Cell, Vol. 60, 1009-1017,
March
23, 1990, Copyright
0 1990 by Cell Press
Segregation of Recombined Chromosomes Meiosis I Requires DNA Topoisomerase II David Rose, Winston Thomas, and Connie Holm Department of Cellular and Developmental Biology Harvard University Cambridge, Massachusetts 02138
Summary To understand better the similarities and differences between meiosis and mitosis, we examined the meiotic role of DNA topoisomerase II, an enzyme that is required mltotlcally to disentangle sister chromatids at the time of chromosome segregation. In meiosis, we found that topolsomerase II is required only at the time of nuclear division. When cold-sensitive fop2 mutants are induced to sporulate at the restrictive temperature, they undergo premeiotlc DNA synthesis and commitment to meiotic levels of recombination but fail to complete the first meiotic nuclear division. The introduction of a mutation blocking recombination relieves the requirement for topoisomerare II in meiosis I. These results suggest that topoisomerase II is required at the time of chromosome segregation in meiosis I for the resolution of recombined chromosomes. Introduction The processes of both meiosis and mitosis require that chromosomes be replicated faithfully and segregated precisely in microtubule-mediated nuclear divisions. There are, however, important differences between the two processes. In contrast to the single mitotic nuclear division, meiosis comprises two chromosome segregation events. Premeiotic DNA synthesis also differs from premitotic DNA synthesis, as manifested in part by its extended duration (Williamson et al., 1983). In addition, meiotic and mitotic recombination require different gene products (for review, see Haynes and Kunz, 1981) and occur at different levels. Study of the effects of single mutations on both mitosis and meiosis is frequently informative. For example, mutations in the structural gene for 8-tubulin confer similar mitotic and meiotic phenotypes, blocking both mitosis and sporulation (Huffaker et al., 1988). Mutations in the RADSO gene, on the other hand, have strikingly different meiotic and mitotic phenotypes. tad50 mutants fail to undergo meiotic recombination (Game et al., 1980) but in mitosis are hyper-recombinant and deficient in DNA repair (Haynes and Kunz, 1981). These studies point out important similarities and differences between mitosis and meiosis and also provide a more complete picture of the function of the gene product examined. With these studies as a background, we compared the roles of DNA topoisomerase II in mitosis and meiosis. The role of DNA topoisomerase II in mitosis is well understood. DNA topoisomerase II acts by making a double-
in
strand break in a DNA molecule, passing another doublestranded segment through the break, and then resealing it (Brown and Cozzarelli, 1979; Liu et al., 1980). In this way, DNA topoisomerase II can disentangle intertwined DNA molecules and can relax both positively and negatively supercoiled DNA (Baldi et al., 1980; Hsieh, 1983; Hsieh and Brutlag, 1980; Kreuzer and Cozzarelli, 1980; Liu et al., 1981). In yeast, temperature-sensitive top2 mutants allowed to progress through mitosis at the restrictive temperature suffer a high level of inviability, which is accompanied by elevated levels of chromosome breakage and nondisjunction (Holm et al., 1985, 1989; Uemura and Yanagida, 1988; Uemura et al., 1987). Inviability can be prevented by treatment with the microtubule-destabilizing drug nocodazole (Holm et al., 1985) indicating that the loss in viability is specifically associated with cells attempting to segregate their chromosomes in the absence of topoisomerase II. These observations have led to the hypothesis that topoisomerase II facilitates chromosome segregation in mitosis by resolving entangled sister chromatids, which are intertwined to some extent following the completion of DNA synthesis (Sundin and Varshavsky, 1980, 1981; DiNardo et al., 1984; Weaver et al., 1985). Because topoisomerase II is required for the resolution of intertwined sister chromatids in mitosis, we anticipated that it might play an analogous role in meiosis. In particular, the second meiotic nuclear division is very similar to a mitotic nuclear division in that each involves the segregation of sister chromatids. Meiosis I, on the other hand, differs from both meiosis II and mitosis in that it involves the segregation of homologous chromosomes. Because homologous chromosomes are not replicative sisters, topoisomerase