The sql Mutant of Saccharomyces cereukiae Arrests in ...The sql Mutant of Saccharomyces cereukiae Arrests in Pachytene and Is Deficient in Meiotic Recombination Daniel X. Tishkoff,

Copyright 0 1995 by the Genetics Society of America

The s q l Mutant of Saccharomyces cereukiae Arrests in Pachytene and Is Deficient in Meiotic Recombination

Daniel X. Tishkoff, * Beth Rockmill, G. Shirleen Roedert and Richard D. Kolodner * *Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Department o Baological Chemistry and Molecular

Pharmacology, Hamard Medical School, Boston, Massachusetts 021 15 and f Department of Biology, Yale University, New Haven, Connecticut 06520-8103

Manuscript received April 1, 1994 Accepted for publication October 1, 1994

ABSTRACT Strand exchange protein 1 (Sepl ) from Saccharomyces meuisiae promotes homologous pairing of DNA

in vitro and sepl mutants display pleiotropic phenotypes in both vegetative and meiotic cells. In this study, we examined in detail the ability of the sepl mutant to progress through meiosis I prophase and to undergo meiotic recombination. In meiotic return-to-growth experiments, commitment to meiotic recombination began at the same time in wild type and mutant; however, recombinants accumulated at decreased rates in the mutant. Gene conversion eventually reached nearly wild-type levels, whereas crossing over reached 15-50% of wild type. In an assay of intrachromosomal pop-out recombination, the sepl, dmcl and rad51 single mutations had only small effects; however, pop-out recombination was virtually eliminated in the sepl dmcl and sepl rad51 double mutants, providing evidence for multiple recombination pathways. Analysis of meiotic recombination intermediates indicates that the sepl mutant is deficient in meiotic double-strand break repair. In a physical assay, the formation of mature reciprocal recombinants in the sepl mutant was delayed relative to wild type and ultimately reached only 50% of the wild-type level. Electron microscopic analysis of meiotic nuclear spreads indicates that the seplA mutant arrests in pachytene, with apparently normal synaptonemal complex. This arrest is RADPindepen- dent. We hypothesize that the Sepl protein participates directly in meiotic recombination and that other strand exchange enzymes, acting in parallel recombination pathways, are able to substitute partially for the absence of the Sepl protein.

H ETERODUPLEX DNA is a central intermediate in homologous recombination in all current models ( PETES et al. 1991 ) . The most extensively stud- ied protein that catalyzes the formation of hetero- duplex DNA is the RecA protein of Escherichia coli. Muta- tions in the recA gene result in pleiotropic phenotypes, including severe defects in recombination (reviewed by SMITH 1988) . In vitro, RecA protein promotes a number of reactions, including the transfer of one strand of a linear DNA duplex to a homologous single-stranded circular DNA (KO~ALCZYKOWSKI 1991; RADDING 1991; WEST 1992). Strand exchange protein 1 (Sepl ) , a 175- kD protein from Saccharomyces cerevisiae, was purified to homogeneity based on its ability to promote a similar strand exchange reaction in vitro ( KOLODNER et al. 1987; DYK~TRA et al. 1990). Like R e d , Sepl promotes strand exchange between a linear duplex DNA and a circular single strand as well as the renaturation of homologous single-stranded DNA and the formation of paranemic joints ( KOLODNER et al. 1987; HEYER et al. 1988; DEX~TRA et al. 1990; CHEN et al. 1994). However, Sepl does not require ATP, and it has been shown to contain an intrin-

Curresponding author: Richard Kolodner, Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, 44 Binney St., Bos- ton, MA 02115.

Genetics 139: 495-509 (February, 1995)

sic 5 to 3 exonuclease activity that is required to initiate strand exchange ( KOLODNER et al . 1987; JOHNSON and KOLODNER 1991 ) . The exonuclease activity of Sepl is slow and nonprocessive, with a turnover number of 20 nucleotides per minute and a processivity of 45 nucleotides on duplex DNA (JOHNSON and KOLODNER 1993). For Sepl to initiate strand exchange, the linear duplex must have a single-stranded tail 220 nucleotides in length; this single-stranded tail can be produced by Sepl or an exogenous exonuclease. The Sepl nuclease activity is dispensable for the strand displacement and branch migration phases of the strand exchange reaction (JOHNSON and KOLODNER 1993). Recently, LIU and GILBERT (1994) have shown that SEPl encodes a nuclease specific for G4 tetraplex DNA, suggesting that Sepl protein may act in Grich sequence-mediated recombination and telomere function.

Sepl is representative of a recently identified class of homologous pairing proteins that do not require ATP but do require an intrinsic or associated exonuclease for strand exchange. These proteins include the Drosophila Rrpl protein, the human HPP-1 protein and the yeast Dpal proteins, which are strand transfer enzymes with intrinsic exonuclease activities (Rrpl and HPP-1) or which require an exogenous exo-

496 D. X. Tishkoff rt nl.

nuclease activity (Dpa l ) (HALBROOK and MCENTEE 1989; MOORE and FISHEI. 1990; FISHEL et al. 1991; SANDER et al. 1993; R. FISHEL, personal communication) . Recent studies have demonstrated that a combination of E;. coli ExoVIII and RecT protein (which are encoded by the same operon) promotes strand exchange in vitro ( HALL rt al. 1993; HALL and KOLOUNER 1994). In these reactions, ExoVIII provides a 5’-3’ exonuclease and RecT functions in homologous pairing. The role of ExoVIII ( the recli gene product) and RecT in recombination is well established (for review, see Kol,(_)DNER rt a[. 1993).

Recently, the W 5 I and DMCI genes of yeast have been shown to encode proteins with significant sequence homology to each other arid to the bacterial RecA protein (BISHOP et nl. 1992; SHINOHAM rt nl. 1992). However, neither the Rad51 nor the Dmcl protein has yet been shown to promote RecA-like pairing reactions in vitro. KAD51 belongs to the RAD52 epistasis group of mutants, which are deficient in the repair of DNA damage induced by ionizing radiation. All mem- bers of this group display a characteristic array of pleiotropic phenotypes, including sporulation defects and reductions in mitotic and meiotic recombination (for review, see RENICK 1987). The dmcl null mutant arrests i n a late stage of meiosis I prophase and displays a moderate defect in commitment to meiotic recombination (BISHOP rt d . 1992) . In experiments that physi- cally monitor recombination intermediates and products, both the rad51 mutant and the dmrl mutant show defects in their ability to repair double-strand breaks (DSBs) and to form mature recombinants (BIsFIoP rt (11. 1992; SHINOFIAKA et al. 1992) . Sepl protein is known to promote strand exchange, whereas Dmcl and Rad51 proteins are suspected to promote strand exchange by virtue of their homology to RecA. A mutation in any one of these genes does not completely eliminate meiotic recombination. Perhaps Dmcl, Rad51 and Sepl have partially redundant functions and act in parallel pathways of meiotic recornbination.

Determining the in -0iuo role ( s ) of Sepl is compli- cated 11s the fact that the .wpl null mutation is pleiotropic. The S f P l gene has been isolated independently in several laboratories studying very different aspects of yeast cell metabolism (for review, see Kt:hRSEYand KIP- LIM; 1991 ) . The S I P 1 gene is identical to the KKMl gene, which was identified as a mutation that affects nuclear fusion (KIM rt nl. 1990), to KAR5, which was isolated as a mutation that stabilizes a plasmid with a defective replication origin ( KIPLING rt a 2 . 1991 ) , and to XlWl , which was isolated as a gene encoding a 5 ‘ to 9 ’ exoribonuclease ( LARIMER and STEVENS 1990) . These diverse phenotypes indicate that Sepl functions in other cellular processes as well as recombination.

