MOLECULAR AND CELLULAR BIOLOGY, June 1993, p. 3445-34550270-7306/93/063445-11$02.00/0Copyright © 1993, American Society for Microbiology

Meiosis-Specific Arrest Revealed in DNA Topoisomerase II MutantsDAVID ROSE AND CONNIE HOLM*

Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Avenue,Cambridge, Massachusetts 02138

Received 9 December 1992/Returned for modification 12 January 1993/Accepted 26 March 1993

Although the processes of mitosis and meiosis are similar, there is evidence for fundamental regulatorydifferences between the two. To examine these differences, we have compared the meiotic phenotype of DNAtopoisomerase II mutants with their previously described mitotic phenotype (C. Holm, T. Goto, J. Wang, andD. Botstein, Cell 41:553-563, 1985). top2 mutants in meiosis show no defects in the latest detectable stages ofrecombination, yet they arrest prior to spindle establishment at meiosis I. Fluorescence and electronmicroscopy reveal that top2 mutants exhibit wild-type levels of meiotic chromosome condensation and formmorphologically normal synaptonemal complex but are delayed in the exit from pachytene. Arrested cellsretain viability and form colonies if transferred to mitotic medium. Our results suggest that the top2 meioticarrest is regulatory in nature. This arrest may have evolved to ensure the resolution of fortuitous tanglesbetween nonhomologous chromosomes.

The processes of mitosis and meiosis share many goalsand mechanisms, and comparison of the two can oftenprovide information about each process. Overall, both pro-cesses require the accurate replication of DNA followed bythe precisely regulated segregation of chromosomes. In eachcase, chromosome segregation is mediated by a microtubule-based spindle, and the products of nuclear division aresubsequently partitioned into progeny cells. Although meio-sis II is very similar to mitosis, meiosis includes an addi-tional specialized division, meiosis I, in which recombina-tion between homologs is followed by reductionalsegregation of chromosomes. It is likely that the execution ofthis specialized division requires both structural and regula-tory proteins that are meiosis specific; many meiosis-specificgenes have, in fact, been identified (4, 13, 15, 22).One way of elucidating the similarities and differences

between mitosis and meiosis is to compare the mitotic andmeiotic phenotypes conferred by single mutations. It is notsurprising, for example, that mutations in the CDC13 genethat cause mitotic cells to arrest at the transition betweenDNA synthesis and mitosis (48) also confer arrest in meiosis(43, 47). It is revealing, however, that while cdc13 mutantsarrest in mitosis after the spindle has formed, cdc13 mutantsin meiosis arrest earlier, before the establishment of ameiotic spindle. This difference in terminal morphologysuggests that while the structural requirements of mitosisand meiosis are similar, there are significant regulatorydifferences between the two modes of division.

Direct evidence for a regulatory difference between mito-sis and meiosis comes from studies comparing the mitoticand meiotic phenotypes caused by mutations in the CDC28gene, which encodes the kinase component of maturation-promoting factor (3, 10, 18). Most cdc28 mutations causecells to arrest in mitosis at Start, prior to the initiation ofDNA synthesis (19). In meiosis, however, these same mu-tations cause cells to arrest after DNA synthesis, atpachytene (42). These results indicate that while CDC28 mayplay a regulatory role in both mitosis and meiosis, itsprimary mitotic control point is at Start, while the corre-sponding point of regulation in meiosis is at pachytene.

* Corresponding author.

Unfortunately, most existing mutations are not amenableto this type of comparative analysis. The majority of existingmeiotic mutations cause cells to complete an aberrant mei-osis that produces inviable spores (for example, radSO [34],spoll [14], mer2 [12], and hopl [22]); only a small number ofmutations that cause a uniform arrest in meiosis (and mightthereby reveal meiotic regulatory mechanisms) have beenidentified (e.g., dmcl [4]). Furthermore, because most ge-netic screens for meiotic mutants have selected for muta-tions that do not confer a mitotic phenotype, comparison ofthe mitotic and meiotic effects of the resulting mutations hasbeen impossible. While existing meiotic mutations haveproven invaluable in elucidating the meiosis-specific pro-cesses of chromosome pairing, synapsis, and recombination,most are not well suited to the comparison of the regulatorymechanisms that govern mitosis and meiosis.

It is especially informative to compare the mitotic andmeiotic phenotypes conferred by mutations in a gene thatencodes a protein of known enzymatic activity, such asDNA topoisomerase II. Topoisomerase II acts by making adouble-strand break in a DNA molecule, passing anotherdouble-stranded segment through the break, and then reseal-ing the break (7, 32). In this way, topoisomerase II disen-tangles intertwined DNA molecules and relaxes both posi-tively and negatively supercoiled DNA (2, 27, 28, 31, 33). Inmitosis, topoisomerase II activity is required specifically atthe time of chromosome segregation for the resolution ofintertwinings between sister chromatids. In the absence oftopoisomerase II activity, cells proceed with spindle polebody separation and nuclear division (23). However, be-cause intertwinings between sister chromatids cannot beresolved, nuclei elongate but cannot divide normally, leadingto elevated levels of chromosome breakage and nondisjunc-tion (24). In spite of these deficiencies in nuclear division,many cells proceed with cytokinesis, pinch off the nucleus inthe neck of the bud, and rapidly lose viability (23). Thus, itis clear that in mitosis, no mechanism exists for preventingcell cycle progression in the absence of topoisomerase IIactivity.