Ii may not be required for their separation. However, topoisomerase II may be required for other processes in meiosis I, such as premeiotic DNA synthesis, initiation of meiotic recombination, or the resolution of recombined DNA molecules. We report here that DNA topoisomerase II is required for the meiotic nuclear divisions. Cold-sensitive top2 mutants induced to sporulate at the restrictive temperature undergo premeiotic DNA synthesis and commitment to meiotic levels of recombination but arrest before the first meiotic nuclear division. Our results suggest that DNA topoisomerase II is required for the resolution of recombined chromosomes at the time of chromosome segregation in meiosis I. Results DNA Topoisomerase II Is Required for Sporulatlon We first determined that topoisomerase II is required for the completion of sporulation, which includes both the process of meiosis and the encapsulation of meiotic products in spore walls. TCP2+/7?F2+ and top2cs/top2cs strains were induced to sporulate at both the permissive temperature (25%) and the restrictive temperature (10%). Although TCR?+/Topr strains exhibited only a slight reduction in
Cell 1010
Table 1. Sporulation Frequencies TOP2+/7Of2+ Strains
of top2/top2
and
Sporulation
TOP2 +/TOP2 top2/top2
Days at Restrictive
Temperature
Frequency
Strain
25’C
10%
CH1131 (TOP2+/TOP2+) CH1123 (top2-13/top2-73) CH995 (top2-74/top2-14) CH1393 (top2-I 7/top2-7 7)a
0.55 0.53 0.56 0.41
0.40 0.03 0.03 0.16
a lop2-7 7 mutants have a leaky cold-sensitive cell cycle as well as in sporulation.
Q +
phenotype
-I
in the mitotic
Oi 0
final sporulation frequency at 10% fop2%op2cs mutants showed a dramatic reduction in sporulation at the restrictive temperature (Table 1). These results indicate that topoisomerase II is required for the completion of sporulation. DNA Topoisomerase II Is Required Specifically at the Time of the Meiotic Nuclear Divisions To determine the time in sporulation during which topoisomerase II is required, we performed two complementary temperature-shift experiments. In the first experiment, TOP2+ITOP2+ and fop2Wop2cs cells were induced to sporulate at the restrictive temperature. Aliquots of the sporulating cultures were shifted at intervals to the permissive temperature and allowed to complete sporulation, and the final sporulation frequencies of the shifted cells were then determined. Shifting to permissive temperature permits sporulation in fop2Vtop2cs cells, provided that the shift occurs before topoisomerase II is irreversibly required; sporulation does not occur in top2Vtop2”~ cells shifted to permissive temperature any later than the time at which the requirement for topoisomerase II begins. Thus, the point at which a shift to permissive temperature no longer ailows sporulation marks the beginning of the interval during which topoisomerase II is required in sporulation. As expected, in TOP2+/TO/W cells the final sporulation frequency was independent of the time of the shift, 40% in each case (Figure 1). In contrast, in top2cs/fop2cs cells wild-type levels of sporulation were attained only if the cells were shifted on or before day 6; in the interval between day 6 and day 6, final sporulation frequencies dropped rapidly. This result indicates that cells enter the interval during which topoisomerase II is required between day 6 and day 6. To determine the stages in meiosis represented by days 6, 7, and 6, cells fixed at the time of the shift were stained with the DNA stain DAPI and examined by fluorescence microscopy (Figure 2). TOP2+/TOP2+ cells were mononucleate on day 6. On days 7 and 6, a progressively greater number of cells had undergone both nuclear divisions. Thus, the beginning of the requirement for topoisomerase II in meiosis corresponds to the beginning of the meiotic nuclear divisions in KR!?VTOP2+ cells. Consistent with this observation, top2cs/top2cs cells were indistinguishable from TOP2+/72%?+ cells on day 6 but failed to undergo nuclear divisions on days 7 and 6. Instead, the cells
10
Figure 1. Sporulation Frequencies to Permissive Temperature
20
of Cultures
Shifted from Restrictive
Strains CH1123 (top2-73/top2-73:+) and CH1131 (TOfzI/TOP2+:i+) were induced to sporulate at the restrictive temperature, and aliquots were shifted at intervals to the permissive temperature and allowed lo complete sporulation. Final sporulation frequencies of the shifted aliquots are plotted as a function of the time at which they were shifted from restrictive temperature lo permissive temperature.
