during the early part of this work, Joe Wagstaff and Tom. Petes for ... R. Roth. 1980. The role of radiation(rad) genes in meiotic recombination in yeast. Genetics ...
Vol. 1, No. 10
MOLECULAR AND CELLULAR BIOLOGY, OCt. 1981, p. 891-901 0270-7306/81/100891-11$02.00/0
Recombinationless Meiosis in Saccharomyces cerevisiae ROBERT E. MALONEI2* AND R. EASTON ESPOSITO2 Department ofMicrobiology, Loyola University Medical Center, Maywood, Illinois 60153'; and Department ofBiology, University of Chicago, Chicago, Illinois 606372
Received 9 February 1981/Accepted 8 July 1981
We have utilized the single equational meiotic division conferred by the spol31 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980; Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spol31 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.
One approach to the study of genetic recombination and its role in meiosis that has been used successfully in Drosophila and in a number of higher plants has been the analysis of mutations that alter the properties of recombination (1). Data from a number of eucaryotic organisms indicate that genetic exchange during meiosis is required for proper chromosome pairing and disjunction in meiosis I. Mutations that reduce the frequency of meiotic recombination generally result in high levels of nondisjunction leading to aneuploid meiotic products. The yeast Saccharomyces cerevisiae is an excellent organism in which to study genetic recombination and meiosis, in part because it provides the opportunity to correlate genetic, cytological, and biochemical observations. It has until recently proven difficult to identify and study recombination-deficient (Rec-) mutants in yeasts. S. cerevisiae has at least 17 chromosomes (25); thus, in the absence of proper pairing and disjunction in meiosis I, the probability of a meiotic product (ascospore) receiving at least one of every chromosome is extremely low. Mutations creating deficiencies in meiotic recombination would therefore be expected to lead almost exclusively to the formation of inviable ascospores.
The primary experimental method used to define meiotic Rec- mutations in yeast takes advantage of the observation by Sherman and Roman (24) that yeast cells briefly exposed to medium that induces meiosis and sporulation can be stimulated to undergo high levels of intragenic recombination and yet return to mitotic growth. This approach was used to demonstrate that cells can be induced for meiotic levels of both gene conversion and crossing-over in the absence of commitment to a meiosis I reductional division (8, 9). It was concluded that although recombination is necessary for a proper meiosis I, meiotic levels of recombination are not sufficient for, and do not have to be followed by, a reductional division. A number of candidates for meiotic Rec- mutations have been analyzed in this way, including sporulation-defective (spo) mutations, cell division cycle (cdc) mutations, and radiation-sensitive (rad) mutations that also affect mitotic recombination or ascospore viability (1, 8, 13, 16, 21, 23). Game et al. (13) and Prakash et al. (21) have recently found that strains containing either the rad6-1, rad50-1, rad52-1, or rad57-1 mutation exhibit no or little increase in the frequency of recombination at several loci measured after an expo891
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MOL. CELL. BIOL.
MALONE AND ESPOSITO
sure to sporulation medium. It was concluded on this basis that all of these Rad- mutations are also meiotic Rec- mutations. There are several potential problems with the use of the return to growth protocol to define a meiotic Rec- mutation. Perhaps the most serious is the possibility that a mutation resulting in a meiotic block after recombination might prevent cells from returning to mitotic growth. Such a mutation would appear to be a meiotic Rec- as well as exhibiting a loss of colony-forming ability, although it did not inactivate a function involved in recombination. In fact, substantial losses in viability after exposure to sporulation medium were observed for all of the rad mutations except rad6-1 (13, 21). Hence, although the behavior of these Rad- mutants is consistent with the notion that they are defective in meiotic recombination, altemative interpretations of the results are still possible. We have attempted to bypass the requirement for recombination for successful completion of meiosis by the use of a mutation (spol3-1) which eliminates the meiosis I reductional division. This potentially can allow us to directly examine the effect of rad6-1, rad50-1, and rad52-1 on meiotic recombination in meiotic products (i.e., ascospores). The spo13-1 mutation has been demonstrated to confer the following (Spo-) phenotype (17): (i) early events in meiosis I (i.e., deoxyribonucleic acid synthesis and genetic recombination) occur normally, but no reductional segregation occurs; (ii) these events are usually followed by a single equational meiosis II division; and (iii) the asci formed contain two diploid or near-diploid spores. Since there should, a priori, be no requirement for pairing of homologs for an equational division, we hoped to obtain viable meiotic products even in the absence of recombination. Our approach has been to con-
RM56 RM57
RM58 RM59
RM60 RM64 RM69
(5) for general genetic nomenclature as well as those of Plischke et al. (20) for yeasts are used. Phenotypes are designated by having their first letter capitalized and are not italicized. For example, the wild-type RAD50 gene confers a Rad+ phenotype, whereas the recessive mutant allele rad50-1 confers a Rad- phenotype. Yeast strains. The relevant genotypes of the strains used are described in Table 1. The strains carrying the rad6-1 rad50-1, and rad52-1 mutations were obtained from L. Prakash (University of Rochester). To develop relatively isogenic strains and to construct the combinations of markers necessary to monitor recombination, all strains containing rad mutations were backcrossed at least three times with Rad+ laboratory stocks selected for high sporulation backgrounds. The strain carrying the spol3-1 mutation was obtained in a similar genetic background from S. Klapholz (University of Chicago). Most experiments described were carried out in two different diploid strains to minimize effects due to strain differences. The linkage relationships (20) of the genetic markers used are as shown in Fig. 1. Media and techniques. YPD medium consists of 20 g of glucose, 20 g of peptone, and 10 g of yeast extract per liter. YPA is YPD with 20 g of potassium acetate substituted for glucose. The recipes for all other media have been described previously (14). MMS plates are YPD plates containing 0.01% MMS (Eastman Kodak Co.). Standard techniques were used
TABLE 1. Genotypes of strains Genotype
Diploid RM15
struct double mutants of the rad mutants and spo13-1, to determine whether such double mutants generate viable spores after meiosis, and to analyze recombination in the spores. The feasibility of this technique has been independently demonstrated by the analysis of another putative meiotic Rec- mutant (spoll-i) which produced viable ascospores without recombination in the presence of spol3-1 (S. Klapholtz, Ph.D. thesis, University of Chicago, 1980). MATERLALS AND METHODS Nomenclature. The suggestions of Demerec et al.