In meiosis, top2 mutants behave very differently (40). top2mutants induced to sporulate at the restrictive temperaturesuccessfully complete early meiotic events, such as premei-otic DNA synthesis and commitment to meiotic levels of

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3446 ROSE AND HOLM

TABLE 1. S. cerevisiae strains used in this study

Strain Genotype Reference

CH1212 MTa/MATot his4-539(Am)IHIS4+ leu2-3, 112/LEU2+ Iys2-801(Am)/LYS2+ ura3-52/ura3- Rose et al. (40)52 top2-13Itop2-13

CH1387 MA Ta/MA Ta his4-539(Am)IHIS4+ leu2-3,112/LEU2+ 1ys2-801(Am)/LYS2+ ura3-52/ura3- Rose et al. (40)52 TOP2+/TOP2+

CH1389 MA Ta/MAoTa his4-539(Am)/HIS4+ leu2-3,112ILEU2+ 1ys2-801(Am)/LYS2+ ura3-52/ura3- Rose et al. (40)52 top2-171top2-17

CH1951 MATa-URA3-pBR322-AL4Ta/MA4To-URA3-pBR322-AL4Ta his4-B::LEU2+/his4-X::LEU2+ This studyleu2-3, 112/leu2-3, 112 1ys2-801(Am)/LYS2+ ura3-52/ura3-52 TOP2+/TOP2+

CH1952 MA4Ta-URA3-pBR322-AL4Ta/MATa-URA3-pBR322-AL4Ta his4-B::LEU2+/his4-X: :LEU2+ This studyleu2-3, 112/leu2-3, 112 Iys2-801(Am)/LYS2+ ura3-52/ura3-52 top2-13/top2-13

CH1953 MA4Ta-URA3-pBR322-AL4Ta/ASTa-URA3-pBR322-M4Ta his4-B::LEU2+/his4-X: :LEU2+ This studyleu2-3, 1121leu2-3, 112 1ys2-801(Am)/LYS2+ ura3-52/ura3-52 top2-17/top2-17

recombination. Whereas top2 mutants in mitosis attemptnuclear division and cytokinesis, top2 mutants in meiosisarrest prior to meiosis I, with no evidence of nuclearelongation. These phenotypic differences may simply reflectsome physical difference between mitosis and meiosis, suchas variation in spindle strength. Alternatively, they mayreflect a fundamental regulatory difference between the twoprocesses.To distinguish between these possibilities, we have looked

in detail at the meiotic phenotype of top2 mutants in Sac-charomyces cerevisiae. Our results suggest that the top2meiotic arrest is regulatory in nature, implying that theremay be a meiosis-specific regulatory mechanism that pre-vents the initiation of chromosome segregation in the ab-sence of topoisomerase II activity. This regulatory mecha-nism may have evolved to ensure the resolution of fortuitousintertwinings between nonhomologous chromosomes.

MATERIALS AND METHODS

Media. Presporulation medium (YEPA) consisted of 2%potassium acetate, 1% yeast extract, and 2% Bacto Peptone(Difco). Sporulation medium was 0.3% potassium acetate indistilled water. YEPD medium consisted of 1% yeast ex-tract, 2% Bacto Peptone, 2% Bacto Agar, and 2% glucose.

Strains and plasmids. All S. cerevisiae strains used in thisstudy are listed in Table 1. The strains used for reciprocalrecombination assays are congenic strains derived by trans-formation of a single pair of parental strains. Diploid strainCH1952 was constructed as follows: the haploid parentalstrains WTX107-6c and WTX107-25b were transformed withplasmids containing the top2-13 mutation and with his4heteroallelic plasmids as described previously (40). Theresulting top2-13 his4 strains were then transformed withplasmid pRHB23-4 (kindly provided by Rhona Borts and JimHaber). Integration of pRHB23-4 creates a tandem duplica-tion of the mating type locus. Integration was verified bySouthern hybridization (44) (data not shown). The resultingderivatives of WTX107-6c and WTX107-25b were thencrossed to yield diploid strain CH1952. For diploid strainCH1953, construction was the same as for CH1952, exceptthat the initial transformation of the haploid parental strainswas with a plasmid bearing the top2-17 mutation. Theresultant diploid is thus top2-17/top2-17. The construction ofCH1951 was also identical, except that transformation with atop2 plasmid was not done, and the resultant diploid wasTOP2+/TOP2+.Standard genetic techniques were employed in the han-

dling of all strains (41).

Presporulation and sporulation. Cells were grown over-night at 30°C in presporulation medium to a density of 4 x107 to 1 x 108 cells per ml. Cells were then centrifuged,washed with water or sporulation medium, centrifuged, andresuspended in the original culture volume of sporulationmedium. Aliquots of the sporulating cultures were distrib-uted into culture tubes on New Brunswick Rollordrums orinto flasks in a New Brunswick Gyrotory water bath shakerat either the permissive (25 or 30°C) or restrictive (10°C)temperature. To ensure that the restrictive temperature wasproperly maintained, we used a VWR 2020 low-temperatureincubator with the defrost cycle inactivated.