remained mononucleate, and the nuclei adopted an abnormal morphology. The failure of fop2cs/top2cs cells to undergo the first nuclear division at the restrictive temperature indicates that topoisomerase II is required for meiosis I. The complementary temperature-shift experiment defined the time at which the requirement for topoisomerase II ends. In this experiment, TOR?WOf2+ and top2cWop2cs cells were induced to sporulate at the permissive temperature. Aliquots of the sporulating cultures were shifted at intervals to the restrictive temperature and allowed to complete sporulation, and the final sporulation frequencies of the shifted aliquots were then determined. In top2Wop2cs cells, sporulation is disrupted by a shift to restrictive temperature if the shift occurs before the end of the period during which topoisomerase II is required. Cells shifted later than this time are not affected by the shift and sporulate at wild-type levels. The point at which a shift to restrictive temperature no longer disrupts sporulation marks the end of the interval during which topoisomerase II is required. In TOP2WOf2+ cells, the final sporulation frequency was independent of the time of the shift, as expected (Figure 3). In contrast, top2Vtop2cs cells shifted to restrictive temperature at or before 12 hr sporulated at very low levels; cells shifted after 12 hr showed a progressive increase in sporulation frequency, reaching wild-type levels by 30 hr. This result indicates that cells complete the interval during which topoisomerase II is required between 12 and 30 hr. To determine the stage in meiosis represented by the interval from 12 to 30 hr, cells fixed at the time of each shift were stained with the DNA stain DAPI and examined using fluorescence microscopy (data not shown). At 12 hr, both TOP2+/7UP2+ and top2Vtop2c~ cultures contained almost exclusively mononucleate cells. In the interval from 12 to 30 hr, however, a progressively greater number of cells had undergone both nuclear divi-
The Meiotic 1011
Figure
Role of DNA Topoisomerase
2. Morphology
of top2/top2
II
and TOFV+/TOP2+
Cells Undergoing
Meiosis
at the Restrictive
Temperature
In the experiment described in Figure 1, strains CH1131 (7Cf2+/7Df~: A, B, and C) and CH1123 (top2-13/top2-13: D, E, and F) were induced to sporulate at the restrictive temperature. Samples were fixed and stained with DAPI after 6 days (A and D), 7 days (6 and E), and 6 days (C and F) at the restrictive temperature. The cell in (B) with the X-shaped nuclear morphology has completed meiosis I and is in the middle of meiosis II, The cells in (C) are tetranucleate, but not all nuclei are in the focal plane. The bar represents 5 Wm.
sions and were tetranucleate. This observation demonstrates that the end of the interval during which topoisomerase II is required corresponds to the completion of the meiotic nuclear divisions. DNA Topoisomerase II Is Not Required for Early Events in Meiosis I To define the role of topoisomerase II in meiosis I more precisely, we examined the effect of cold-sensitive top2
”
-D +
1
01
' 0
I IO
Hours
' at
I 20
'
Permissive
Figure 3. Sporulation Frequencies save to Restrictive Temperature
I 30
TOP2 +/TOP2 topz/topz
-
I 40
+
'
I 50
Temperature
of Cultures
Shifted
from
Permis-
Strains CH1123 (top2-13/top2-734 and CH1131 (TOrWTOP2+:& were induced to sporulate at the permissive temperature, and aliquots were shifted at intervals to the restrictive temperature and allowed to complete sporulation at the restrictive temperature. Final sporulation frequencies of the shifted aliquots are plotted as a function of the time at which the culture was shifted from permissive to restrictive temperature.