met13-c trp5-c leul-c ade6 a rad50-1 ade2-1 lys2-1 tyrl-2 his7-1 CAN)' ura3-1 hisl-315 + metl3-c cyn2r trp5-c leul-c + ade5 metl3-d CYH2' trp5-d leul-d a rad50-l ade2-1 lys2-1 tyrl-l his7-2 can)' ura3-313 + a rad50-1 ade2-1 lys2-1 tyrl-2 his7-1 CAN)' ura3-1 cyh2' trp5-c leul-c a rad5O-) ade2-1 Iys2-1 tyr)-) his7-2 can)' ura3-313 ade5 CYH2 trp5-d leui-d trp5-d leul-d + CYH2a ura3-313 ade5 ade2-1 lys2-) tyr)-) his7-2 can)' r-
a
a
ade2-1 lys2-2 tyrl-2 his7-1 CANI' ura3-1
+
cyh2'
CAN)' ura3-1
+
cyh2' met13-c trp5-d leul-c
ade2-1 lys2-1 tyrl-l his7-2 canl' ura3-313 ade5 CYH2' metl3-d trp5-d leul-1 +
a
a a a a a a a
ade2-1 lys2-2 tyrl-l
+
rad5O-l spol3-1 ade2-1 lys2-1 tyrl-l his7-2 canl' ura3-313 ade5 metl3-d CYH2 trp5-d leul-d + cyh2' trp5-c leul-c rad50-1 spol3-1 ade2-1 lys2-2 tyrl-l his7-1 CAN)' ura3-1 + + CYH2' ade5 ura3-313 his7-2 tyrl-l ade2-1 lys2-1 spol3-1 rad5O-l canl' trp5-d leul-d rad5O-a spol3-1 ade2-1 lys2-2 tyrl-l + CAN)' ura3-1 + metl3-c cyh2' trp5-c leul-c spol3-1 ade2-1 lys2-2 tyrl-l his7-1 CAN)' ura3-1 + metl3-c cyh2' trp5-c kul-c spol3-1 ade2-1 Iys2-1 tyrl-l his7-2 canl' ura3-313 ade5 metl3-d CYH2' trp5-d leul-d a spol3-1 ade2-1 lys2-2 tyrl-l his7-1 CAN)' ura3-1 ade5 metl3-c cyh2' trp5-c kul-c a spol3-1 ade2-1 lys2-1 tyrl-l his7-2 can)' ura3-313 ade5 met13-d CYH2' trp5-d leul-d
VOL. 1, 1981
rad50-1 AND RECOMBINATION II
dtyrl
lys2 80
rad5O
25
V canl
ura3
40
50 VII
ade5
XIII
cyh2
metl3
87
MAT
50
52
IIV
III
his7
23
893
5
o
rad6 35
VIII leul
trp5
11
spol3 10
17
3
rad52 20 FIG. 1. Linkage relationships ofgenetic markers used.
for sporulation, dissection, testing of auxotrophic requirements, and prototroph selection of diploids (14). Determination of mitotic recombination frequencies. All diploids were freshly constructed before each experiment. Single colonies were picked into 1 ml of water, counted, and inoculated into YPD culture medium at a concentration of 20 cells per ml. The culture was grown at 300C to a cell density of approximately 2 x 107 cells per ml. Cells were then centrifuged, washed twice in 0.1 M tris(hydroxymethyl)aminomethane-0.1 M ethylenediaminetetraacetic acid (pH 6.1) and twice in water, sonicated briefly, and plated at various dilutions on complete medium, media lacking various auxotrophic requirements, or media, containing the drugs cycloheximide or canavanine. Plates were scored after 3 days of incubation at 300C. Determination of meiotic intragenic recombination frequencies. YPD cultures were inoculated as above and were grown to an approximate concentration of 2 x 106 cells per ml. The cultures were then divided into two parts: one part was washed and resuspended in YPA, and the other was left in YPD. At 2 x 107 cells per ml, the YPD culture and part of the YPA culture were plated as above to determine frequencies of mitotic recombination. The remaining YPA culture was washed and suspended in sporulation medium (SPIII-21) to induce meiosis and sporulation. The sporulation culture was vigorously aerated at 300C for 48 h, at which time the culture was plated to determine the frequencies of meiotic gene conversion. (No significant differences were observed between the YPD and YPA cultures for mitotic recombination. The YPA values are used in Tables 5 and 9.) Determination of mitotic mutation frequencies. The strains used to measure mutation frequencies were homozygous homothallic diploids. Cultures were inoculated, grown, and analyzed as described above. Prototrophic revertants were detected after 3 to 4 days of growth at 300C, as were forward mutations to Canr (canavanine resistance). A second class of Lys' colonies appeared on the selective plates after 8 to 10 days of incubation. These were easily distinguished from true Lys' revertants by their smaller size, and proved to contain ochre suppressors of lys2-1 or Iys22.