Fluorescence and electron microscopy. Surface-spread nu-clei were prepared by the method of Dresser and Giroux (9),as modified by Padmore et al. (37a). Samples (20 ml) ofsporulating cultures were harvested by centrifugation andresuspended in 2-ml portions of 200 mM Tris, pH 7.9.Dithiothreitol was added to a final concentration of 20 mM,and cells were incubated at room temperature for 2 min.Following incubation, cells were pelleted and resuspended in1 ml of 50 mM Tris-0.5 M KCl; 16 ,ul of Zymolyase solution(20 mg of Zymolyase 100T per ml, 2% glucose, 50 mM Tris,pH 7.9) were then added. Cells were then incubated at 30°Cfor 25 min. Following incubation, spheroplasts were pelletedand resuspended in 2 ml of a solution of 1 M sorbitol, 0.5 mMMgCl2, 1 mM EDTA, and 0.1 M morpholineethanesulfonicacid (MES), pH 6.4. Spheroplasts were then pelleted again,and 1 ,u of 1 M phenylmethylsulfonyl fluoride in dimethylsulfoxide was added directly to the pellet. Spheroplasts werelysed by the addition of 600 ,ul of a solution of 0.5 mMMgCl2, 1 mM EDTA, and 0.1 M MES, pH 6.4, and fixed bythe addition of 4.2 ml of 0.4% paraformaldehyde, pH 7.Fixed nuclei were dispensed onto washed, plastic-coatedglass slides. After the nuclei were allowed to settle for 10min, the fixative was drained and 300 ,ul of fresh fixativewere added. After 5 min, the fixative was drained and slideswere washed with 0.4% Photoflo (Kodak) and air dried.

For electron microscopy, spread preparations werestained by the method of Howell and Black (26). Electronmicroscopy was carried out with a Philips EM301 set at 60kV. For each sample, 50 nuclei were photographed. Themorphology of each nucleus was subsequently determinedfrom the photograph. Each nucleus received two scores, onefor spindle pole body morphology (single, partially dupli-cated, fully duplicated, or separated), and one for chromo-somal morphology (synaptonemal complex [SC] present orabsent). For SC morphology, all samples were scored twiceand an average score for each time point was obtained toensure accuracy. In both wild-type and mutant cultures, the

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MEIOTIC ARREST IN TOPOISOMERASE II MUTANTS 3447

efficiency of spindle pole body staining was variable fromsample to sample, with some nuclei showing no spindle polebody or spindle staining. To correct for this deficiency instaining, the graphs describing spindle pole body morphol-ogy present each class as a percentage of the number of cellsthat showed any spindle pole body staining. In addition, forthe data shown in Fig. 2 and 3, wild-type scores from day 16to day 20 were normalized to account for the fact that 40% ofthe culture had formed spores by day 16 (data not shown)and were thus not subject to scoring, since packaged nucleiare not visible in spread preparations. Since there is no sporeformation in mutant cultures, no similar normalization oftop2 scores was necessary.For fluorescence microscopy, spread preparations were

stained with 1 p,g of 4',6-diamidino-2-phenylindole (DAPI)per ml, 1 mg ofp-phenylenediamine per ml, 2 mM Na2CO3,23 mM CHNaO3, 0.1x phosphate-buffered saline (10 mMKH2PO4, 40 mM K2HPO4, 150 mM NaCl, 15 mM NaN3) in90% glycerol. Samples were examined with a Zeiss Axiophotand photographed with Kodak Technical Pan Film.Recombination assays. The production of mature recombi-

nant DNA molecules was assessed by the method of Borts etal. (6). Recombination was monitored in a top2-13Itop2-13strain (CH1952), a top2-17Itop2-17 strain (CH1953), and awild-type strain (CH1951). The general results were thesame for top2-13 and top2-17 strains, and only the results forthe top2-13 strain are presented in detail. Aliquots (25 mleach) of sporulating cultures were removed and fixed by theaddition of 25 ml of cold ethanol and 1 ml of 500 mM EDTA.Fixed samples were then stored at -20°C. DNA was isolatedfrom fixed cells by the method of Davis et al. (8) withmodifications described elsewhere (1). Isolated DNA wasdigested with BglII and separated electrophoretically on0.6% agarose gels. Recombinant fragments were detected bygel hybridization (38), using the 1,932-bp XmnI fragment ofpBR322 (5) as a probe.

V'iability assays. Mitotic viability was assayed in a top2-13/top2-13 strain (CH1212), a top2-17Itop2-17 strain (CH1389),and a TOP2+/TOP2+ strain (CH1387). The general resultswere the same for top2-13 and top2-17 strains. Aliquots (1 mleach) of each sporulating culture were removed and soni-cated with a Branson Sonifier. Individual unbudded cellswere then micromanipulated in a grid pattern onto YEPDplates. Plates were incubated at the permissive (25°C) orrestrictive (10°C) temperature, and cells were subsequentlyscored for their ability to form colonies (at the permissivetemperature) or to produce at least one bud (at the restrictivetemperature). For cells shifted to the permissive tempera-ture, viability was also assayed by plating cells on YEPDplates, counting colonies, and comparing the number ofcolonies observed to the number expected based on hemo-cytometer counts. Plating and micromanipulation analysesyielded comparable results.

RESULTS

Reciprocal recombination proceeds normally in top2 mu-tants. Although commitment to meiotic levels of recombina-tion occurs normally in top2 mutants (40), arrest in meiosis Icould be caused by a defect later in the recombinationpathway. One possibility is that top2 mutants undergo arrestbecause of a physical block in the process of recombinationitself. To test this possibility, we employed the method ofBorts et al. (6) to compare the production of covalentlyrecombined DNA molecules (mature recombinants) in top2mutants and wild-type strains. In this assay, diploid strains

homozygous for a tandem duplication of the mating typelocus are constructed by plasmid integration (Fig. 1A). Theduplicated AMT genes flank pBR322 sequence and the S.cerevisiae URA3 gene. Because of a naturally occurringrestriction site polymorphism between the MATa and MATaalleles, such a strain contains two pBR322-containing BglIIfragments: a 9-kb fragment from the MA4Ta chromosome,and a 31-kb fragment from the MATa chromosome. Areciprocal recombination event occurring anywhere alongthe length of the 9-kb MATa fragment will give rise to twonovel fragments (a 27-kb fragment and a 14-kb fragment).While the 27-kb recombinant fragment is not resolved fromthe 31-kb parental fragment under standard electrophoreticconditions, the 14-kb recombined fragment is easily sepa-rated from other hybridizing fragments. It has previouslybeen demonstrated that 95% of the recombination eventsdetected in this assay are reciprocal in nature (6); thus, theassay primarily measures crossover events, with only aminor contribution from gene conversion.

