events in meiosis. Our results demonstrate that several EE gene products required for the initiation of recombination are also involved in controlling the proper timing of reductional division, which suggests that these two unique meiotic processes do communicate with each other. The nature of the transient signal proposed in this article is under further investigation.

	ACKNOWLEDGMENTS

We thank James E. Haber for providing plasmids pL32 and pJH10. We thank Jan Fassler and Stuart Haring for reading and commenting on the paper and John Nau for technical assistance. This work was supported by National Institutes of Health grant R01-GM36846 to R.E.M.

Manuscript received August 25, 1998; Accepted for publication February 9, 1999.

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