RESULTS
Does the spo13-1 mutation bypass the meiotic defects of rad6-1, rad50-1, and
rad52-1? The data in Table 2 demonstrate that a number of independent diploids homozygous for rad6-1, rad50-1, or rad52-1 did not allow the fornation of viable meiotic products. Whereas the rad6-1 mutation allowed essentially no sporulation whatsoever, the rad50-1 and rad52-1 mutations allowed some sporulation, but generated inviable spores. These results confirn the previously reported phenotypes of the rad mutants (4, 12). To determine whether the spol3-1 mutation circumvented these meiotic defects, we constructed double mutants. The rad50-1/ rad50-1 spol3-1/spol3-1 diploids were capable of sporulating and produced highly viable spores (Table 2). The sporulation defects of the rad6-1 and rad52-1 mutations were not overcome by spol3-1. Hence, only the rad50-1 mutation was examined in subsequent experiments. Determination of the effect of the rad5O1 mutation on spontaneous mitotic recombination. Hunnable and Cox (15) previously reported that ultraviolet-induced mitotic gene conversion was not reduced by rad50-1, whereas Game et al. (13) concluded that spontaneous mitotic gene conversion at the hisl locus was normal. To determine whether spontaneous mitotic recombination was generally unaffected by the presence of the rad50-1 mutation and to establish the basal level of recombination occurring in cultures before meiosis, we measured the spontaneous frequencies of intragenic recombination (i.e., gene conversion) and intergenic recombination (i.e., crossing-over) at several loci on three chromosomes. The former was monitored by the production of prototrophs at heteroallelic loci, and the latter was monitored by homozygosis of recessive drug resistance markers due to crossing-over between the locus and its centromere (Table 3). In contrast to our initial expectation, the data suggested that the rad50-1 mutation generally increased spontaneous mitotic recombination. To determine whether the Canr and Cyhr colonies obtained in this experiment arose from
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MOL. CELL. BIOL.
MALONE AND ESPOSITO
mitotic crossmg-over or gene conversion, we examined their genotypes in more detail. If Canr
and Cyhr colonies are produced by crossing-over, then other markers on the same chromosome arm should be homozygous since multiple exchanges are infrequent in mitosis. If they are TABLE 2. Sporulation and spore viability of Rad and Rad+ diploidsa No. of inSpores depend- Sporulation % No. Diploid genotype ent dip(5 asci) Viaexamloids exble ined amined
Spo+ diploids rad6-1 rad6-1 rad50-1 rad50-1 rad52-1 rad52-1 RAD RAD
Spo- diploids rad6-1 spol3-1 rad6-1 spol3-1 rad50-1 spol3-1 rad50-1 spol3-1 rad52-1 spol3-1 rad52-1 spol3-1
4
0.23 ± 0.31
0
10
4
9.7± 2.8
0
48
4
12± 5.4
0
48
4
82± 7.1
91
100
2
0.20 ± 0.05
0
4
2
52 ± 2
70
220
2
8.7 ± 1.5
0
160
2 52 ± 13 52 346 RADspol3-1 RAD spol3-1 a Sporulation values (number of aaci divided by total number of cells plus buds) and spore viability were determined from at least two clones of each independent diploid. Approximately 200 cells were examined to determine the percent asci; Spo+ strains produce predominantly tetrads, whereas Spo- strains produce predominantly dyads. Diploids described in this table were constructed from intercrosses between segregants of radl RAD diploids in either Spo+ (Table 2A) or Spo- (Table 2B) genetic backgrounds.
produced by gene conversion, then other markers will remain heterozygous unless the gene conversion event is accompanied by an associated crossover. Approximately 50% of the Cyhr colonies were homozygous for one or both of the centromere proximal loci leul and trp5; greater than 90% of the Cyhr colonies were homozygous for the distal ade5 locus. Although only the proximal heteroallelic ura3 locus was present on chromosome V, approximately 20% of the Canr colonies were homozygous for ura3. The genotypes of Canr and Cyhr colonies were generally similar for both the wild type and the Rad+ diploid. We conclude that the Canr and Cyhr colonies reflect crossing-over and that spontaneous mitotic crossing-over is elevated by the presence of rad50-1. To determine whether mutation contributed to the elevated prototroph frequencies observed in the presence of rad50-1, the reversion frequency of several of the alleles used to monitor mitotic gene conversion was measured (Table 4). In the presence of rad50-1 none of the heteroalleles used to measure recombination at leul, ura3, or Iys2 exhibited mutation frequencies sufficiently high to explain the observed recombination frequencies. Although the reversion frequencies in the Rad- diploids were slightly elevated over wild-type levels, they were still several orders of magnitude below the observed recombination frequencies. The observed increases in prototroph frequencies in the presence of rad50-1 must therefore be due to elevated gene conversion. Since the Iys2-1 and lys22 alleles are suppressible, it was also possible to measure forward mutation to ochre suppressors; the data in Table 4 indicate that rad50-1 did not increase the frequency of forward mutations. We also observed the appearance of Canr colonies
TABLE 3. Effect of rad50-1 on spontaneous mitotic recombination Mean frequency of recombinantsa Diploid genotype
Drug-resistant colo-
Prototrophs x 104CFUb
No. of culleul-c leul-12
trp5-c trp5-d 3.5
met13-cc metl3-d
ura3-1 ura3-313
his7-1 his7-2
canl' CANiP
cyh2' CYH2'
3.5 12.2 6 1.3 5.8 0.41 0.26 rad50-1 rad50-1 1.2 0.042 1.3 0.088 6 0.86 0.65 0.38 RAD50 RAD50 2.9 6.1 9.8 15 4.6 1.5 5.4 Relative increased a Values shown are the geometric means of frequencies determined for all cultures. Three cultures of two independent diploids were examined. Prototroph frequency is a monitor of intragenic recombination, and the frequency of drug-resistant colonies reflects intergenic mitotic recombination. The rad5O-1/rad5O-1 diploids were RM56 and RM57; the RAD50/RAD50 diploids were RM15 and RM58. b CFU, Colony-forming units. 'The Rad- and Rad+ means for the metl3 locus are taken from only three cultures. d Represents ratio of rad5O-1/rad5O-1 to RAD50/RAD50.