Crossover events were measured in TOP2+ITOP2+ andtop2ltop2 mutant strains homozygous for duplications of themating type locus. These strains were induced to sporulateat both the permissive temperature (25°C) and the restrictivetemperature (10°C). Aliquots of the sporulating cultureswere removed at 36 h at the permissive temperature and at14 days at the restrictive temperature and were fixed forsubsequent DNA isolation. Isolated DNA was digested withBglII, subjected to agarose gel electrophoresis, and hybrid-ized with radioactively labelled pBR322 DNA (Fig. 1B). Inboth strains at both temperatures, the recombinant productis clearly visible; densitometric analysis reveals that in allcases, the recombinant band contains 3 to 5% of all pBR322-hybridizing DNA (data not shown). These data indicate thattopoisomerase II activity is not required for the productionof mature recombinants and that the top2 mutant arrestoccurs after the completion of recombination. Thus, the top2mutant arrest does not result from a failure in reciprocalrecombination.

top2 mutants arrest before establishment of the meioticspindle. Because topoisomerase II activity is not required forthe production of mature recombinants, the top2 meioticarrest cannot be attributed to a block in recombination.What, then, is the nature of this arrest? One possibility isthat meiotic cells, like mitotic cells, initiate chromosomesegregation but are physically blocked by unresolved chro-mosomal intertwinings. In this case, cells should undergoarrest after the formation of the meiotic spindle, as inmitosis. Alternatively, meiotic cells may undergo a regula-tory arrest before chromosome segregation, thus preventingthe DNA damage, nondisjunction, and death that are prod-ucts of an abortive attempt at chromosome segregation inmitosis (23, 24). Arrested cells thus might contain duplicatedbut unseparated spindle pole bodies. To distinguish betweenthese two possibilities, TOP2+ITOP2+ and top2/top2 cellswere induced to sporulate at the restrictive temperature.Under these conditions, cultures in this strain backgroundsporulate with an efficiency of 40 to 60% (40). Aliquots of thesporulating cultures were removed at intervals, and thespindle morphologies of cells at each time point were deter-mined by electron microscopic analysis of spread meioticnuclei (Fig. 2). At To, nuclei in both cultures contain single,unduplicated spindle pole bodies. As meiosis proceeds, thenumber of nuclei containing duplicated spindle pole bodiesincreases. In the wild-type culture, nuclei containing dupli-cated but unseparated spindle pole bodies reach a maximumof 32% on day 12. However, in wild-type cells, this stage is

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3448 ROSE AND HOLM

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transient; in later time points, the number of cells in thisclass decreases to 7%, as cells proceed with spindle polebody separation and chromosome segregation. In contrast,top2 mutants show a progressive accumulation of cells withduplicated but unseparated spindle pole bodies reaching amaximum of 59% on day 20, with no appreciable decrease.Because the sporulation efficiency in these cultures is only40 to 60%, this result demonstrates that virtually all of themeiotic cells in the culture have this morphology. Thus, top2mutants arrest in meiosis with duplicated, unseparated spin-dle pole bodies; unlike mitotic cells, meiotic cells do notproceed with spindle pole body separation and chromosomesegregation in the absence of topoisomerase II activity.

top2 mutants are delayed in the exit from pachytene. Tounderstand the nature of the meiotic arrest exhibited by top2mutants, we examined the kinetics of SC morphogenesis intop2 mutant and wild-type cells, using the same spreadpreparations that were used for spindle pole body analysis.At each time point, the percentage of nuclei which containSCs was determined by electron microscopy (Fig. 3). Inwild-type cultures at To, nuclei show no detectable SCstructure, as expected. As meiosis progresses, however, SCassembly takes place, and by day 6 the number of nucleicontaining SCs peaks at 34%. In pachytene nuclei from day6, SC is full length and continuous, and both lateral elementsare clearly visible (Fig. 4A, C, and E). After day 6, thenumber of nuclei containing SCs drops rapidly as cellsproceed with meiosis, reaching a level of approximately 6%by day 12. Thus, in wild-type cells, pachytene is a relativelytransient stage; SC assembly is followed closely by disas-sembly.

In contrast, the kinetics of SC formation in top2 mutantssuggest that they are delayed in the exit from pachytene (Fig.3). Like wild-type nuclei, top2 mutant nuclei contain nodetectable SC structure at To, and there is an increase in thenumber of nuclei containing SCs by day 6. However, incontrast to wild-type cultures, the SC does not immediatelydisassemble; instead, the number of nuclei containing SCsremains high as late as day 22 (20%). Thus, it appears thattop2 mutants are delayed in the exit from pachytene. Al-though the timing of this delay suggests that the top2 meioticarrest may be caused by some deficiency in SC morphogen-esis, pachytene nuclei from this interval are morphologicallynormal at a gross level by several criteria (Fig. 4B, D, andF). First, SC in top2 mutants is apparently continuous, with