mutations on two early meiotic events, premeiotic DNA synthesis and commitment to meiotic levels of recombination. To determine whether cold-sensitive fop2 mutations grossly affect the kinetics of premeiotic DNA synthesis, strains were induced to TOP2VTopzc and top2%op2cs undergo meiosis at both restrictive and permissive temperatures. Aliquots of the sporulating cultures were removed at intervals, stained with propidium iodide, and analyzed by flow cytometry. The kinetics of DNA synthesis, as revealed by this analysis, are not grossly affected by cold-sensitive top2 mutations (data not shown). Another early meiotic process in which topoisomerase II might play a role is meiotic recombination. Since lop2V top2cs cells failed to complete sporulation, we were unable to measure recombination by looking at the genotype of meiotic products. However, a return-to-growth protocol can be employed to examine commitment to meiotic levels of recombination in strains unable to complete meiosis (Sherman and Roman, 1963). To assay recombination, all strains used in the return-to-growth experiment were heteroallelic for mutations in the HIS4 gene. Although these diploid strains are initially His-, a recombination event between the two mutations can yield a wild-type HIS4+ gene. Thus, the production of prototrophs serves as an assay of recombination frequency. To determine whether topoisomerase II is required for commitment to meiotic levels of recombination, we compared commitment to recombination in TOP,!WTOP2+ and top2Wop2c~ strains at both permissive and restrictive temperatures. Cells were induced to begin sporulation, aliquots of the sporulating cultures were plated at intervals at the permissive temperature, and the frequency of histidine prototrophs was determined for each time point. The
Cdl 1012
top2/top2 + VI+ Q +
TOP2 +/Top2 topz/top2
f?ADSO/RADSO
&nucleate Tetranucleate Multinucleate
+
1011 0
2
Days
4
6
at Restrictive
8
10
Temperature
0 Days
Figure 4. Commitment to Meiotic Levels of Recombination in top2/top2 and TOP2+/TOf2+ Strains Strains CH1391 (TOP2+/TOP2+ his4-X/his4-8: -B) and CH1392 (top0 73/fop2-73 his4- X/hisCB:+) were induced to sporulate at the restrictive temperature. Aliquots of the sporulating cultures were removed at intervals and tested for the production of His+ recombinants. The number of histidine prototrophs per lo6 colony forming units (c.f.u) is plotted as a function of time.
kinetics and magnitude of commitment to meiotic levels of recombination were comparable in top2V~op2cS and Top2c/ToP2+ strains at the restrictive temperature (Figure 4). As shown in Figure 4, the t0p2~~lfop2c~ strain showed a slightly lower final recombination frequency than the TOP2+/ToP2+ control; however, a similar difference was seen at the permissive temperature (data not shown). Thus, topoisomerase II appears unnecessary for commitment to meiotic levels of recombination. DNA Topoisomerase II Is Requimd for the Segregation of Recombined Chromosomes While topoisomerase II is not required for commitment to meiotic levels of recombination, it may be required at some later stage of recombination or for the resolution of recombined chromosomes. To test this possibility, we compared meiosis in TOP2+/Top2c and top2Vtop2cS strains bearing or lacking a rad50 mutation, which blocks meiotic recombination (Game et al., 1980) but allows fairly normal progression of meiosis at the light microscope level (Farnet et al., 1988). Cells were induced to sporulate at either the permissive or restrictive temperature, and at intervals they were scored for nuclear morphology. top2V top2cS RADSO+/RAD50+ cells failed to undergo the first nuclear division, and most cells remained mononucleate (Figure 5, top). In contrast, top2cs/top2cs rad5Okad50 cells were initially mononucleate, but the frequency of binucleate cells increased on day 5 to ~10% (Figure 5, bottom). A similar level of binucleate cells was seen in TOP2+/7UP2+ md5Ohd50 strains (data not shown). Thus, in a fo@Vt0p2~~ rad5Ohd50 strain, progression through meiosis I is unaffected by the presence of a top2 mutation. After completing meiosis I, t0p2cV~p~~ rad5Ohd50 cells went on to express a unique phenotype. Instead of successfully completing meiosis II and producing tetranucleate cells, they gave rise to a novel class of cells that con-
10
5
at
Restrictive
top2hop2
0
rad50had50
5
Days
at
Temperature
Restrictive
10 Temperature