895
rad50-1 AND RECOMBINATION
VOL. 1, 1981
effect in preventing recombination after expoto sporulation medium (13), to verify its phenotype in our strains, and to provide a control for the double-mutant studies with spol3-1. The same diploids used in the mitotic recombination studies were exposed to sporulation medium for 48 h. Cells were then plated on appropriate selective media and examined for their frequency of gene conversion and crossing-over (Table 5). The Rad+ cells showed typical meiotic increases in recombination. In contrast, the Rad- cultures displayed almost no induction of meiotic recombination. These results are in agreement with those previously reported (13). As expected, no Rad- asci were viable; the surviving vegetative cells exhibited negligible increases in recombination over the mitotic background (Table 6). The Rad+ vegetative cells, however, showed the large increases expected for the induction of meiotic recombination. The
from diploids which were homozygous CAN1/ CAN18 in both Rad+ and Rad- cells. Since the resistant allele is recessive, these events may be due to mutation associated with crossing-over or with chromosome loss. We conclude that the rad50-1 mutation leads to increased general spontaneous mitotic crossing-over and gene conversion. If the rad50-1 mutation results in the loss of a functional gene product, then that product cannot be essential for spontaneous mitotic recombination. However, since the mutation leads to increased amounts of exchange, the wild-type RAD50 product, although not required for mitotic recombination, may participate in some aspect of the exchange process. Examination of rad50-1 cells after exposure to sporulation medium. Recombination was examined at several loci on three chromosomes to determine the generality of the rad5O-
1
sure
TABLE 4. Effect of rad50-1 on mutation in homozygous diploidsa
Diploid genotype
ura3-1
Mutation frequency x 107 leul-12 lys2-2 lys2-1
kul-c
ura3-313
sup
lys2-2
sup"
lys2-1
CANI'
1.2 19 8.2 0.63 1.1 0.71 0.87 0.71 0.70 rad50-1 rad50-1 19 1.1 0.20 8.9 0.43 0.96 0.18 0.16 0.43 RAD50 RAD50 a The reversion frequencies of several alleles used in testing recombination were analyzed in homoallelic Rad+ and Rad- diploids. Values shown are geometric means of three cultures of each diploid. Colonies capable of growth on canavanine presumably represent forward mutation to canavanine resistance associated with chromosome loss or crossing-over since canavanine resistance is recessive to sensitivity. b It was possible to distinguish LYS' revertants from forward mutations to transfer ribonucleic acid suppressors (see text).
TABLE 5. Recombination frequencies of Rad- diploids after exposure to sporulation mediuma Culture
rad5o-1 Mitotic Meiotic Relative increase"
X Resistant colonieF 10U Intragenic recombinants x 10' CFU CFU__ cyh2 canl his7 ura3 metl3 leul trp5 _
1.0 2.4 2.4
_
_
__
4.3 5.8 1.3
_
_
_
_
2.5 5.4 2.2
_
_
_
__
0.21 0.96 4.6
_
_
_
_
0.16 0.67 4.2
_
_
__
_
5.3 18 3.4
_
_
_
_
_
_
1.7 1.8 1.1
RAD50 1.2 0.78 0.10 0.89 0.78 Mitotic 730 680 6.5 230 90 Meiotic 1,068 567 65 258 115 Relative increase' a Rad- and Rad+ diploids were analyzed for intragenic and intergenic recombination at the loci shown. The heteroalleles used are described in Table 2. A single culture was divided into two portions; one was plated immediately for mitotic recombination frequencies, and the other was sporulated and plated after 48 h in sporulation medium. The frequency of drug-resistant colonies per colony-forming unit (CFU) is a monitor of intergenic recombination in mitotic cells; in meiotic cultures it represents a combination of intergenic recombination occurring in surviving diploid cells and segregation of chromosomes in haploids. The Rad+ diploid sporulated 61%, whereas the Rad- diploid sporulated 4%. The Rad- diploid was RM56, and the Rad+ diploid was RM58. b Represents ratio of meiotic culture to mitotic culture.
896
MALONE AND ESPOSITO
MOL. CELL. BIOL.