The S W Z null mutant has been characterized previously for various phenotypes related to recombination

(DYKSTRA et al. 1991; TISHKOFF et nl. 1991). Mitotic phenotypes include slow growth, a decrease in the ability to recover from damage induced by ultraviolet light and a slight decrease (two- to threefold) in gene conversion ( TISHKOFF et al. 1991 ) . During meiosis, sejjl mutants display a defect in sporulation and a reduction in spore viability. Cells from the sepl mutant undergo premeiotic DNA replication with wild-type kinetics, but they arrest before the first meiotic division (TISHKOFF rt al. 1991). Meiotic recombination in s $ l strains was measured in return-to-growth experiments, in which cells are induced to undergo meiotic recombination and then returned to vegetative growth. Wild type and the srpl mutant reached the point of commitment to meiotic recombination at the same time (TISHKOFF P[ (11. 1991 ) . After extended incubation in sporulation medium, the s $ l mutant displayed a slightly higher than wild-type level of recombination between his4 heteroal- leks (TISHKOFF rl nl. 1991). In contrast, DYKSTKA et (11. ( 1991 ) observed a modest (two- to threefold) reduction in meiotic gene conversion at HIS1 and M E 2 , but the strains used were not isogenic to SK1.

In this study, we demonstrate that the sepl mutant arrests in the pachytene stage of prophase with fully synapsed chromosomes. In a physical assay, the .sej)l mutant is delayed in the production of reciprocal crossover products and the final level of recombinants generated is reduced compared with wild type. Physical monitoring also indicates that DSB repair is delayed and very inefficient. These observations are consistent with the view that Sepl plays a role in meiotic recombination.

MATERIAIS AND METHODS

Yeast strains and genetic methods: The S. cermisiar strains used in this study (listed in Table 1 ) are isogenic heterothal- lic SK1 derivatives except for RKYl340, RKYl951, BR2495, RKYl405, BR2908 and BR2909. RKYl340 was constructed by mating the haploid strains, NLBL1 and NLBL3 (ESPOSI~’C> and BROWN 1990), and selecting diploids. RISYl9.51 was constructed by introducing an s rp lA :: URA3 mutation into NI.BL1 and NLBL3, mating the resulting transformants and selecting diploids. BK2495 is the result of mating the haploids, BR1373-6D and BR1919-8B, as described by Ro(:L(MII.I. and R O ~ F . K (1990) . RKYl405 was constructed by introducing the wplA :: C W 3 mutation into BR1373-6D and BR1919-8B and mating the resulting transformants to generate a diploid. BR2909 is a His’ derivative of RKY1405 generated by mitotic recombination. BR2908 was constructed by mating .~po13::l/RA3 derivatives of BR1373-6D and BRlY19-8B and then selecting a His’ mitotic recombinant from the diploid. Strains novel to this study were constructed by standard methods (SHERMAN rt nl. 1983) .

Yeast cells were transformed using the lithium acetate procedure ( p r o rl al. 1983) . s@IA denotes strains in which all of the EcoRI fragments internal to the SEPI gene are replaced by the U r n 3 gene. The q$lA::URA3 nutation w d S intro- duced into S . C f 7 W z 5 z ~ k P strains using an Asd digest of pKDK227 (see section on plasmids). Proper substitution of the mutant allele was verified by a PCR method described elsewhere ( REE- NAN and KOI.OI)NEK 1992).

Meiotic Recombination in sepl

TABLE 1

497

Yeast strain list

Strain Genotype

mn 340

m n 9 5 1

mn 672

w n 9 5 2

~ ~ n 9 5 3

~ ~ n 9 5 4

~ ~ n 9 5 5

RKYI 956

mn308

mn 957

mn958

m n 9 5 9

w n 7 0 6

wn 707

RKyl960

BR2495

mn 405

BR2908

BR2909

MATO ade5 SEPl metl3-c q h 2 trpS LEUl ade6 clyS lys2-2 tyrl-2 his7-l (lde2-I ura3-1 CAN1 MATa ADE5 SEPl metl3-d CYH2 TRP5 leu1 ADE6 CLYS lys2-I tyrl-l his7-2 ade2-l ura3-I canl MATa ade5 seplA::URA3 metl3-c q h 2 trp5 LEUl ade6 cly8 ' lys2-2 tyrl-2 his7-1 & ura3-l CAN1 MATa M E 5 s&lA::URA3 metl3-d CYH2 TRP5 leu1 ADE6 CLYS hs2-1 tyrl-1 his7-2 ade2-1 ura3-1 canl MATa leu2::hik his4-B::ADEZ::his4-X ura3 lys2 ho::LYS2 ade2 MATa leu2::hisG his4-B urn3 lys2 ho::LYSZ ade2 MATa 1euZ::hisG his4-B::ADEZ::his4-X urn3 lys2 ho::LYS2 acle2 seplA::URA3 MATa leu2::hisG his4-B 1 1 r d lys2 ho::LYLS2 add seplA::URA3 MATO leu2::hisG his4-B::ADEZ::his4-X ura3 lys2 ho::I,YS2 ade2 dmc1A::LElJZ MATa leu2::hisG his4-B urn3 lys2 ho::LYSZ adr2 dmclA::l,EU2 MATa leu2::hisG his4-B::AnEZ::his4-X u r d lys2 ho::l,YS2 n d ~ 2 SP~I~A: :CVL~ dm~lA: : lXU2 MATa leu2::hisG his4-B urn3 lys2 ho::LYSZ ade.2 seplA::UUA3 dmrlA::l,ElJ2 MATa leu2::hisG his4-B::ADEZ::his4-X ura3 lys2 ho::l,YS2 ade2 rnd5lA::his(XJRA3-his(; MATa leu2::hisG hi~4-B urn3 lys2 ho::LYS2 ade2 rad5lA::hisGlJRA 3-hisG MATa leu2::hisG his4-B::ADEZ::his4-X urn3 lys2 ho::LYSZ adr.2 seplA::URA3 rad5ln::hisGURA3"hisC MATa leu2::hisG his4-B ura3 lys2 ho::LYSZ ndc2 S Q ~ ~ A : : C ~ R A ~ md5IA::hisGURA3-hisC; MATa leu2::hisG his4-X ura3 lys2 ho::I,YS2 MATa leu2::hisG his4-B zwa3 lys2 ho::LYS2 MATa leu2::hisG his4-X z~rajr lys2 h0::LY.Q s@lA::URA3 MATa h2::hisG his4-B u r d lys2 ho::I.YSZ seplA::URA3 MATa leu2::hisG his4-X urn3 lysZ ho::LYS2 dmclA::LEU2 MATa leu2::hisG his4-B ura3 lys2 ho::LYS2 dmclA::lXU2 MATa leu2::hisC his4-X ura3 lys2 ho::LYS2 sepIA::URA3 dmc1A::LElJZ MATa leu2::hisG his4-B ura3 lys2 ho::LYS2 seplA::URA3 dmclA::LElJ2 MATa leu2::hisG HlS4::LEUZ ura3 lys2 ho::LYSZ MATa leu2::hisG his4-X::LEUZ-URA3 urn3 lys2 ho::LYS2 MATa leu2::hisG HIS4::LEUZ ura3 lys2 ho::LYS2 sepl::TnlOLUK79-2 MATa lm2::hisG his4-X::LEU2-URA3 ura3 lys2 ho::LYS2 s@l::TnlOLUK79-2 MATa leu2::hisG his4-X::IXU2-URA3 IL7723 lys2 ho::LYSB seplA::URA3 rad9::IXUZ MATa leu2::hisG HlS4::UUZ ura3 lys2 h0::LY.U seplA::URA3 rad9::IElJZ MATa leu2-3,112 his4-260 ARG4 thrl-1 ade2-1 ura3-1 trpl-l CYHlO MATa leu2-17 his4-280 arg4-8 thrl-4 adr2-1 urn3-1 t7f11-289 cyhl0 MATa leu2-3,112 his4-260 ARG4 thrl-l ade2-1 um3-I t?$l-l CYHlO seplA::URA3 MATa leu2-27 his4-280 arg4-8 thrl-4 ade2-1 wa3-1 t@l-289 qhlO s@IA::UKA3 MATa leu2-3,112 h i4260 spol3::URA3 ARG4 thrl-I anp2-l ura3-l trpi-l CYHIO MATa leu2-17 HIS4 spo13::URA3 arg4-8 thrl-4 add-1 ura3-1 trpl-289 q h l 0 MATa leu2-3,112 his4-260 A R M thrl-I ade2-1 ura3-1 trpl-1 CYHl0 s@lA::UKA3 MATa ku2-27 HIS4 arcr4-8 thrl-4 add-1 u r d 1 trbl-289 rvhlO s&lA::nRA3