Figure 5. Frequencies of Nuclear Morphologies Observed in top2/ top2 RAD5OlRAD50 and top2ltop2 rad5Olrad50 Strains Undergoing Meiosis at the Restrictive Temperature Strains CH1212 (top2-Wtop2-13 RAD5OIRAD50: top) and CH1218 (top2-73/top2-73 rad5OAhd5OA: bottom) were induced to sporulate at the restrictive temperature. Aliquots of the sporulating cultures were removed at intervals, fixed, and stained with DAPI. The nuclear morphologies of the fixed samples were determined by fluorescence microscopy. The frequencies of binucleate, tetranucleate, and multinucleate cells are presented as a function of time. As indicated in Table 1, the meiotic phenotype of top2%op2cS strains was somewhat leaky; consistent with this obsetvation, a small number of binucleate and tetranucleate cells were formed even at the restrictive temperature. At the permissive temperature, top2/top2 RAD5O/RAD50 strains gave rise to ~50% tetranucleate cells, while top2kop2 red5Okad50 strains gave rise to -2O%-25% tetranucleate cells. At the permissive temperature, all strains produced
March
23, 1990, Copyright
0 1990 by Cell Press
Segregation of Recombined Chromosomes Meiosis I Requires DNA Topoisomerase II David Rose, Winston Thomas, and Connie Holm Department of Cellular and Developmental Biology Harvard University Cambridge, Massachusetts 02138
Summary To understand better the similarities and differences between meiosis and mitosis, we examined the meiotic role of DNA topoisomerase II, an enzyme that is required mltotlcally to disentangle sister chromatids at the time of chromosome segregation. In meiosis, we found that topolsomerase II is required only at the time of nuclear division. When cold-sensitive fop2 mutants are induced to sporulate at the restrictive temperature, they undergo premeiotlc DNA synthesis and commitment to meiotic levels of recombination but fail to complete the first meiotic nuclear division. The introduction of a mutation blocking recombination relieves the requirement for topoisomerare II in meiosis I. These results suggest that topoisomerase II is required at the time of chromosome segregation in meiosis I for the resolution of recombined chromosomes. Introduction The processes of both meiosis and mitosis require that chromosomes be replicated faithfully and segregated precisely in microtubule-mediated nuclear divisions. There are, however, important differences between the two processes. In contrast to the single mitotic nuclear division, meiosis comprises two chromosome segregation events. Premeiotic DNA synthesis also differs from premitotic DNA synthesis, as manifested in part by its extended duration (Williamson et al., 1983). In addition, meiotic and mitotic recombination require different gene products (for review, see Haynes and Kunz, 1981) and occur at different levels. Study of the effects of single mutations on both mitosis and meiosis is frequently informative. For example, mutations in the structural gene for 8-tubulin confer similar mitotic and meiotic phenotypes, blocking both mitosis and sporulation (Huffaker et al., 1988). Mutations in the RADSO gene, on the other hand, have strikingly different meiotic and mitotic phenotypes. tad50 mutants fail to undergo meiotic recombination (Game et al., 1980) but in mitosis are hyper-recombinant and deficient in DNA repair (Haynes and Kunz, 1981). These studies point out important similarities and differences between mitosis and meiosis and also provide a more complete picture of the function of the gene product examined. With these studies as a background, we compared the roles of DNA topoisomerase II in mitosis and meiosis. The role of DNA topoisomerase II in mitosis is well understood. DNA topoisomerase II acts by making a double-
in
strand break in a DNA molecule, passing another doublestranded segment through the break, and then resealing it (Brown and Cozzarelli, 1979; Liu et al., 1980). In this way, DNA topoisomerase II can disentangle intertwined DNA molecules and can relax both positively and negatively supercoiled DNA (Baldi et al., 1980; Hsieh, 1983; Hsieh and Brutlag, 1980; Kreuzer and Cozzarelli, 1980; Liu et al., 1981). In yeast, temperature-sensitive top2 mutants allowed to progress through mitosis at the restrictive temperature suffer a high level of inviability, which is accompanied by elevated levels of chromosome breakage and nondisjunction (Holm et al., 1985, 1989; Uemura and Yanagida, 1988; Uemura et al., 1987). Inviability can be prevented by treatment with the microtubule-destabilizing drug nocodazole (Holm et al., 1985) indicating that the loss in viability is specifically associated with cells attempting to segregate their chromosomes in the absence of topoisomerase II. These observations have led to the hypothesis that topoisomerase II facilitates chromosome segregation in mitosis by resolving entangled sister chromatids, which are intertwined to some extent following the completion of DNA synthesis (Sundin and Varshavsky, 1980, 1981; DiNardo et al., 1984; Weaver et al., 1985). Because topoisomerase II is required for the resolution of intertwined sister chromatids in mitosis, we anticipated that it might play an analogous role in meiosis. In particular, the second meiotic nuclear division