increase in prototrophs in the vegetative celLs suggests that, after long exposures to sporulation medium, even cells which fail to complete spor-
ulation have been induced for meiotic recombination (Table 6). Mitotic and meiotic recombination in Rad- Spo- diploids. To examine the effect of rad50-1 on meiotic recombination in ascospores, we examined the genotypes of the spores produced in rad50-1/rad50-1 spol3-1/spol3-1 diploids. To establish the amount of recombination occurring before meiosis, the level of mitotic recombination in Rad- Spo- diploids was determined. Table 6 indicates that mitotic gene conversion and crossing-over in a spo13-1 background were generally elevated by rad50-1, just as they were in the wild-type background. This provides additional support for the conclusion
that rad50-1 elevates mitotic recombination. The spol3-1 mutation itself did not substantially affect mitotic recombination (compare Tables 3 and 7). Meiotic crossing-over was examined in RadSpo- and Rad+ Spo- diploids by dissecting and analyzing dyad (two-spored) asci. Klapholz and Esposito (17) have demonstrated that spol3-1/ spol3-1 diploids undergo normal meiotic levels of recombination followed primarily by an equational division of centromeres. Aberrant segregation of chromosomes resulting in monosonic and trisomic spore pairs was also observed. The expected dyad phenotypes for an equational segregation with and without exchange, as well as one type of aberrant segregation, are shown in Fig. 2. The data in Table 8 clearly indicate that the rad50-1 mutation essentially abolished
TABLE 6. Properties of Rad- and Rad' vegetative cells and asci after 48 h in sporulation mediuma Cell type
rad5o-1 rad50-1 Vegetative Asci
Fraction of
0.96 0.04
% Recombinant (no.) at:
No. examined
99 50
No. viable
32
canl
cyh2
6 (2)
9 (3)
leul
0
ob
RAD50 RAD50 0.39 80 77 19 (15) 17 (13) 20 (16)c Vegetative 0.61 50 41 (20) 45 (22) 24 (12)" Asci 49b The sporulation cultures described in Table 4 spread on YPD plates, and vegetative cells or asci were isolated by micromanipulation and allowed to grow into colonies. Viable colonies were analyzed for recombination or segregation events (or both) at canl, cyh2, or leul. The diploids used were the ones in Table 5. b For an ascus to generate a viable colony, only one of the four spores need be viable. c The reason for the high frequency of leul recombinants in vegetative cells of the Rad+ strain is that homoallelic Leu- recombinants are caused by crossing-over between leul and the centromere. In a normal plating experiment (Table 4) only Leu' recombinants caused by gene conversion are detectable. a
TABLE 7. Spontaneous mitotic recombination in rad50-1 spo13-1 and RAD50 spol3-1 diploidsa Mean frequency of recombinantsb Diploid genotype
tures
Prototrophs x 104 CFU
Resistant colonies x
103
leul ura3 canl trp5 lys2 cyh2 3 3.5 rad50-1 spol3-1 3.9 0.61 1.0 4.1 33 rad50-1 spol3-1 3 0.8 1.3 0.044 2.4 0.76 0.088 RAD50 spol3-1 RAD50 spol3-1 Relative increase' 2.9 3.0 14 5.4 6.0 23 a Values shown are the geometric means of colony forming units (CFU) from three cultures for each genotype. The rad5O-1/rad5O-1 spol3-1/spol3-1 diploid was RM59, and the RAD50/RAD50 spol3-1/spol3-1 diploid was RM64. b The heteroalleles and drug resistance markers used to determine intragenic (prototrophic) and intergenic (drug-resistant colonies) recombination were the same as those listed in Table 3. The alleles used for the Iys2 locus were Iys2-1 and lys2-2. 'Represents ratio of rad50-1/rad50-1 spol3-1/spol3-1 to RAD50/RAD50 spol3-1/spol3-1.
rad50-1 AND RECOMBINATION
VOL. 1, 1981 MEIOTIC SEGREGATION A. EQUATIONAL: NO EXCHANGE I-c
. I-c
897
DYAD GENOTYPE SPORE B PHENOTYPE SPORE A
r r
I-d
S
I-d
S
1,3 a 2,4
IrI-d-c
r
I-c
r
S
1-d
S
1-c
r
1-d
S
~
r
I-
1,4 a 2,3
B. EQUATIONAL: EXCHANGE
I-c
r
15 a 2,4 1-dS ~
M
1-d
S
~
1I,4a&2,3
1-c
r
I-d
S
a
I-c I-d
I-c
r
I-c
r
a
1d -
5
1-d
S
r
S
C. ONE TYPE OF ABERRANT
SEGREGATION I-c r_
Ii-iLiI rij S 1-dIY
I a 2,3,4
I-d
A
FIG. 2. Descriptions of genotypes of spores obtained in dyads after (A) equational segregation, (B) equational segregation with a crossover between the centromere and the gene locus, and (C) one type of aberrant segregation. In (C), one homolog fails to divide and segregates as a unit to one pole, giving rise to one 2n 1 and one 2n + 1 spore (cf. reference 17). The symbols P, R, and A refer to dyad phenotypes: P, parental dyad; R, recombinant dyad; and A, aberrant dyad. The notations 1-c and 1-d refer to different auxotrophic alleles of the same gene (e.g., leul-c and leul-d). The notations r and S refer to drug-resistant and drug-sensitive alleles respectively, of the same gene (e.g., cyh2r and CYH2r). Recombination between the gene locus and the centromere gives rise to dyads with spores which are homoallelic for each auxotrophic allele and homozygous for the drug-resistant and drug-sensitive alleles.
meiotic crossing-over; only one dyad diagnostic of recombination was observed. By extrapolation from the Rad+ Spo- data, 154 recombination-type dyads were expected among the RadSpo- dyads. Thus, crossing-over was reduced at least 150-fold by rad50-1. The data in Table 8 also indicate that the rad50-1 mutation reduces the frequency of aberrant dyads (see below). Does rad50-1 also reduce meiotic gene conversion in a spol3-1 background? The data in Table 9 indicate that RAD50 is, indeed, required for meiotic gene conversion. The gene conversion frequencies observed in the control Rad+ Spo- diploid were quite similar to those observed in normal Rad+ Spo+ strains (compare Tables 5 and 9). Whereas the Spo- diploid exhibited a 25- to 100-fold increase over the miotic background after 48 h in sporulation medium, the Rad- Spo- strain gave no increase. Thus, rad50-1 eliminates meiotic gene conversion as well as crossing-over. The cultures described in Table 9 were also separated by microdissection
into two populations: asci and vegetative cells (Table 10). The asci produced by the Rad- Spocells, as expected, were quite viable; on the other hand, the surviving vegetative cells were somewhat less viable than the Rad+ Spo- control. The frequency of recombinants in the vegetative cells appeared to be somewhat higher than the frequencies measured in the total population in
the direct plating experiment. If this difference is real, it may reflect the fact that the vegetative cells in this experiment were permitted to grow on nonselective medium before testing.
DISCUSSION The rad5O series of mutations were originally isolated as mutations which conferred the phenotype of X-ray sensitivity (12). Many of these mutations have in addition been reported to cause (i) a reduction in radiation-induced mitotic recombination, (ii) reduced spontaneous mitotic and meiotic recombination, and (iii) lowered spore viability and reduced sporulation ability.
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MALONE AND ESPOSITO
MOL. CELL. BIOL.