All strains listed above are isogenic SKI derivatives except for RKYl340, RKkl951, BR249.5, RK11405, BR2908 and BR2909. RKYl340 and RKYl951 were constructed from haploid strains (NLBLI and NLBL3) obtained from MI(:€fAEI. ESPOSITO (ESPOSITO and BROWN 1990). BR2495, RKYl405, BR2908 and BR2909 were constructed from haploid strains, BR1373-6D and BR1919-8B (ROCKMILL and ROEDER 1990). dcmlA mutants, rad9mutants and strains used for physical recombination assays were constructed using haploid SKI strains obtained from DOLIGIAS BISHOP and NANCY KIXKNER (see WVII:KIAI.S .AND METHODS).

~_____

The seplA rad5lA double mutant (RKYl956) was constructed by mating seplA::URA3 strains to rad51A::URA3 strains [AKMOl and AKYl02, obtained from A. SHINOIiAKA ( SHINOHARA et al. 1992) ] , sporulating the resulting diploids and dissecting tetrads. Ura+ spores from tetrads that segre- gated 2 2 for uracil auxotrophy were analyzed to confirm the presence of the rad5lA mutation using the zymolyase test previously described to test for the rad50 mutation ( TISHKOFF et al. 1991 ) . Appropriate spore progeny were crossed to each other to generate sep lA rad5lA homozygous diploids. To construct seplA dmclA strains (RKYl954 and RKYl959), seplA::URA3 haploids were mated to dmc1A::LEUZ haploids (NKYl425 and NKYl426, obtained from D. BISHOP and N. KLECKNER, Department of Biochemistry and Molecular Biol-

ogy, Harvard University) and the resulting diploids were sporulated and tetrads dissected. Appropriate Urd+ Leu.' spore progeny were mated together and diploids were isolated. The sep1A rad9 strain (RKYl960) was constructed by mating seplA:: IJIU3 strains with rad9::LEU2 strains obtained from D. BISHOP and N. KI.EC:KNF.K (NKYl.517 and NKYl518), sporulating the resulting diploids and dissecting tetrads; appropriate Ura+ Leu' progeny were matrd together and diploids were isolated. RKM706 was constructed by mating NKY692 with NKYl092 (obtained from N. KI.E.(:KNER) and isolating diploids. To construct RKYl707, sepl ::TnlOLUK79-2 haploids (see TISHKOFF et al. 1991) were mated to NKY692 and NKYl092; appropriate spore progeny were mated and diploids isolated.

498 D. X. Tishkoff et nl.

Media: Rich ( W D ) , minimal and synthetic complete media were prepared as described by SHERMAN et al. ( 1983) . YPA is 1% potassium acetate, 2% peptone and 2% yeast extract. Sporulation medium (SPM) is 3% potassium acetate and 0.02% raffinose. For experiments involving RKYl340 and RKYl951, media were prepared as described by ESPOSITO and GOLIN (1977). Strains BR2495, RKYl405, BR2908 and BR2909 were grown in WAD medium ( W D plus 0.3 mhl adenine) and sporulated in 2% potassium acetate.

Sporulation and recombination assays: Diploid cells were sporulated at 30" in liquid SPM after growth in YPA to a concentration of -2 X 10' cells/ml as described by ALANI et al. (1990). Meiotic return-to-growth experiments were performed as described previously ( TISHKOFF et al. 1991 ) . Mitotic recombination rates were determined as previously described, except that 5 colonies were analyzed rather than 11 ( TISHKOFF et nl. 1991 ) .

Plasmids, enzymes and related methods: Restriction endo- nucleases were purchased from New England Biolabs (Bev- erly, M A ) and used according to the recommendations of the manufacturer. T4 DNA ligase was purified using an unpublished procedure of R.D.K. E. coli cells were transformed using the calcium chloride method ( MANIATIS et al. 1982) .

Plasmids were constructed using standard procedures. The SEPI deletion plasmid, pRDK227, was constructed by in- serting a HindIII/XbaI fragment containing the SEPI gene into the HindIII/XbaI sites in the polylinker of pBluescript SK+ (Stratagene). This plasmid was digested with EmFQ to delete the region of the SEPl gene encoding amino acids 38- 1300 (see TISHKOFF et al. 1991), and a Hind111 fragment containing the URA3 gene, to which EcoRI adaptors had been ligated, was inserted to create pRDK227.

Southern blot hybridizations were performed as described by CHURCH and GI1,BER.r ( 1984) except that a pressure blotter (Stratagene) was used for transfer of DNA from agarose gels to genescreen ( DuPont NEN) as described by the manufacturer. DNA probes for the physical analysis of DSBs and crossover products at the HIS4-IEU2 hotspot have been previously described ( CAO et al. 1990). Probes were labeled with "I' using the random primer method ( FEINBERG and VOGEISTFIN 1988).

Physical analysis of meiotic timecourses: DSBs and physical recombinants were detected in cells from sporulating cultures as described by SHINOHARA et al. (1992). DNA was isolated from meiotic cultures using a slight modification of the procedure of SHERMAN et al. (1983). Southern blots of gels for physical analyses were imaged and quantitated on a Molecular Dynamics PhosphorImager.

Cytology: Cells were grown to late stationary phase in rich medium for 24 (=PI) or 48 (seplA) hr and then pelleted by centrifugation and resuspended in seven times the original volume of sporulation medium. After 15,22 and 42 hr incubation at 30" with shaking, nuclei were spread according to the protocol of DRESSER and GIROL'X (1988).