is very similar to a mitotic nuclear division in that each involves the segregation of sister chromatids. Meiosis I, on the other hand, differs from both meiosis II and mitosis in that it involves the segregation of homologous chromosomes. Because homologous chromosomes are not replicative sisters, topoisomerase Ii may not be required for their separation. However, topoisomerase II may be required for other processes in meiosis I, such as premeiotic DNA synthesis, initiation of meiotic recombination, or the resolution of recombined DNA molecules. We report here that DNA topoisomerase II is required for the meiotic nuclear divisions. Cold-sensitive top2 mutants induced to sporulate at the restrictive temperature undergo premeiotic DNA synthesis and commitment to meiotic levels of recombination but arrest before the first meiotic nuclear division. Our results suggest that DNA topoisomerase II is required for the resolution of recombined chromosomes at the time of chromosome segregation in meiosis I. Results DNA Topoisomerase II Is Required for Sporulatlon We first determined that topoisomerase II is required for the completion of sporulation, which includes both the process of meiosis and the encapsulation of meiotic products in spore walls. TCP2+/7?F2+ and top2cs/top2cs strains were induced to sporulate at both the permissive temperature (25%) and the restrictive temperature (10%). Although TCR?+/Topr strains exhibited only a slight reduction in
Cell 1010
Table 1. Sporulation Frequencies TOP2+/7Of2+ Strains
of top2/top2
and
Sporulation
TOP2 +/TOP2 top2/top2
Days at Restrictive
Temperature
Frequency
Strain
25’C
10%
CH1131 (TOP2+/TOP2+) CH1123 (top2-13/top2-73) CH995 (top2-74/top2-14) CH1393 (top2-I 7/top2-7 7)a
0.55 0.53 0.56 0.41
0.40 0.03 0.03 0.16
a lop2-7 7 mutants have a leaky cold-sensitive cell cycle as well as in sporulation.
Q +
phenotype
-I
in the mitotic
Oi 0
final sporulation frequency at 10% fop2%op2cs mutants showed a dramatic reduction in sporulation at the restrictive temperature (Table 1). These results indicate that topoisomerase II is required for the completion of sporulation. DNA Topoisomerase II Is Required Specifically at the Time of the Meiotic Nuclear Divisions To determine the time in sporulation during which topoisomerase II is required, we performed two complementary temperature-shift experiments. In the first experiment, TOP2+ITOP2+ and fop2Wop2cs cells were induced to sporulate at the restrictive temperature. Aliquots of the sporulating cultures were shifted at intervals to the permissive temperature and allowed to complete sporulation, and the final sporulation frequencies of the shifted cells were then determined. Shifting to permissive temperature permits sporulation in fop2Vtop2cs cells, provided that the shift occurs before topoisomerase II is irreversibly required; sporulation does not occur in top2Vtop2”~ cells shifted to permissive temperature any later than the time at which the requirement for topoisomerase II begins. Thus, the point at which a shift to permissive temperature no longer ailows sporulation marks the beginning of the interval during which topoisomerase II is required in sporulation. As expected, in TOP2+/TO/W cells the final sporulation frequency was independent of the time of the shift, 40% in each case (Figure 1). In contrast, in top2cs/fop2cs cells wild-type levels of sporulation were attained only if the cells were shifted on or before day 6; in the interval between day 6 and day 6, final sporulation frequencies dropped rapidly. This result indicates that cells enter the interval during which topoisomerase II is required between day 6 and day 6. To determine the stages in meiosis represented by days 6, 7, and 6, cells fixed at the time of the shift were stained with the DNA stain DAPI and examined by fluorescence microscopy (Figure 2). TOP2+/TOP2+ cells were mononucleate on day 6. On days 7 and 6, a progressively greater number of cells had undergone both nuclear divisions. Thus, the beginning of the requirement for topoisomerase II in meiosis corresponds to the beginning of the meiotic nuclear divisions in KR!?VTOP2+ cells. Consistent with this observation, top2cs/top2cs cells were indistinguishable from TOP2+/72%?+ cells on day 6 but failed to undergo nuclear divisions on days 7 and 6. Instead, the cells
10
Figure 1. Sporulation Frequencies to Permissive Temperature
20
of Cultures
Shifted from Restrictive
Strains CH1123 (top2-73/top2-73:+) and CH1131 (TOfzI/TOP2+:i+) were induced to sporulate at the restrictive temperature, and aliquots were shifted at intervals to the permissive temperature and allowed lo complete sporulation. Final sporulation frequencies of the shifted aliquots are plotted as a function of the time at which they were shifted from restrictive temperature lo permissive temperature.