TABLE 8. Recombination in Rad- Spo- and Rad+ Spo- dissected dyadsa No. of dyads (P:R:A)
ade5
trp5
cyh2
II
V
VII
Diploid genotype
leul
canl
ura3
III
lysl 42:0:0
his7
MAT
23:0:0 62:0:3 63:0:2 63:0:1 42:0:0 64:0:1 rad50-1 spol3-1 61:1:3 63:0:0 rad50-1 spol3-1 35:13:10 41:13:4 29:22:6 31:17:8 29:20:7 RAD50 spol3-1 21:11:2 31:14:13 27:21:7 29:22:4 RAD50 spol3-1 a Dyads were dissected, spores were germinated, and the resulting colonies were analyzed for phenotype. Only dyads containing two viable spores are listed in this table. Dyads were classified into three types: P, dyads in which both spores had phenotypes diagnostic of an equational division; R, dyads in which the two spores had phenotypes diagnostic of an equational division plus a crossover between the locus and the centromere; A, aberrant dyads which did not fit either the R or P classification. Roman numerals over the loci monitored for exchange refer to the chromosome on which the markers are located. See text for further discussion. The rad501/rad50-1 spol3-1/spol3-1 diploids were RM59 and RM60, and the RAD50/RAD50 spo13-1/spo1l-1 diploids were RM64 and RM69.
TABLE 9. Recombination frequencies in Rad- Spo- and Rad+ Spo- diploids after exposure to sporulation mediuma x Resistant colonies 10F
Intragenic recombinants x 104 CFU
CFU
Culture leul
rad50-1 spol3-1 rad50-1 spol3-1 Mitotic Meiotic Relative increaseb
trp5
ura3
Iys2
canl
cyh2
3.7 4.1 1.1
0.44 1.5 3.3
0.86 0.91
21 21
3.7 3.5
2.9 3.7 1.3
1.1
1.0
0.95
RAD50 spol3-1 RAD50 spol3-1 1.4 Mitotic 1.3 0.040 3.6 0.67 0.16 140 350 7.7 1.0 350 190 Meiotic 100 269 Relative increaseb 48 25 97 284 a Rad- Spo+ and Rad+ Spo- diploids were exposed to sporulation medium and analyzed as described in Table, 5. The Rad- Spo- diploid sporulated 26%, whereas the Rad+ Spo- diploid sporulated 58%. The alleles used to monitor recombination are described in Tables 3 and 7. The diploids used were those described in Table 8. CFU, Colony-forming units. b Represents ratio of meiotic culture to mitotic culture.
TABLE 10. Properties of vegetative cells and asci in Rad- Spo- diploids after 48 h in sporulation mediuma % Recombinant (no.) at: Fraction of No. viable No. examined Cell type
rad50-1 spol3-1 rad50-1 spol3-1 Vegetative Asci
total
0.74 0.26
97 52
31
48b
canl
cyh2
leul
3 (1) 3 (1)
6 (2) 3 (1)
3 (1) 0 (0)
RAD50 spol3-1 RAD50 spol3-1 12 (8) 67 19 (13) 0.42 96 19 (13) Vegetative 15 (5) 42 32 (11) 34b 20 (7) 0.48 Asci a The sporulation cultures described in Table 9 were spread on YPD plates, and vegetative cells and asci were isolated by micromanipulation and allowed to grow into colonies. Viable colonies were tested for recombination events at canl, cyh2, or leul. The diploids used were those described in Table 9. b For an ascus to generate a viable colony, only one of the four spores need be viable.
VOL. 1, 1981
Although it is unlikely that all X ray-sensitive mutations will prove to affect recombination, it would not be surprising if many did, since recombination is likely to play a role in the repair of X-ray damage (22). All of these observations lead to the notion that "meiotic" recombination functions might be utilized in the repair of X-ray damage in mitotic cells. The purpose of these experiments was to determine whether the reduction in spore formation as well as the high spore inviability observed in rad6-1 rad50-1, and rad52-1 strains resulted from deficiencies in meiotic recombination. We thus attempted to bypass the requirement for recombination for proper meiosis I segregation by the use of a mutation, spol3-1, which causes cells to undergo a single, predominantly equational division during meiosis (17). Although the recombination defect conferred by the rad50-1 mutation is not eliminated, viable meiotic products (i.e., spores) are formed in the presence of spol3-1. However, neither rad6-1 nor rad52-1 strains form viable spores in the spo13-1 background. From analysis of recombination in meiotic products we conclude that rad50-1 is a general meiotic Rec- mutation. The effect of the spol2-1 mutation on the ability of the three rad mutants to undergo meiosis and sporulate was also examined (data not shown). spol2-1 diploids have a phenotype similar to spol3-1 diploids: they exhibit wildtype levels of meiotic recombination, undergo a single equational division, and produce two diploid spores (17). None of the three rad mutants was able to sporulate and form viable products in the presence of the spol2-1 mutation. Studies on the interaction of spo12-1 and spol3-1 with another meiotic Rec- mutation, spoll-i, have also led to the conclusion that recombination is not required for viable spore production in a spol3-1 background but is essential in the presence of spo12-1 (Klapholz, Ph.D. thesis). We observed essentially no meiotic recombination in the presence of the rad50-1 mutation after exposure to sporulation medium or directly in ascospores. Meiotic crossing-over is reduced at least 150-fold, and meiotic gene conversion is not increased over the mitotic background level (Tables 8 and 9). Since meiotic recombination was impaired at all loci and in all intervals examined on four different chromosomes, we conclude that the RAD50 gene product plays a central role in the meiotic recombination process. The behavior of rad50-1 is in contrast to the cog and rec mutations in Neurospora (3) as well as the con mutations in yeasts (10), all of which seem to confer locus-specific effects on meiotic recombination. Furthermore, the data presented
rad50-1 AND RECOMBINATION
899
here support the widely held view that meiotic gene conversion and crossing-over in yeasts represent events generated by the same general recombination mechanism. It has been calculated that gene conversion events and their associated crossovers are sufficient to account for all recombination in meiosis (11). Since a single mutation (rad50-1) eliminated both conversion and crossing-over, at least one common step must be shared between them. A study of aberrant dyads formed in spol3-1 diploids indicated that many of them contain one trisomic (2n + 1) and one monosomic (2n - 1) spore (17). Since aberrant segregation was not observed in the viable spores produced by spoll-1 spol3-1 diploids it was proposed that recombination might be responsible for aberrant segregation (Klapholz, Ph.D. thesis). This is supported by the present observation that rad50-1 not only abolished meiotic recombination but also reduced the frequency of aberrant dyads (Table 8). Campbell and Fogel (2) and Liras et al. (18) have demonstrated that mitotic recombination can be associated with increased chromosome loss; thus, high levels of recombination may generally interfere with the process of equational chromosome division. The fact that the rad50-1 mutation reduces recombination more than it does the frequency of aberrant