RESULTS

Commitment to meiotic recombination is reduced in the sepl mutant: A sepl deletion mutation was intro- duced into a strain in which gene conversion can be measured at multiple loci and crossing over can also be assessed. Meiotic recombination was assayed in return-to-growth experiments in which cells are intro- duced transiently into starvation medium and then returned to growth medium, after the commitment to

meiotic recombination but before the commitment to meiotic chromosome segregation. In the strains used (RKYl340 and "951, Table 1 ) , intragenic recombination (gene conversion) between heteroalleles at the HIS7 and LYS2 loci produces His+ and Lys' recombinants, respectively. Single-site conversion at the hetero- zygous CYH2 locus generates cycloheximide-resistant ( Cyh R, cyh2/ ryh2) recombinants; at a lower frequency, Cyh' recombinants can also be generated by double crossovers. Crossing over in the CYH2-7Rp5 interval on chromosome VZI can produce recombinants that are CyhK and white due to homozygosis of the cyh2 and nde5 markers, respectively. (The parental diploid makes colonies that are red in color due to the homozygous nde2 mutation.) Single-site convertants and crossovers were selected on medium lacking tryptophan and leu- cine to demand the presence of both copies of chromosome VII.

In both wild type and the sepl mutant, gene convertants and crossover products were first detected at 6.5 hr after introduction into sporulation medium. After 11 hr of sporulation, gene conversion in the s e p l A mutant was 28% or less of wild type (Table 2 ) . Crossing over was reduced even more severely to 15% of the wild-type level. Between 24 and 48 hr, gene conversion in the mutant continued to increase, whereas gene conversion in wild type remained constant (wild-type cells have completed sporulation by 24 h r ) . By the 48-hr time point, gene conversion in the mutant reached 56- 88% of the wild-type level. Data for the 2 4 and 48-hr time points for the single-site conversion and crossover classes of recombinants are not shown in Table 2, because the selection used to assay these recombinants assumes that all cells are diploid. Because wild type sporulates to produce haploids but the s@lA mutant does not, comparisons between wild type and mutant are impossible at late times. At the 11-hr time point shown in Table 2, wild type has not yet committed to sporulation.

To further examine the effect of the sef11A mutation on crossing over, two additional strains were used to measure exchange in the HIM-MA7interval on chromosome I I I . The SEPI strain used is homozygous for a disruption of the SPOI3 gene ( KLAPI

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sepl null mutation reduced crossing over slightly less than twofold, compared with the sevenfold reduction reported in Table 2. The difference between these results might reflect variation in the effect of the sepl null mutation in different genetic intervals or in different strain backgrounds. Alternatively, it might result from the fact that the cells analyzed for crossing over in Table 3 were preselected for meiotic gene conversion (at THR4) and thus represent a subset of cells that have entered meiosis. Finally, the difference might be due to the fact that in the experiment in Table 3, sample cells were harvested at a late time point in meiosis, whereas the experiment in Table 2 measures crossing over only at a relatively early time point. The results presented in Table 2 and Figures 2 and 3 indicate that recombination in the sepl mutant probably continues to increase while sepl mutants are held in sporulation medium, whereas in wild-type strains recombination plateaus after a short period.

The sepl null mutation has little effect on mitotic recombination: To determine whether different types of mitotic recombination are affected by the sq lA mutation, strains RKYl340 and RKYl951 were used to measure the mitotic rates of prototroph formation at the heteroallelic loci, HIS7, LYS2, TYRl and h4ET13. In addition, single site conversion at CYH2 and homozygosis of the recessive c u d , cyh2 and ade5 markers were measured. The sepIA mutant showed only slight differ- ences from wild type. In almost all cases, recombination was decreased about twofold in the mutant (data not shown ) , consistent with the results reported previously ( TISHKOFF et al. 1991 ) .

The sepl mutation affects meiotic intrachromosomal recombination: Because commitment to meiotic interchromosomal recombination is reduced in the seplA mutant, it was of interest to examine the effect of the s@lA mutation on intrachromosomal exchange. To assay intrachromosomal events, SK1 diploids were used in which one homologue of chromosome ZIZ contains directly repeated HIS4 genes separated by the ADE2 gene; one copy of HIS4 carries the his4-X allele and the other carries the hzs4-B mutation. The other chromosome III homologue contains a single HIS4 gene carrying the his4-B allele. Recombination between the his4 alleles on the same chromosome can lead to deletion of the ADE2 gene, resulting in His+ colonies that are red in color ( i . e . , Ade-) . These pop-outs can result either from reciprocal crossing over or from single- strand annealing ( NICKLOFF et al. 1989; SUCAWARA and WER 1992) . Intra- or interchromosomal gene conversion events generate His+ colonies that are white ( i . e . , Ade + ) ; less frequently, interchromosomal crossing over also generates white His+ colonies.

After 7 hr of sporulation (Table 4), the frequency of popouts in the seplA mutant was only 23% of that observed in wild type, but convertants were increased

500 D. X. Tishkoff et al.

TABLE 3

Meiotic crossing over in sepl diploids

Strain His- a His- a Recombination (genotype) His" nm His' a His' a (doubles) (reductional) Total frequency (%)

BR2908 (SEPl) 32 31 18 3 1 1 282 39.4 BR2909 ( s @ l A ) 27 12 8 0 1 284 25.7

After 24 hr in sporulation medium, threonine prototrophs of strains BR2908 (SEPI) and BR2909 ( s e p l A ) were selected. The frequency of prototrophs was 2.7 X 10" for wild type and 1.5 X for mutant. The resulting (diploid) colonies were scored for histidine prototrophy and mating type. His- nm colonies were classified as single recombinants and the number of these was doubled to account for HZS4/HZ.S4 MATa/MATa cells. His' a spores were counted as single recombinants, except that the number was reduced by the number of His- a colonies (reductional segregants), because there should be an equal number of HZM/HZS4 MATa/MATa reductional segregants that are not recombinant. The Hist a colonies were classified as single recombinants except that the number was reduced by the number of His- a double recombinants, because there should be an equal number of HIS4/HZS4 MATa/MATa double recombinants. To obtain the total number of spores carrying two recombinant chromatids (due to four-strand double exchanges), the number of His- a colonies was doubled to account for the reciprocal HZLS4/HZS4 MATa/MATa recombinants. Recombination frequency was calculated by summing the single crossovers and twice the number of double crossovers and dividing by the number of colonies examined. The number of recombinants was divided by the number of colonies examined (rather than the number of chromatids) to account for the fact that equational chromosome segregation following recombination generates detectable recombinants only half of the time. nm, nonmater; a, mating type a; a , mating type a.

slightly (1.6-fold). This differential effect suggests that SEPl plays a more important role in meiotic pop-out recombination than in meiotic gene conversion. After 24 hr of sporulation, the induction of both pop-outs and convertants was increased severalfold in the s @ l A mutant compared with wild type (Table 4) , suggesting that both pop-outs and gene convertants accumulate after meiotic arrest in s e p l A strains. It should be noted that the wild-type SK1 strain used here completes sporulation between 12 and 18 hr in sporulation medium.