_ 31 kb

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FIG. 1. Reciprocal recombination in top2/top2 and TOP2+/TOP2+ strains. (A) Structures of the mating type locus in strainsused to assay reciprocal recombination. Duplications of the matingtype locus were created by plasmid integration as described byBorts et al. (6). Recombination (denoted by the large X) producesnovel restriction fragments. See text for a complete description. S.cerevisiae genomic DNA is denoted by a straight line; pBR322 DNAis denoted by a wavy line. B, BglII site. (B) Autoradiographsshowing the production of recombined DNA molecules in top2/top2and TOP2+/TOP2+ strains. Strains CH1951 (TOP2+ITOP2+) andCH1952 (top2-13Itop2-13) were induced to sporulate at both thepermissive temperature (25°C) and the restrictive temperature(10°C). Aliquots of the sporulating cultures were removed at To, at36 h at 25°C, and at 14 days (14d) at 10°C. Genomic DNA wasisolated and cut with BglII. pBR322-hybridizing fragments weredetected by gel hybridization (38). The 31- and 9-kb bands are

parental fragments. The 14-kb band is a recombined fragment; a27-kb recombined fragment is not resolved from the 31-kb parentalband. Lanes shown for each strain are from different parts of a singlegel, which accounts for variations in electrophoretic mobility.

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MEIOTIC ARREST IN TOPOISOMERASE II MUTANTS 3449

70

X. -6 ---- top2-13/top2-13(A.60- ToP2VrIOP2 +

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30-

40-

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U{0 10 20 30

Days @ 1 OOCFIG. 2. Spindle pole body morphology of TOP2+/TOP2+ and

top2-13/top2-13 strains undergoing meiosis at the restrictive temper-ature. Strains CH1387 (TOP2+/TOP2+ and CH1212 (top2-131top2-13) were induced to sporulate at the restrictive temperature (100C).Aliquots of the sporulating cultures were removed at intervals, andspread nuclei were prepared and silver stained. The spindle polebody morphologies of the spread samples were determined byelectron microscopy. Spindle pole staining was variable; only thosenuclei with visible spindle pole bodies were scored. The percentagesof nuclei containing duplicated but unseparated spindle pole bodies(SPB) are presented as a function of time at the restrictive temper-ature.

no gaps. Second, the number and contour length of mutantSC segments is indistinguishable from that seen in wild-typenuclei. Finally, the distance between lateral elements iscomparable in mutant and wild-type nuclei. Thus, while theresolution of this analysis is not sufficient to detect smallimperfections in SC structure, it is clear that top2 mutantsform grossly normal SCs.While top2 mutants are clearly delayed at pachytene,

dissolution of the SC eventually takes place. Late in meiosis,we observed an accumulation of nuclei with no SC structurecontaining duplicated but unseparated spindle pole bodies(Fig. 4G and H). This morphological class increased from 2to 30% of the population in the interval between day 18 andday 22 (data not shown). While this morphology is alsoobserved transiently in wild-type cultures, it appears so latein top2 mutant cultures that it may be the result of nonspe-cific degradation of the SC.

top2 mutants are not deficient in chromosome condensation.The fact that top2 mutants are delayed in the exit frompachytene suggests that they may be deficient in some aspectof SC morphogenesis. Alternatively, the delay might be dueto a deficiency in another process that occurs at the sametime. One possibility that is consistent with studies of mitoticchromosome condensation in Schizosaccharomyces pombe(45) is that topoisomerase II is required for meiotic chromo-some condensation. To test this possibility directly, wecompared the level of condensation in pachytene nuclei fromwild-type and top2 mutant cultures by staining spread prep-arations with the DNA stain DAPI. In these preparations,individual chromosomes are clearly visible (Fig. 5). Thelevels of chromosome condensation in wild-type (Fig. 5A toC) and mutant (Fig. 5D to F) pachytene nuclei appear to beidentical. There are no consistent differences in either thelength or thickness of the chromosomes from the two

C)-U- TOP247TOP2+U)30-

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Days @ 1OCFIG. 3. SC morphogenesis of TOP2+/TOP2+ and top2-13/

top2-13 strains undergoing meiosis at the restrictive temperature.Strains CH1387 (TOP2+/TOP2+) and CH1212 (top2-131top2-13)were induced to sporulate at the restrictive temperature (10°C).Aliquots of the sporulating cultures were removed at intervals, andspread nuclei were prepared and silver stained. The SC morpholo-gies of the spread samples were determined by electron microscopy.The percentages of nuclei containing SCs are presented as a functionof time at the restrictive temperature.

strains. Thus, we conclude that top2 mutants are not grosslydeficient in meiotic chromosome condensation.

Meiotically arrested top2 mutants retain mitotic viability. Iftop2 mutants undergo a regulatory arrest in meiosis, thenone would expect that the arrest might be reversible, be-cause an irreversible arrest could not have conferred aselective advantage during evolution. However, previousexperiments have demonstrated that the top2 meiotic arrestis irreversible; once arrested, mutant cells are unable toresume and complete meiosis even when shifted to thepermissive temperature (40). We reasoned that meioticallyarrested cells, although incompetent to leave the arrest viameiosis, might be able to escape the arrest by entering themitotic cell cycle. To test this possibility, TOP2+ITOP2+and top2ltop2 cells were induced to sporulate at the restric-tive temperature (10°C). Cells were held at the restrictivetemperature for 15 to 45 days, giving them ample time toarrest (in the case of top2 mutants) or to complete meiosis (inthe case of wild-type cells). Individual unbudded cells werethen transferred to vegetative plates by micromanipulation,and the cells were incubated at the permissive temperatureand scored for their ability to form colonies. The overallviabilities of TOP2+/TOP2+ and top2-13Itop2-13 cells underthese conditions were virtually identical (85% [n = 131] and86% [n = 180], respectively). Thus, meiotically arrested top2mutants remain capable of leaving the meiotic arrest viamitosis at the permissive temperature. Subsequent analysishas demonstrated that the resulting colonies are composedof diploid cells (data not shown); thus, it appears thatarrested cells enter the mitotic cell cycle at the G2-Mtransition.