dyads suggests that there may be other processes which contribute to the formation of aberrant dyads. Alternatively, the residual level ofmeiotic recombination in rad50-1 spol3-1 strains may be sufficient to create a low level of improper segregation. Spontaneous mitotic recombination in the presence of rad50-1 is not diminished; thus, its reduction of recombination is specific for meiosis. Similar results have been found for spoll-i (Klapholz, Ph.D. thesis). In contrast, the rad52-1 mutation appears to reduce both meiotic and mitotic recombination (13, 19, 21). These results provide evidence that different gene products are required for mitotic and meiotic recombination although some gene products are utilized in both processes. Paradoxically, although rad50-1 does not reduce mitotic recombination, it nevertheless affects the process. The rad50-1 mutation confers a moderate hyperrec phenotype in mitosis, both for spontaneous gene conversion and crossing-over. The fact that rad52-1 (19), reml-l (14), and rad50-1 all affect both crossing-over and conversion in mitosis argues that the relationship between gene conversion and crossing-over in mitosis may be similar to that in meiosis. The available data on the mitotic association of these two recombination events support this view (7).
900
MALONE AND ESPOSITO
The fact that the rad50-1 mutation stimulates mitotic recombination, even though it eliminates meiotic recombination, raises the interesting question as to the possible function of the wildtype gene product. Such a phenotype has been proposed for an enzyme whose function it is to specifically cleave half-chiasmata or "Holliday structures" (7). Esposito (6) has presented evidence that a large portion of mitotic recombination in yeast occurs at the two-strand stage (or at least in unreplicated regions of chromosomes), whereas it has been well established that meiotic recombination occurs at the four-strand stage. Resolution of the Holliday structure in mitosis before replication would lead to loss of reciprocal recombinants, whereas such resolution at the four-strand stage would be required for subsequent proper chromosome disjunction in meiosis I. Therefore, a mutant which failed to cleave Holliday structures would lead to a meiotic hyporec but a mitotic hyperrec phenotype (7). Is the rad50-1 strain such a mutant? Two observations suggest that it is not: first, assuming that recombination occurs at the fourstrand stage in a spo13-1 meiosis, meiotic levels of recombination without cleavage of Holiday structures should lead to difficulties in equational segregation (e.g., a three-strand double crossover). These in turn should lead to higher levels of aberrant segregation and spore inviability in rad50-1/rad50-1 spol3-1/spol3-1 diploids. Instead, the Rad- Spo- strain has greatly reduced levels of aberrant segregation and increased spore viability. Second, failure to cut Holliday structures should not affect, a priori, the frequency of meiotic gene conversion. Yet the rad50-1 mutation eliminates meiotic gene conversion in meiotic products and in vegetative cells induced for meiotic recombination. Thus, it appears unlikely that the RAD50 gene product is an endonuclease specific for cutting Holiday structures. A second hypothesis to explain the contrasting meiotic and mitotic phenotypes of the rad50-1 mutation is that the RAD50 gene product is normally required for repair of spontaneously occuring mitotic lesions which are recombinogenic. Thus, the elimination of such repair would elevate spontaneous mitotic recombination. It is likely that the RAD50 gene product plays some role in mitosis since rad50-1 cells grow at a considerably slower rate than do wild-type cells. A third possibility is that there may be more than one recombination system acting in mitotic cells. For example, one system may be that which produces recombinant products between homologous chromosomes, and another might be one which acts on exchange between sister
MOL. CELL. BIOL.
chromatids. These systems may share some common functions with each other and also with meiotic recombination. It is interesting to note that both meiotic and sister strand recombinations occur in duplicated deoxyribonucleic acid molecules (in G2), whereas mitotic recombination between homologs appears to occur between unreplicated chromosomes. Thus, one hypothesis to explain the phenotype observed is that the RAD50 gene product is utilized in both meiotic recombination and in mitotic sister strand exchange but not in mitotic recombination between homologs. If sister strand exchange normally competes with mitotic homolog exchange from some limiting shared functions, then inactivation of sister strand exchange by the rad50-1 mutation could result in the elevation of exchange between homologs. We are currently testing this hypothesis by e-amining the effects of rad5O-1 on mitotic sister strand exchange. The requirement for the RAD6 and RAD52 genes for viable spore production was not bypassed by the spol3-1 mutation, even though the rad50-1 and spoll-1 data indicate that recombination per se is not required for a spo13-1 meiosis. Three possible explanations for the RAD52 and RAD6 requirements in a spo13-1 meiosis are: (i) the RAD6 and RAD52 products may play a required role in meiotic processes other than, or in addition to, recombination; (ii) the absence of the RAD6 and RAD52 products during meiosis may lead to deoxyribonucleic acid damages which result in defects in subsequent meiotic steps; and (iii) the rad52-1 and rad6-1 mutations may block recombination at an intermediate stage where equational segregation is disturbed. In this scheme, rad50-1 would lead to an early recombination block which would not interfere with equational segregation. Preliminary data on the behavior of the triple mutant rad50-1 rad52-1 spol3-1 strain suggests that it is capable of sporulation and produces viable, nonrecombinant spores; this provides support for the argument that rad50-1 acts before rad52-1. Experiments are also in progress to determine whether rad50-1 can allow rad6-1/rad6-1 spo13-1/spol3-1 diploids to sporulate. ACKNOWLEDGMENTS We express our appreciation to Sue Klapholtz, who provided much unpublished data, the strain carrying the spol3-1 mutation, and excellent criticism of the manuscript. We would also like to thank Michael Esposito for helpful discussions during the early part of this work, Joe Wagstaff and Tom Petes for their criticisms and comments on the manuscript, and Rita Vanags for preparation of the figure. This work was supported by Public Health Service grant GM-23277-02 from the National Institutes of Health and
VOL. 1, 1981
C.C.R.C. grant PHSCA-19265 (project 508) to R.E.E.; R.E.M. was supported by Public Health Service postdoctoral fellowship GM-05965-03 from the National Institutes of Health.