SEPI, DMCI and RALl51 may function in parallel pathways of meiotic intrachromosomal recombination: Because the d m c l A and seplA mutants have similar meiotic phenotypes and both genes are thought to encode strand exchange enzymes, it was of interest to determine whether the seplA and d m c l A mutations have synergistic effects on recombination. When commitment to meiotic intrachromosomal recombination was measured (Table 4 ) , the induction of pop- out recombinants was found to be reduced significantly in the double mutant relative to wild type and the single mutants. After 7 hr of sporulation, the meiotic frequency of pop-outs in the double mutant was 30-fold less than wild type and 76- and 7-fold less than the dmclA and s@lA single mutants, respectively (Ta- ble 4). After 24 hr of sporulation, the s @ l A d m c l A double mutant reached a frequency of pop-outs 8-fold lower than wild type and 20- and 25-fold lower than the d m c l A and seplA single mutants, respectively (Ta- ble 4 ) . In the assay used, the sep lA dmc lA double mutant showed a less severe defect in gene conversion events than in meiotic pop-out events. Compared with wild type, the induction of gene convertants in the double mutant was reduced eightfold at 7 hr and twofold at 24 hr (Table 4 ) . In addition, the sef11A dmclA

double mutant displayed an absolute defect in sporulation, whereas the single mutants sporulated poorly (Table 4 ) .

Because DMCl and RAD51 are thought to encode redundant functions (BISHOP et al. 1992; SHINOHARA et al. 1992), it was also of interest to determine whether the s+l and rad51 mutations act synergistically. Similar to the s e p l A d m c l A double mutant, the seplA rad5lA double mutant displays a more severe decrease in the meiotic frequency of pop-outs than the corresponding single mutants. After 7 hr of sporulation, the frequency of pop-outs was decreased 17-fold relative to wild type, 10-fold relative to rad51A and 4fold relative to seplA (Table 4 ) . At the same time point, the sep1A rad51A double mutant showed less than a sixfold decrease in the meiotic frequency of gene convertants compared with wild type (Table 4) . These results suggest that the s @ l A r a d 5 l A double mutant, like the sep1A dmclA double mutant, displays a more severe defect in meiotic pop-out events than in gene conversion events.

Both the RAD51 gene and the SEPl gene are ex- pressed during vegetative growth; therefore, it was of interest to measure mitotic intrachromosomal recombination in the seplA rad51A double mutant. Although a slight decrease in the rate of formation of both pop- outs and intrachromosomal gene convertants was observed in the seplA single mutant relative to wild type, there was no significant difference between the rad51A single mutant and the .sqblA m d 5 1 A double mutant (Table 5 ) . Both ra t l5 lA and .seplA rad5lA displayed a 50- to 100-fold decrease in the rate of formation of convertants relative to wild type, whereas the rate of formation pop-outs was elevated slightly, consistent with previous results for rad51A mutants ( SHINOHARA et al. 1992). These results indicate that the SEPZ and W 5 1

Meiotic Recombination in st’l,I

B.

RKY 1706 ( S E P I / S E P I ) RKY 1707 I s e p l / , s e n I ~ 0 2 4 6 8 1 0 M O 2 4 6 8 1 0 1 2 2 4

C.

RKY I706 ( S E P I / S E P I _ ) Bl(Y 1707 (.SCn//StWl) 0 2 4 6 8 1 0 M 0 2 4 6 8 10

gene products are not required for mitotic pop-out recombination.

Meiotic interchromosomal gene conversion in s@lA dmclA strains: The results just presented demonstrate that the s ~ p l A and dmclA mutations have a synergistic effect on a specific class of meiotic intrachromosomal recombination events. To examine the effect of the seplA d m c l A double mutant on meiotic interchromosomal gene conversion, a s ~ p l A d m c l A double mutant carrying HIS4 heteroalleles was constructed and analyzed in return-to-growth experiments (Table 6 ) . At early time points (4..5 and 7 h r ) , the frequencies of His’ recombinants in the double mutant were similar to those of the spj,lA single mutant. At 24 hr, the frequency in the double mutant was only slightly less than that of the d m c l A single mutant. These results suggest that the synergistic decrease in recombination resulting

FIGLIKE 2.-DSR repair and physical rccomhinant formation in sd)1 mutants. RKYl706 and RKYl707 were sporulated synchronously and genomic DNA isolated from samplcs takcn at the times indicated was digested with IhmHI ( A ) , A t 1 (R) or X/mI ( C ) . Southern blots shown are representatives o f two independent experiments that yielded similar results

I DSB I1 with all three digests. Numbers above lanes indicate hours of incubation in SI”; M indicates markers generated by digestion of bacteriophage lambda DNA with HindllI. P, parental RnmHI or Ad fragments; DSR I, major DSR fragment; DSR 11, minor DSR fragment; PI and P2, parental XhoI fragments; R1 and R2, recombinant X/mI fragments (see Figurc 1 ) . Probes and fragment sizes arc shown in Figure 1.

I P

: DSB I

from the combination of s q l A and d m c l A mutations is limited to meiotic intrachromosomal pop-out events.

This experiment revealed a differencc between the s(.lIA single mutant and the sPf,lA dmc1A double mutant in terms of the kinetics of induction of His’ recombinants (Table 6) . In the s@IA mutant, recombinant5 increased dramatically ( 145-fold) between 7 and 24 hr. However, in the dmclA single mlltant and in thc sqlA dmclA double mutant, recombinants increased only four- to sixfold after 7 hr. This result suggests that the sqhlA mutant arrests at a point in meiosis at which recombination can continue to occur, whereas the d m c l A and seplA dmclA mutants do not. Thus, the dmclA mutation is epistatic to the .yhIA mutation with regard to this aspect of phenotype.

Meiotic DSBs are not repaired efficiently in s@l mutants: The metabolism of meiotic DSBs was investi-

D. X. Tishkoff et nl.

30 -

20 -

10 -

O& 0 1 0

Hours in SI"

20

FIGURE 3,"Quantitation of physical recombinant formation in sepl mutants. Recombinant XhoI fragments in the gel shown in Figure 2C were quantitated using ImageQuant soft- ware on a Molecular Dynamics PhosphorImager. In addition, two lanes not shown in Figure 2C, containing 12- and 24hr time points, were quantitated. Percent recornbirlants is the ratio of recombinant XhoI fragments (R1 + R2) to total DNA (P1 + R1 + R! + P 2 ) .

gated in sepl mutants using a strain (RKM707) containing a genomic HLS4-IXU2 construct that has been shown to act as a meiotic recombination hotspot (also see CAO et al. 1990). During early meiotic prophase, two DSBs occur at the HZS4-LLU2 locus. These can be detected by digesting genomic DNA with either BnmHI or PstI and monitoring the formation of diagnostic fragments by Southern blotting (Figure 1; and also see CAO et al. 1990). The diagnostic fragments disappear later in prophase ( PADMORE et nl. 1991 ) , presumably because DSBs are processed into subsequent recombinatiou in-

termediates. The DSB ends are initially discrete, but the 5' ends are degraded to varying extents, whereas the 3' ends remain stable ( KLECKNER et al. 1991 ) .

In the wild-type strain (RKYl706) in which genomic DNA was digested with BnmHI, both DSB fragments were visible after 2 hr in sporulation medium and had disappeared almost completely by 6 hr (Figure 2A) , consistent with previously published results ( CAO et nl. 1990; PADMOKE et nl. 1991; BISHOP et al. 1992; SHINO- HAKA st nl. 1992). In the s e p l A mutant, the DSBs did not appear until 4 hr in sporulation medium. They continued to accumulate up to 8 hr to a level that appeared to be greater than the maximum level seen in wild type and they remained approximately at this level through the 12-hr time point (Figure 2A).