Meiotically arrested cells can revert to a mitotic cell cyclewhen they are shifted to vegetative medium and returned tothe permissive temperature; which of these two conditions isthe signal that triggers the shift to the mitotic cell cycle? Onepossibility is that arrested cells are poised at a point inmeiosis at which topoisomerase II activity is necessary; inother words, arrested cells would be "waiting" for the

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MEIOTIC ARREST IN TOPOISOMERASE II MUTANTS 3451

FIG. 4. Nuclear morphologies of TOP2+ITOP2+ and top2ltop2 strains undergoing meiosis at the restrictive temperature. Strains CH1387(TOP2+ITOP2+) and CH1212 (top2-131top2-13) were induced to sporulate at the restrictive temperature (10°C). Aliquots of the sporulatingcultures were removed at intervals, and spread nuclei were prepared and silver stained. (A, C, and E) TOP2+ITOP2+ pachytene nuclei; (B,D, and E) top2-13Itop2-13 pachytene nuclei; (G and H) top2-131top2-13 nuclei in which the SC has broken down. The SC, spindle pole bodies,and nucleolus stain positively in these preparations; microtubules exclude stain. Spindle pole bodies are not visible in all nuclei. In pachytenenuclei (A to F), the SC consists of long, continuous stretches of paired lateral elements. In some nuclei, notably in panels B and E, chromatinis visible as a halo surrounding the SC. Later in meiosis, top2 mutants undergo dissolution of the SC (G and H). In these nuclei, chromatinis visible as a diffuse area of staining; no visible SC structure remains, and the spindle pole bodies, visible as paired black spots surroundedby an area of reduced staining, are fully duplicated but have not separated. Bar, 5 ,um.

return of topoisomerase II activity, and they would be ableto initiate mitosis as soon as this stimulus is provided. In thiscase, cells incubated at the restrictive temperature in vege-tative medium would remain as arrested, unbudded cells. Analternative possibility is that in response to a deficiency intopoisomerase II activity the cells at the arrest point havemade a regulatory decision to abort meiosis and revert tomitotic growth. If this possibility is true, then the arrested,unbudded cells are waiting only for nutritional conditionswhich support vegetative growth; thus, they will initiatebudding when shifted to vegetative medium, even at therestrictive temperature.To distinguish between these possibilities, we performed a

micromanipulation experiment in which single, unbudded,

meiotically arrested cells were transferred to vegetativemedium at the restrictive temperature. Since top2 mutantswill not form colonies at the restrictive temperature, cellswere scored microscopically in this experiment for theirability to form at least one bud, indicating that they hadrecovered from the meiotic arrest and initiated a mitotic cellcycle. top2 mutants showed a recovery frequency of 82%, alevel comparable to that seen in both mutant and wild-typecultures allowed to recover at the permissive temperature.This analysis demonstrates that the exit of meiotically ar-rested top2 mutants to the mitotic cell cycle does not requiretopoisomerase II activity; a shift to vegetative medium issufficient. Since these cells are incabable of completingmeiosis at either the permissive or restrictive temperature~~~~~~~~~~~'~

FIG. 5. Meiotic chromosome condensation in TOP2+/TOP2+ and top2/top2 strains undergoing meiosis at the restrictive temperature.Strains CH1387 (TOP2+ITOP2+) and CH1212 (top2-13Itop2-13) were induced to sporulate at the restrictive temperature (10°C). Aliquots ofthe sporulating cultures were removed at intervals, and spread nuclei were prepared and stained with the DNA stain DAPI to visualizechromatin. Each photograph shows a single nucleus. (A to C) wild-type pachytene nuclei; (D to F) top2/top2 pachytene nuclei. Bar, 5 ,um.

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(40), it appears that these cells have made a regulatorydecision to abort meiosis and to proceed with mitosis as soonas nutritional conditions improve.

DISCUSSION

We have examined the meiotic phenotype conferred bymutations in the structural gene for DNA topoisomerase II.top2 mutants successfully complete the latest detectablestage of recombination, the production of mature reciprocalrecombinant DNA molecules. The mutants form full-length,cytologically normal SC, and DAPI staining of meioticspread preparations reveals no gross deficiency in chromo-some condensation. However, top2 mutants are delayed inthe exit from pachytene and arrest prior to spindle pole bodyseparation. While this meiotic arrest is not reversible, cellscan escape the arrest via mitosis, forming colonies upontransfer to vegetative medium at the permissive tempera-ture.The top2 meiotic arrest appears to be regulatory. A com-

parison of the mitotic and meiotic phenotypes conferred bytop2 mutations strongly suggests that the meiotic arrest isregulatory in nature. In mitosis, cells are unable to resolveintertwinings between sister chromosomes, but they none-theless proceed with nuclear division and cytokinesis. As aresult, cells display elevated frequencies of chromosomebreakage and nondisjunction, and they rapidly become invi-able (23, 24). Thus, there is clearly no mitotic regulatorymechanism that protects the cell by preventing cell cycleprogression in the absence of topoisomerase II activity. Themeiotic phenotype of top2 mutants is different in at least twoimportant respects. First, while mitotic cells rapidly loseviability, meiotically arrested top2 mutants remain fullyviable, even when held at the arrest point for as long as 30days. This result suggests that the progress of meiosis is notphysically impeded, as in mitosis, but rather that cells havebecome arrested before suffering significant damage to theirDNA. Second, top2 mutants arrest in meiosis before spindlepole body separation. A priori, there is no reason to expectthat a defect in DNA topology conferred by a top2 mutationshould physically prevent spindle pole bodies from separat-ing. In fact, topoisomerase II activity is clearly not aprerequisite for spindle pole body separation; spindle estab-lishment occurs successfully in the absence of topoisom-erase II activity both during mitosis (23) and in meiotic cellsif recombination has been blocked (40). An economicalhypothesis is that the deficiency in topoisomerase II activitytriggers a meiosis-specific regulatory mechanism that sec-ondarily blocks spindle pole body separation.