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ray sensitive mutants in yeast. Mutat. Res. 24:281-292.
Game, J. C., T. S. Zamb, R. Braun, M. Resnick, and R. Roth. 1980. The role of radiation(rad) genes in meiotic recombination in yeast. Genetics 94:51-68. 14. Golin, J., and M. Esposito. 1977. Evidence for joint genic control of spontaneous mutation and genetic re13.
LITERATURE CITED 1. Baker, B., A. Carpenter, M. Esposito, R. E. Esposito, combination during mitosis in Saccharomyces. Mol. and L, Sandler. 1976. The genetic control of meiosis. Gen. Genet. 150:127-135. Annu. Rev. Genet. 10:53-134. 15. Hunnable, E. G., and B. Cox. 71. The genetic control of 2. Campbell, D., and S. Fogel. 1977. Association of chrodark recombination in yeast. Mutat. Res. 13:297-309. mosome loss with centromere adjacent mitotic recom- 16. Kassir, Y., and G. Simehen. 1978. Meiotic recombinabination in a yeast disomic haploid. Genetics 85:573tion and DNA synthesis in a new cell cycle mutant of 585. Saccharomyces cerevisiae. Genetics 90:49-68. 3. Catcheside, D. G. 1974. Fungal genetics. Annu. Rev. 17. Klapholtz, S., and R. E. Esposito. 1980. Recombination Genet. 8:279-307. and chromosome segregation during the single division 4. Cox, B., and J. Parry. 1968. The isolation, genetics, and meiosis in spol2-1 and spol3-1 diploids. Genetics 96: survival characteristics of UV light sensitive mutants in 589-611. yeast. Mutat. Res. 6:37-55. 18. Liras, P., J. McCusker, J. Mascolis, and J. Haber. 5. Demerec, M., E. Adelberg, A. J. Clark, and P. Hart1978. Characterization of a mutation in yeast causing man. 1966. A proposal for a uniform nomenclature in nonrandom chromosome loss during mitosis. Genetics bacterial genetics. Genetics 54:61-76. 88:651-671. 6. Esposito, M. 1978. Evidence that spontaneous mitotic 19. Malone, R. E., and R. E. Esposito. 1980. The RAD52 recombination occurs at the two-strand stage. Proc. gene is required for homothallic interconversion of matNatl. Acad. Sci. U.S.A. 75:44364441. ing types and spontaneous mitotic recombination in 7. Esposito, M., and J. Wagstaff. 1981. Mechanisms of yeast. Proc. Natl. Acad. Sci. U.S.A. 77:503-507. mitotic recombination. In J. Strathern, J. Broach, and 20. Plischke, M., R. von Borstel, R. Mortimer, and W. B. Jones (ed.), Molecular biology of the yeast SacchaCohn. 1976. Genetic markers and associated gene prodromyces. Cold Spring Harbor Press, Cold Spring Haructs in S. cerevisiae. In G. Fasman (ed.), Handbook of bor, N.Y. biochemistry and molecular biology. CRC Press, Cleve8. Esposito, R. E., and M. Esposito. 1974. Genetic recomland. bination and commitment to meiosis in Saccharomyces. 21. Pralash, S., L Prakash, W. Burke, and B. MonteProc. Natl. Acad. Sci. U.S.A. 71:3172-3176. lone. 1980. Effects of the RAD52gene on recombination 9. Esposito, R. E., D. Plotkin, and M. Esposito. 1974. The in Saccharomyces cerevisiae. Genetics 94:31-50. relationship between genetic recombination and com- 22. Remick, M. A. 1976. The repair of double strand breaks mitment to chromosomal segregation at meiosis, p. 277in DNA: a model involving recombination. J. Theor. 285. In R. Grell (ed.), Mechanisms in recombination. Biol. 59:97-106. Plenum Publishing Corp., New York. 23. Schild, D., and B. Byers. 1978. Meiotic effects of DNA 10. Fogel, S., and R. Roth. 1974. Mutations affecting meiotic defective cell division cycle mutations in Saccharomygene conversion in yeast. Mol. Gen. Genet. 130:189ces cerevisiae. Genetics 90:49-68. 201. 24. Sherman, F., and H. Roman. 1963. Evidence for two 11. Fogel, S., R. Mortimer, K. Lusnak, and F. Tavares. types of allelic recombination in yeast. Genetics 48:2551978. Meiotic gene conversion: a signal of the basic 261. recombination event in yeast. Cold Spring Harbor 25. Wickner, R. 1979. Mapping chromosomal genes of SacSymp. Quant. Biol. 43:1325. charomyces cerevisiae using an improved genetic map12. Game, J., and R. Mortimer. 1974. A genetic study of Xping method. Genetics 92:803-821.