In a separate experiment, genomic DNA was digested with Pstl, which yields smaller diagnostic restriction fragments than BamHI (see Figure 1 ) . In this case, the kinetics of DSB formation in the mutant were similar to wild type (Figure 2B) ; DSB formation was not delayed as significantly as in the experiment involving BnmHI (Figure 2A). In wild-type cells, the PStI fragments (especially the smaller 3.9 kbp fragment) appear smeared on Southern blots, presumably due to hetero- geneity in length caused by variable 5 to 3 ' resection of strands that have their 5 ends at the DSB site (Figure 2B) (also see BISHOP el nl. 1992). In sepl cells, the A t 1 fragments diagnostic of DSBs appeared to be as heterogeneous as those of wild type (Figure 2 B ) , indicating that the exonuclease activity of the Sepl protein is not required for the generation of single-stranded tails at the sites of meiotic DSBs. At the 10- and 12-hr time points, the signal for the 3.9 kbp PstI fragment appeared to be decreased relative to the signal for the 6.2 kbp PstI fragment (Figure 2B). At the 24hr time point, the 3.9 kbp fragment was no longer visible and

TABLE 4

Commitment to meiotic intrachromosomal recombination

Frequency of His+ Ade- Pop-outs Frequency of His+ Ade' (x

Strain (relevant convertants ( X 10 ")

% 5% genotype) 0 hr 7 hr 24 hr 0 hr 7 hr 24 hr Sporulated Viable

~ ~ 1 6 7 2 (.YEPI) 5.0 150.0 (100) 180.0 (100) 10.0 160.0 (100) 137.0 (100) 90.0 5.0 RKYl952 ( sep1A) 1.9 35.0 (23) 600.0 (333) 11.2 260.0 (163) 800.0 (584) 9.0 1.3 RKYl953 ( d m c l A ) 12.0 370.0 (247) 480.0 (267) 7.1 190.0 (119) 240.0 (175) 0.15 0.8 RKYl955 ( r d 5 1 A ) 14.0 88.0 (59) 58.0 (32) 1.2 13.0 (8) 63.0 (46) 9.1 0.2 RKn954 ( S e p l A d m c l A ) 2.2 4.9 (3) 24.0 (13) 2.7 19.7 (12) 70.6 (52)

Meiotic Recombination in sepl 503

TABLE 5 Rates of mitotic intrachomosomal recombination in s @ l A

and d 5 1 A mutants

Rate of Rate of Strain (relevant Ade- His+ Ade+ His+

genotype) Popouts convertants

my1672 (SEPI) 1.4 X 2.2 X 1 0 - ~ m n 9 5 2 (sepln) 7.9 X 1.0 X RKYl955 ( rad5 lA) 5.7 X 10-5 2.5 X 10” RKYl956 (seplArad5lA) 6.3 X 4.0 X 10”

All strains carry his4-B::ADE2::his4-Xon one copy of chromosome I11 and his4-B on the homologue.

the amount of the 6.2 kbp fragment was reduced. This loss or reduction may be due in part to resection of the DSBs beyond the PstI sites so that the fragments can no longer be cleaved by the restriction enzyme. In addition, some of the DSBs may be processed to generate mature reciprocal recombinants (as described in the following section ) .

To determine whether the inability of sepl mutants to repair meiotic DSBs is specific to the artificial HIS4- LEU2 construct, DSB formation was examined at a natu- rally occurring DSB site upstream of the ARG4 gene (SUN et al. 1989). The results were similar to those seen at the HIS4-LEU2 locus (data not shown; H. SUN and R. D. KOLODNER, unpublished data), suggesting that the effect of the sepl mutation on DSB metabolism is not due to some unusual feature of the HIS4-LEU2 construct.

The results of these experiments indicate that the resection of DSBs is normal in the sepl mutant. How- ever, there is a delay in the formation of DSBs and there is a more pronounced delay in the processing of DSBs to subsequent recombination intermediates or products. In the sefI2 mutant, meiotic crossing over is delayed

and inefficient: The production of mature recombinants at the HIS4-LEU2 locus can be monitored by the

generation of diagnostic XhoI fragments (Figure 1 ) (also see CAO et al. 1990). The recombinant products detected are thought to result almost exclusively from reciprocal exchange rather than gene conversion ( CAO et al. 1990) and are presumed to initiate with the DSBs observed earlier (reviewed by KLECKNER et al. 1991 ) . In the wild-type strain, recombinants (R1 and R2 XhoI fragments) appeared after 4 hr of sporulation and reached a maximum level of 25% of the total DNA after 6-8 hr of sporulation (Figures 2C and 3 ) , consistent with previously published results ( CAO et al. 1990; PAD- MORE et al. 1991; BISHOP et al. 1992; SHINOW et al. 1992). The decrease in the total amount of DNA recov- ered from wild-type cells after the 6 h r time point (Fig- ure 2 ) is due to the increasing difficulty in isolating DNA as spore walls form; this effect is not seen in sepl strains because they do not sporulate. In the sepl strain, recombinants were not detectable until the Ghr time point, at a level 10-fold less than in wild-type cells at the same time point (Figures 2C and 3 ) . After 24 hr of sporulation, sepl cells reached a maximum level of recombinants that was 40-50% of the maximum level seen in wild type (Figure 3) . These results suggest that the sepl mutant is delayed in the formation of reciprocal recombinants and that the final level of recombinants generated is reduced even after prolonged incubation under sporulation conditions.

Cytological analysis of sq2A mutants demonstrates meiotic arrest in pachytene: Fluorescence microscopy of propidium iodide-stained meiotic sepl cultures has shown that sgbl mutants fail to complete either of the two meiotic divisions, arresting in meiosis as mono- nucleate cells ( TISHKOFF et al. 1991). To determine more precisely the stage of meiotic prophase in which the s@lA mutant arrests, spreads of meiotic nuclei were stained with silver nitrate and examined in the electron microscope. The strains used for this analysis do not sporulate as rapidly as the SKI strains used for the other experiments reported in this paper.

During prophase of meiosis I, homologous chromo-

TABLE 6 Commitment to interchromosomal gene conversion in s @ l A d m l A strains

His’ recombinants ( X

Strain (relevant genotype) 0 hr 4.5 hr 7 hr 24 hr

m n 3 0 8 (SEPI) 1.6 12.3 (100) 92.0 (100) 220.0 (100) my1957 ( s e p l n ) 0.8 3.5 (29) 8.3 (9) 1200.0 (545) RKM958 (dmc lA) 0.9 18.6 (151) 52.0 (57) 194.0 (88) RKy1959 (seplAdmclA) 0.7 1.9 (15) 14.2 (15) 79.5 (36) Cells were sporulated synchronously as described in the MATERIALS AND METHODS and aliquots were plated

onto complete medium and complete medium minus histidine at the times indicated. Time points represent hours after transfer to sporulation medium. Each meiotic value represents the meiotic frequency minus the mitotic (0 hr) frequency. Numbers in parentheses indicate the frequency of recombination in the mutatnt as a percent of the wild-type level.