top2 mutants are unusual in that they revert from theinitiation of meiosis to a state in which only mitotic divisionis possible. Wild-type cells become committed to the com-pletion of meiosis around the time of spindle pole bodyseparation (16, 17, 25, 36); after this point, cells will com-plete meiosis even if shifted to vegetative medium. top2mutants clearly arrest prior to becoming committed to thecompletion of meiosis, because they are able to revert tomitotic growth if shifted to vegetative medium. However,the arrested cells are no longer competent to completemeiosis, even if shifted to the permissive temperature (40).Thus, top2 mutants undergo a novel commitment event:cells have not merely failed to commit to the completion ofmeiosis but have become committed to not dividing meioti-cally. Thus, it appears as if a regulatory switch is thrown atthe top2 meiotic arrest point, aborting meiosis and commit-ting the cells to a mitotic division.

Because top2 mutants exhibit a delay in the exit frompachytene, but ultimately undergo SC dissolution, it isimportant to consider which of these phenotypes providesthe most information about topoisomerase II function. Tem-perature-shift experiments have revealed that meiotic cellsfirst require topoisomerase II activity at approximately 6 to8 days at 10°C (40), which corresponds roughly to the time ofpachytene, well before the dissolution of the SC that occursaround day 20. For this reason, we view pachytene as theprimary arrest point for top2 mutants, and see the dissolu-tion of SCs as nonspecific degradation. Previous evidencesuggests that pachytene is an important point of regulatorycontrol in meiosis. Mutations in the DMCI gene, whichconfer defects in recombination, cause cells to arrest at astage that may correspond to wild-type pachytene on thebasis of spindle pole body morphology, although SC forma-tion is not complete (4). More important, mutations in theStart genes CDC28, CDC36, and CDC39 also confer arrest atpachytene, with fully normal SC (42). This result raises theinteresting possibility that the arrests caused by dmcl andtop2 mutations are mediated by previously identified genesthat play a central regulatory role in the mitotic cell cycle.What defect is responsible for the arrest? Because top2

mutants in meiosis undergo arrest prior to spindle formation,the top2 meiotic arrest cannot be attributed to a defect inchromosome segregation per se. Moreover, the fact thattop2/top2 rad5O/rad5O mutants (in which chromosome pair-ing, synapsis, and recombination are blocked) successfullycomplete the first meiotic nuclear division (40) stronglysuggests that top2 mutants undergo arrest in response to adefect in meiotic prophase. What is the nature of this defect?Unfortunately, no morphological abnormality is visible atthe electron microscopic level in top2 mutants prior to thearrest point. We must, therefore, propose that the top2meiotic arrest is caused by a defect that is beyond theresolution of the techniques we have employed. In theabsence of any visible defect, we recognize several possibleroles that topoisomerase II might play prior to the meioticarrest point. We will discuss four possible roles for topo-isomerase II in prophase and our reasons for favoring thelast one.

First, topoisomerase II could be a structural component ofmeiotic chromosomes, and top2 mutations may disrupt thisrole. Topoisomerase II has been described as a majorcomponent of the chromosome scaffold, a residual structureisolated by extraction of mitotic chromosomes (11), althoughrecent in vitro results suggest that this result may be anartifact of extraction conditions (21). In addition, topoisom-erase II has been localized to meiotic chromosome cores inboth chicken (35) and S. cerevisiae (30). This localizationmay reflect a structural role for the enzyme. Alternatively, itmay be a result of the preferential binding of topoisomeraseII to crossed DNA molecules (49); such molecules might bemore abundant in the chromosome core, where the concen-tration of DNA is higher. If the top2 meiotic arrest werecaused by a structural defect and topoisomerase II was amajor structural component of the scaffold, one might expectsome gross morphological abnormality in top2 mutants.However, neither allele examined showed any defect inchromosomal morphology at either the light or electronmicroscope level. Thus, while top2 mutations may causedefects which are beyond the resolution of these techniques,it seems unlikely that the top2 meiotic arrest reflects a majorstructural role for the enzyme.A second possibility is that the meiotic arrest reflects an

enzymatic role for topoisomerase II in chromosome conden-

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sation. In S. pombe, topoisomerase II activity is required forthe hypercondensation of chromatin seen when chromosomesegregation is blocked genetically, although topoisomeraseII activity is apparently not required for normal levels ofcondensation (45). In meiosis, where chromosomes con-dense more fully than in mitosis, it could be argued thattopoisomerase II might play a role in normal condensation.A meiotic deficiency of the same magnitude as that seenmitotically in S. pombe (where chromosome length is 2- to2.5-fold greater in top2 mutants) should be detectable inDAPI-stained meiotic spread preparations. However, wedetect no difference in the extent of chromosome condensa-tion in wild-type and top2 mutant cells by this method. Thus,any deficiency in chromosome condensation would have tobe an unexpectedly subtle one.