remained mononucleate, and the nuclei adopted an abnormal morphology. The failure of fop2cs/top2cs cells to undergo the first nuclear division at the restrictive temperature indicates that topoisomerase II is required for meiosis I. The complementary temperature-shift experiment defined the time at which the requirement for topoisomerase II ends. In this experiment, TOR?WOf2+ and top2cWop2cs cells were induced to sporulate at the permissive temperature. Aliquots of the sporulating cultures were shifted at intervals to the restrictive temperature and allowed to complete sporulation, and the final sporulation frequencies of the shifted aliquots were then determined. In top2Wop2cs cells, sporulation is disrupted by a shift to restrictive temperature if the shift occurs before the end of the period during which topoisomerase II is required. Cells shifted later than this time are not affected by the shift and sporulate at wild-type levels. The point at which a shift to restrictive temperature no longer disrupts sporulation marks the end of the interval during which topoisomerase II is required. In TOP2WOf2+ cells, the final sporulation frequency was independent of the time of the shift, as expected (Figure 3). In contrast, top2Vtop2cs cells shifted to restrictive temperature at or before 12 hr sporulated at very low levels; cells shifted after 12 hr showed a progressive increase in sporulation frequency, reaching wild-type levels by 30 hr. This result indicates that cells complete the interval during which topoisomerase II is required between 12 and 30 hr. To determine the stage in meiosis represented by the interval from 12 to 30 hr, cells fixed at the time of each shift were stained with the DNA stain DAPI and examined using fluorescence microscopy (data not shown). At 12 hr, both TOP2+/7UP2+ and top2Vtop2c~ cultures contained almost exclusively mononucleate cells. In the interval from 12 to 30 hr, however, a progressively greater number of cells had undergone both nuclear divi-
The Meiotic 1011
Figure
Role of DNA Topoisomerase
2. Morphology
of top2/top2
II
and TOFV+/TOP2+
Cells Undergoing
Meiosis
at the Restrictive
Temperature
In the experiment described in Figure 1, strains CH1131 (7Cf2+/7Df~: A, B, and C) and CH1123 (top2-13/top2-13: D, E, and F) were induced to sporulate at the restrictive temperature. Samples were fixed and stained with DAPI after 6 days (A and D), 7 days (6 and E), and 6 days (C and F) at the restrictive temperature. The cell in (B) with the X-shaped nuclear morphology has completed meiosis I and is in the middle of meiosis II, The cells in (C) are tetranucleate, but not all nuclei are in the focal plane. The bar represents 5 Wm.
sions and were tetranucleate. This observation demonstrates that the end of the interval during which topoisomerase II is required corresponds to the completion of the meiotic nuclear divisions. DNA Topoisomerase II Is Not Required for Early Events in Meiosis I To define the role of topoisomerase II in meiosis I more precisely, we examined the effect of cold-sensitive top2
”
-D +
1
01
' 0
I IO
Hours
' at
I 20
'
Permissive
Figure 3. Sporulation Frequencies save to Restrictive Temperature
I 30
TOP2 +/TOP2 topz/topz
-
I 40
+
'
I 50
Temperature
of Cultures
Shifted
from
Permis-
Strains CH1123 (top2-13/top2-734 and CH1131 (TOrWTOP2+:& were induced to sporulate at the permissive temperature, and aliquots were shifted at intervals to the restrictive temperature and allowed to complete sporulation at the restrictive temperature. Final sporulation frequencies of the shifted aliquots are plotted as a function of the time at which the culture was shifted from permissive to restrictive temperature.