504 D. X. Tishkoff et nl.

6.2 kh

his4-X LEU2

1-93kb -1 URA3 Parent

B 1- 6.8 kh 4 11.8 (P2j

HIS4 LEU2

B + X 16.3 ( R l j I

HIS4 LEU2 urn3 Recombinant

p X X B I I I 1

13.2 (R2j

his4-X LEU2 1 1 1 1 1 1 1 1

I ! ! & = Major DSB site Xhol probe PstI, BnntHI probe = Minor DSB Site

! ! I

FIGURE 1.-Map of the HZWLEU2 locus on chromosome Il l . The two PstI sites shown produce a parental fragment of 12.8 kbp and two DSB fragments of 6.2 and 3.9 kbp. The two BnmHI sites produce a parental fragment of 13.1 kbp and two DSB fragments of 9.3 and 6.8 kbp. Crossing over between the parental homologues leads to recombinants that yield different size XhoI fragments than the parents. The XhoI sites shown in boldface yield either parental fragments (P1 and P2) or recombinant fragments (R1 and R'L) . Also shown are probes and DSB sites. Adapted from C A ~ et al. 1990.

somes synapse with each other to form the synaptonemal complex (SC) . Each SC is composed of two densely staining parallel structures, called lateral elements, and a less densely staining central region. Lateral elements are referred to as axial elements when they are not synapsed. During normal SC assembly, short axial elements appear before any tripartite structure is evident ( PADMORE et al. 1991 ) . During zygotene, individual axial elements elongate and pairs of axial elements join to form short stretches of tripartite SC. Pachytene is defined as the stage in which tripartite structure spans the entire length of each chromosome, forming mature SC. After pachytene, the SCs disassemble rapidly but are thought to pass briefly through an intermediate stage in which the SCs appear "moth-eaten" ( PADMOKE et al. 1991).

After 15 hr in sporulation medium, -85% of wild- type cells were either in the pachytene stage of meiosis or had already completed one or both meiotic divisions (Figures 4 and 5, A-C) . The remaining 15% of the nuclei, which appeared unstructured, were in prophase I either just before or just after pachytene. At 15 hr in an isogenic s@lA mutant, the frequency of nuclei in pachytene was slightly higher than in the wild-type strain (Figure 4 ) and the appearance of the SCs was indistinguishable from that seen in wild type (Figure 5D) . This suggests that sepl mutants reach pachytene

at approximately the same time as wild-type cells. How- ever, sgl strains fail to make the transition from pachytene to meiosis I. At 22 and 42 hr, when almost all wild- type cells had completed meiosis (data not shown) , a significant fraction of sepZ nuclei still displayed synapsed chromosomes and duplicated but unseparated spindle pole bodies (Figure 5F) and only a small fraction had progressed to the meiotic divisions (Figure 4 ) . Based on the limited number of time points examined, the kinetics of SC formation appeared to be similar in wild type and mutant.

In addition to pachytene nuclei, nuclear spreads containing unsynapsed axial elements were observed in the

mutant. These nuclei represent an aberrant class not observed in wild type. Wild-type nuclei sometimes contain lightly stained short axial elements, whose kinetics of assembly are consistent with their being SC precursors ( PADMOKE ~t nl. 1991 ) . In contrast, the axial elements observed in .sef)l nuclei were more heavily stained and often longer than those observed in wild type (Figure 5 , E and G ) . The frequency of nuclei containing axial elements increased with time in sporulation medium, suggesting that these axial elements are the breakdown products of synapsed chromosomes (Figure 4) . Like the nuclei containing SCs, the nuclei containing axial elements contained duplicated but unseparated spindle pole bodies.

These data indicate that the s e p l A mutant arrests in the pachytene stage of meiosis I prophase. Over time, the SC is degraded partially to produce nuclei containing axial elements or completely dissolved to generate nuclei without any obvious SC-related structures.

The meiotic cell cycle arrest point of the s@l mutant is RAD9 independent: The arrest of seP1 mutants in pachytene may indicate that a meiotic cell cycle checkpoint is triggered by a defect in some aspect of cell metabolism that occurs during or before this stage of meiosis. It has recently been shown that r d r l 3 mutants, which arrest in the G2 stage of the mitotic cell cycle, arrest in meiosis after DNA replication but before X: formation or commitment to recombination ( WEBEK and BWKS 1992). Mutation of the M I 9 gene, which is thought to encode a cell cycle checkpoint function (WEINERT and HARTM'E1.L. 1988), alleviates both the mitotic arrest and the meiotic arrest seen in cdr13 mutants ( WEBER and BWKS 1992). To determine whether rad9 also alleviates the meiotic arrest observed in ,s@I mutants, a sepZA rad9 double mutant was constructed and analyzed. In the sepZA rad9 double mutant (RKYl960), 2.7% of the cells sporulated compared with 9.0% in the s e p l A single mutant (RKYl952). Thus, the meiotic arrest observed in s @ l A strains is not alleviated by the rad9 mutation. The mitotic slow growth phenotype of the sepl mutant is also not affected by the rad9 mutation (data not shown).


(A) wild type 15 hrs (C) sepl 22 hrs

Us 32 AE S M MI1

8oT (B) sepl 15 hr

60

40

20

0

U S 3 2 A E S " I I

U s 3 2 AE S M MI1

(D) sepl 42 hrs

40 -

L s 3 2 A E S " I I

FIGURE 4.-Distribution of meiotic nuclei in wild type and sepl . The percentage of nuclei at each stage in meiosis is indicated. (A) Wild type (BR2495) after 15 hr in sporulation medium. ( B ) The sepl strain (RK1405) after 15 ( B ) , 22 ( C ) and 42 ( D ) hr in sporulation medium. At least 100 nuclei were counted at each time point. Nuclei classified as US contain duplicated but unseparated spindle pole bodies and chromatin without any obvious structure. Nuclei classified as SC contain synapsed chromosomes. AE refers to nuclei containing darkly staining axial elements and S represents nuclei with darkly stained condensed chromatin. Nuclei classified as MI contain one pair of separated spindle pole bodies with an intervening region of negatively stained microtubules. MI1 nuclei contain two spindles.

DISCUSSION

The meiotic phenotype of the sepl mutant is consistent with a role for Sepl in recombination: Previously, the Sepl protein was purified and shown to catalyze in vitro homologous pairing reactions similar to those catalyzed by E. coliRecA protein ( KOLODNER et al. 1987; HEYER et al. 1988; DYKSTFU et al. 1990; JOHNSON and KOLODNER 1991, 1993; CHEN et al. 1994). More recently, Sepl has been shown to be representative of a class of eukaryotic and prokaryotic homologous pairing proteins that are distinct from R e d in that they require an intrinsic or associated exonuclease activity required for strand exchange (see the introduction). To better understand the role of Sepl in recombination in vivo, we have used genetic, physical and cytological analyses to examine a variety of meiotic events in the sepl mutant.

The results presented in this communication demonstrate that the s@lA mutant shows a decrease in commitment to meiotic interchromosomal gene conversion and crossing over at early to intermediate time points (Table 2 ) , although they reach the point of commitment at the same time as wild type (Tables 2 and 5 ) (also see TISHKOFF et al. 1991 ) . In addition, DSB repair and particularly the formation of mature reciprocal recombinants are delayed and reduced in efficiency in the sgbl mutant (Figure 2 ) . In both the genetic and physical assays, the levels of recombinants produced approach wild-type levels at late time points. However, at earlier times, recombination is reduced severely. Cy- tological analysis has shown that the sepl mutant prog- resses to pachytene with approximately normal kinetics and then arrests (Figures 4 and 5; also see text in RE- SULTS). To explain these results, we propose that the

.506

A

I). X. Tishkolf P/ (I/.

. .

D 4.t-

. . .. . . . . . - E' . . .