Third, it is conceivable that the meiotic arrest of top2mutants occurs in response to DNA damage caused bymutant topoisomerase II protein. Wild-type topoisomeraseII does make double-strand breaks, and the RAD9 check-point mechanism, which causes mitotic arrest in response todouble-strand breaks (20), has been shown to functionmeiotically (47). However, while we cannot rule out thepossibility that RAD9 acts at multiple times in meiosis, theonly established RAD9-mediated meiotic arrest point is priorto chromosome pairing and synapsis. Thus, the top2 arrest,which occurs after synapsis, is unlikely to be RAD9 depen-dent. In addition, top2 alleles that cause meiotic arrest bymaking unrepaired double-strand breaks would be expectedto be dominant; both of the alleles used in this study are fullyrecessive (unpublished observations).

Finally, an attractive model is one in which topoisomeraseII activity is required for the resolution of fortuitous tanglesbetween nonhomologous chromosomes. Such intertwinings,termed interlocks, are morphologically visible during earlyprophase in a number of organisms (46), and they arebelieved to be a consequence of the imprecise orientation ofchromosomes in the presynaptic nucleus. In many organ-isms, including S. cerevisiae, synapsis is initiated at multiplesites along the length of the chromosome. As a result,nonhomologous chromosomes can become trapped betweensynapsing homologs as synapsis proceeds, forming an inter-lock. Because the resolution of interlocks requires the reso-lution of a DNA-DNA intertwining, it has previously beenproposed that topoisomerase II may play a role in thisprocess (39).Unresolved interlocks would pose a serious problem for

the cell at metaphase I because of the way in which meioticcells align chromosomes in preparation for their segregation(29, 39). It has previously been demonstrated that tensionbetween the kinetochore and microtubule organizing centeris required for proper alignment of chromosomes on thespindle (37). To generate this tension, kinetochores must bephysically connected to one another. Cytological studies inmany organisms suggest that this attachment is produced indifferent ways in mitosis and meiosis. In mitosis, sistercentromeres are associated at the time of metaphase. Aslong as this association between sister centromeres is main-tained, sister kinetochores will attach to spindle fibers thatoriginate at opposite poles, and sister chromatids will disjoinproperly at anaphase. In meiosis, however, homologs inmany organisms are associated not at the centromere but atthe chiasmata; proper spindle attachment is therefore depen-dent upon these physical connections between chromo-somes. Thus, in this specialized division, interlocks betweennonhomologous chromosomes could interfere with properspindle attachment by producing inappropriate connections

between nonhomologous chromosomes. Improper attach-ment would, in turn, result in errors in segregation atanaphase I. Thus, even a small number of intertwiningsbetween nonhomologs would be detrimental to the cell andcould thereby provide a basis for the evolution of a meiosis-specific mechanism to ensure their resolution.While the exact mechanism by which interlocks are re-

solved is obscure, cytological observations provide someclues. First, interlocks decrease in number as synaptonemalcomplex assembly progresses; in silkworms, interlocks arenumerous in early prophase but are completely resolved bymidpachytene (39). This temporal correlation suggests thatSC morphogenesis may drive the resolution of interlocks.Second, small regions of the axial cores have been observedto disassemble during the process of interlock resolution(39). This observation has led to the proposal that theresolution of interlocks occurs in three steps (46). First, theprotein component of the interlock is resolved by the local-ized dissolution of axial cores. Second, topoisomerase IIacts to resolve the remaining DNA-DNA intertwining. Fi-nally, the axial cores are reassembled, and synapsis iscompleted.While this model is appealing, it does not address the issue

of directionality. The reaction that topoisomerase II cata-lyzes is completely reversible; acting alone, the enzyme is aslikely to introduce new intertwinings as to resolve existingones (2, 28, 30). Thus, topoisomerase II can only preferen-tially resolve tangles when directionality is provided exter-nally. How might directionality be imposed in resolvinginterlocks? To address this concern, we propose that thegeneral model be modified so that topoisomerase II acts inconcert with SC reassembly. In this way, the condensationof chromatin that accompanies reassembly of the SC mightprovide directionality for topoisomerase II. This refinedmodel also suggests one mechanism by which the cell mightdetect interlocks. If SC reassembly and interlock resolutionwere mechanically coupled, then blocking resolution byinhibiting topoisomerase II would disrupt SC reassembly,resulting in small discontinuities in the SC; such discontinu-ities might well be beyond the resolution of the cytologicalmethods employed in this study. Kleckner et al. (28) havepreviously proposed that the interruption of SC morphogen-esis by interlocks may act as a cellular signal, triggering apachytene arrest. In the same way, SC discontinuity in top2mutants could serve as a signal to block the initiation ofspindle formation, thus preventing the errors in segregationthat interlocks would cause.The proposed regulatory mechanism is consistent with

existing data and is appealing from a teleological perspec-tive. However, proof that such a mechanism exists willrequire the isolation of mutations which disrupt its function.Mutations of this kind would cause top2 mutants in meiosisto proceed with nuclear division, which would presumablyresult in chromosome breakage, nondisjunction, and death,as in mitosis. Screens designed to isolate these types ofmutations are currently in progress.

ACKNOWLEDGMENTS

We thank Doug Bishop, Mike Dresser, Ruth Padmore, and BethRockmill for advice and many helpful discussions; Adam Driks fortraining and advice in electron microscopy; Dan Branton for the useof his electron microscope; Rhona Borts, for providing plasmids;and Breck Byers, Nancy Kleckner, and Shirleen Roeder for criticalreading of the manuscript.

This work was supported by grants to C.H. from the NationalInstitutes of Health (GM 36510) and the PEW Charitable Trust.

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