mutations on two early meiotic events, premeiotic DNA synthesis and commitment to meiotic levels of recombination. To determine whether cold-sensitive fop2 mutations grossly affect the kinetics of premeiotic DNA synthesis, strains were induced to TOP2VTopzc and top2%op2cs undergo meiosis at both restrictive and permissive temperatures. Aliquots of the sporulating cultures were removed at intervals, stained with propidium iodide, and analyzed by flow cytometry. The kinetics of DNA synthesis, as revealed by this analysis, are not grossly affected by cold-sensitive top2 mutations (data not shown). Another early meiotic process in which topoisomerase II might play a role is meiotic recombination. Since lop2V top2cs cells failed to complete sporulation, we were unable to measure recombination by looking at the genotype of meiotic products. However, a return-to-growth protocol can be employed to examine commitment to meiotic levels of recombination in strains unable to complete meiosis (Sherman and Roman, 1963). To assay recombination, all strains used in the return-to-growth experiment were heteroallelic for mutations in the HIS4 gene. Although these diploid strains are initially His-, a recombination event between the two mutations can yield a wild-type HIS4+ gene. Thus, the production of prototrophs serves as an assay of recombination frequency. To determine whether topoisomerase II is required for commitment to meiotic levels of recombination, we compared commitment to recombination in TOP,!WTOP2+ and top2Wop2c~ strains at both permissive and restrictive temperatures. Cells were induced to begin sporulation, aliquots of the sporulating cultures were plated at intervals at the permissive temperature, and the frequency of histidine prototrophs was determined for each time point. The
Cdl 1012
top2/top2 + VI+ Q +
TOP2 +/Top2 topz/top2
f?ADSO/RADSO
&nucleate Tetranucleate Multinucleate
+
1011 0
2
Days
4
6
at Restrictive
8
10
Temperature
0 Days
Figure 4. Commitment to Meiotic Levels of Recombination in top2/top2 and TOP2+/TOf2+ Strains Strains CH1391 (TOP2+/TOP2+ his4-X/his4-8: -B) and CH1392 (top0 73/fop2-73 his4- X/hisCB:+) were induced to sporulate at the restrictive temperature. Aliquots of the sporulating cultures were removed at intervals and tested for the production of His+ recombinants. The number of histidine prototrophs per lo6 colony forming units (c.f.u) is plotted as a function of time.
kinetics and magnitude of commitment to meiotic levels of recombination were comparable in top2V~op2cS and Top2c/ToP2+ strains at the restrictive temperature (Figure 4). As shown in Figure 4, the t0p2~~lfop2c~ strain showed a slightly lower final recombination frequency than the TOP2+/ToP2+ control; however, a similar difference was seen at the permissive temperature (data not shown). Thus, topoisomerase II appears unnecessary for commitment to meiotic levels of recombination. DNA Topoisomerase II Is Requimd for the Segregation of Recombined Chromosomes While topoisomerase II is not required for commitment to meiotic levels of recombination, it may be required at some later stage of recombination or for the resolution of recombined chromosomes. To test this possibility, we compared meiosis in TOP2+/Top2c and top2Vtop2cS strains bearing or lacking a rad50 mutation, which blocks meiotic recombination (Game et al., 1980) but allows fairly normal progression of meiosis at the light microscope level (Farnet et al., 1988). Cells were induced to sporulate at either the permissive or restrictive temperature, and at intervals they were scored for nuclear morphology. top2V top2cS RADSO+/RAD50+ cells failed to undergo the first nuclear division, and most cells remained mononucleate (Figure 5, top). In contrast, top2cs/top2cs rad5Okad50 cells were initially mononucleate, but the frequency of binucleate cells increased on day 5 to ~10% (Figure 5, bottom). A similar level of binucleate cells was seen in TOP2+/7UP2+ md5Ohd50 strains (data not shown). Thus, in a fo@Vt0p2~~ rad5Ohd50 strain, progression through meiosis I is unaffected by the presence of a top2 mutation. After completing meiosis I, t0p2cV~p~~ rad5Ohd50 cells went on to express a unique phenotype. Instead of successfully completing meiosis II and producing tetranucleate cells, they gave rise to a novel class of cells that con-
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Figure 5. Frequencies of Nuclear Morphologies Observed in top2/ top2 RAD5OlRAD50 and top2ltop2 rad5Olrad50 Strains Undergoing Meiosis at the Restrictive Temperature Strains CH1212 (top2-Wtop2-13 RAD5OIRAD50: top) and CH1218 (top2-73/top2-73 rad5OAhd5OA: bottom) were induced to sporulate at the restrictive temperature. Aliquots of the sporulating cultures were removed at intervals, fixed, and stained with DAPI. The nuclear morphologies of the fixed samples were determined by fluorescence microscopy. The frequencies of binucleate, tetranucleate, and multinucleate cells are presented as a function of time. As indicated in Table 1, the meiotic phenotype of top2%op2cS strains was somewhat leaky; consistent with this obsetvation, a small number of binucleate and tetranucleate cells were formed even at the restrictive temperature. At the permissive temperature, top2/top2 RAD5O/RAD50 strains gave rise to ~50% tetranucleate cells, while top2kop2 red5Okad50 strains gave rise to -2O%-25% tetranucleate cells. At the permissive temperature, all strains produced