FIGIIKE .?.--Electron micrographs of wild-lye and sqt11 meiotic nuclei. ( A X ) Wild type (BR249.5) after I.? hr in sporulation medium; pachytene ( A ) , meiosis I ( R ) and meiosis 11 ( C ) . (D-G) Nuclei from an isogenic .s@l strain (RKYl405) ; pachytene at I5 ( D ) a n d 42 (F) hr ; darkly stained axial elements observed at 1.5 ( E ) and 42 ( G ) hr. In A, E, F and G, arrows points to duplicated hut unseparated spindle pole horlics. In R and C, arrows point t o individual spindle poles. Bar, 1 pm.


Sepl protein participates directly in recombination, but there are other recombination enzymes (perhaps op- erating in parallel recombination pathways) that are able to substitute partially for the lack of Sepl. After the seplA mutant arrests, these other recombination enzymes are able eventually to promote nearly wild- type levels of recombination. In this context, the in vivo defect in the s @ l A mutant is consistent with that expected to result from loss of the known in vitro activities of the Sepl protein.

The modest decrease in the induction of meiotic gene conversion seen in the seplA mutant in Table 2 may appear to contrast with the hyperconversion phenotype for sepl mutants in the SK1 background reported in TISHKOFF et al. (1991). However, the hyperconversion phenotype of sepl mutants is seen only after extended sporulation ( TISHKOFF et al. 1991 ) (Table 6) , and extended time points were not tested in the relatively slowly sporulating strains used in Table 2. It is possible that the frequencies of recombinants would continue to increase after 48 hr in the seplA strain used in Table 2. Alternatively, the meiotic hyperrecombination of the sepl mutant may be specific to the HIS4 locus and/or to the SK1 strain background. DYKSTRA et al. ( 1991 ) did not observe a hyperrecombination phenotype for the sepl mutant even when they analyzed samples taken at late time points in SK1-related strains, in support of the hypothesis that hyperrecombination is unique to the HIS4 locus.

Analysis of sepl mutants provides additional evidence for multiple pathways of recombination in S. w&- iae: Meiotic intrachromosomal pop-out recombination, unlike interchromosomal recombination, can occur by single-strand annealing (NICKLOFF et al. 1989; SUGAWARA and WER 1992). The dmcl mutant shows an elevated level of commitment to meiotic pop-out recombination (BISHOP et al. 1992; this study) ; this ele- vation is thought to reflect the channeling of blocked recombination intermediates into an alternative Dmcl- independent pathway (BISHOP et al. 1992). In this study, we show that commitment to pop-out recombination is modestly reduced at early time points in the s q l A mutant. DYKSTRA et al. (1991) previously reported a threefold reduction in meiotic pop-out recombination in sgbl ( dst2) mutant strains using a his3 dupli- cation. In contrast to the behavior of the sgbl single mutant, meiotic pop-out recombination is eliminated almost completely in the sgblA dmclA and s @ l A r a d 5 l A double mutants (Table 4) . These observations suggest that Sepl acts in a meiotic pop-out pathway that operates in parallel with another pathway requiring the Dmcl and Rad51 proteins. The view that Dmcl and Sepl act in parallel meiotic processes is supported by the observation that a seplA dmclA double mutant shows a more severe defect in sporulation than either single mutant. Although our data suggest that the seplA

dmclA and seplA rad5lA double mutants have more severe defects in pop-out recombination than gene conversion, it is possible that this effect is specific to the assays employed.

The sepl defect triggers arrest in the pachytene stage of the meiotic cell cycle: Previous studies demonstrated that the absence of Sepl protein leads to meiotic arrest before the first meiotic division, but meiotic events lead- ing up to the arrest point, such as DNA replication and commitment to recombination, occur with normal kinetics ( TISHKOFF et al. 1991 ) . In this communication, we show that seplA mutants appear to reach pachytene with approximately normal kinetics but arrest during pachytene when full-length SCs are present and spindle pole bodies are duplicated but unseparated (Figures 4 and 5 ) . The sgbl mutant has also recently been shown to arrest in pachytene in the SK1 strain background ( BAHLER et al. 1994) . sgblA strains arrest uniformly in meiosis with little loss in cell viability (TISHKOFF et al. 1991), suggesting that the arrest is due to triggering of a meiotic cell cycle checkpoint ( HARTWELL and WEIN-

At least two other meiotic mutants, dmcl and z ip l , arrest in prophase of meiosis I (BISHOP et al. 1992; SYM et al. 1993). In both of these mutants, the meiotic arrest is alleviated by a mutation that blocks the initiation of recombination (such as spol l or m e i 4 ) , raising the possibility that the dmcl and zip1 mutants arrest in meiosis in response to the accumulation of an intermediate in the recombination pathway. The sporulation defect of the s q l A mutant is not bypassed by mutations ( e.g., rad50, s p o l l ) that prevent the initiation of recombination ( TISHKOFF et al. 1991; D. X. TISHKOFF, unpublished data), indicating that the meiotic arrest observed in the sgblA mutant cannot be attributed entirely to the failure to process recombination intermediates in the M5Odependent DSB repair pathway.

In light of the meiotic arrest phenotype of the s @ l A mutant, the recombination defect can be interpreted in either of two ways. First, the sgblA mutant may be defective both in recombination and in some other process that is independent of RADS0 and SPOl1, as yet unidentified. Even when the formation of recombination intermediates is prevented, a defect in this other process may trigger the arrest in meiosis. Recombina- tion then continues at a low rate after the arrest point due to parallel recombination pathways. Several observations are consistent with this interpretation. First, the in vitro activities of the Sepl protein suggest that it participates directly in recombination. Second, the multiple mitotic phenotypes of the sgbl mutants make it plau- sible to suppose that the Sepl protein participates in more than one process. Third, the s @ l A mutant a p pears to continue to accumulate recombinants at mid to late time points taken during meiotic return-to-growth experiments, suggesting that it is not blocked at a point

ERT 1989).

508 D. X. Tishkoff et al.

in meiosis that is inhibitory to recombination. Also, note that the top2 and cdc28 mutants undergo normal levels of meiotic recombination even though they arrest in pachytene, indicating that pachytene arrest does not cause recombination defects per se (ROSE and HOLM 1993; N. KLECKNER, personal communication). Fourth, in the s@I mutant, some types of recombination are blocked by mutations that normally do not prevent recombination, indicating that there is at least one Sepl- independent pathway that might account for the resid- ual recombination observed in the sepl mutant.

An alternative interpretation of the s@lA mutant phenotype is that the primary defect is in some process other than recombination and the observed recombination deficiency is an indirect consequence of the failure to progress normally through meiosis. Two aspects of our data argue against this interpretation. First, the decrease in the accumulation of crossover recombinants (Tables 2 and 3; Figure 2C) is greater than can be accounted for easily by the delay in the formation of gene convertants (Tables 2 and 6 ) or by the 2-hr delay in DSB formation (Figure 2B) . Second, the observation that intrachromosomal recombination is decreased much more than interchromosomal recombination in s @ I A d m c l A and seplA r a d 5 l A double mutants (Ta- bles 4 and 6 ) is not easily explained by a general delay in meiotic progression.

We thank ARLEN JOHNSON and ERIC A I ~ I for helpful discussions and critical reading of this manuscript, and we thank MICHAEL KANE for technical support in the construction of the SEPl deletion plasmid. This work was supported by American Cancer Society grant v" 7C to G.S.R. and National Institutes of Health graut GM-29383 to R.D.K.

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Communicating editor: F. WINSTON

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