THE ROLE OF RADIATION (rad) GENES IN MEIOTIC ...rad GENES IN YEAST 55 curs in g538 (Figure 1) and in g550, confirming that the rad mutations studied are recessive with respect to both

THE ROLE OF RADIATION (rad) GENES IN MEIOTIC RECOMBINATION IN YEAST

J. C. GAME1, T. J. ZAMB2, R. J. BRAUN, M. RESNICK3, AND R. M. ROTH

Division of lMicrobiology, National Institute for Medical Research, Mill Hill, London, U . K . and Department of Biology, Illinois Institute of Technology, Chicago, Illinois 60616

Manuscript received February 10, 1979 Revised copy received July 20, 1979

ABSTRACT

In yeast, the functions controlled by radiation-repair genes RAD6, RAD50, RAD52 and RAD57 are essential for normal meiosis; diploids with lesions in these genes either fail to sporulate (rad6) or sporulate but produce inviable spores (rad50, 52, 57) . Since RAD genes may control aspects of DNA metabolism, we attempted to define more precisely the role of each gene in meiosis, especially with regard to possible roles in premeiotic DNA replication and recombination. We constructed diploids singly homozygous for each of the four rad mutations, heteroallelic at his1 and heterozygous for a recessive canavanine- resistance marker. Each strain was exposed to sporulation-inducing conditions and monitored for (1) completion of mitotic cell cycles, (2) cell viability, (3) utilization of acetate for mass increases, (4) premeiotic DNA synthesis, (5) intragenic recombination at his1, and ( 6 ) formation of viable haploid spores. Control strains heterozygous for the rad mutations completed mitosis, metabolized acetate, replicated their DNA, and showed typically high levels of gene conversion and viable-spore formation. The mutant diploids also completed mitosis, utilized acetate, and carried out premeiotic DNA replication. The mutants, however, showed little or no meiotic gene conversion. The rad50, 52 and 57 strains sporulated, but the spores were inviable. The rad6 strain did not sporulate. The rad50, 52 and 57 strains exhibited viabi!ity losses that coincided with the period of DNA synthesis, but not with later meiotic events; the rad6 strain did not lose viability. We propose that the normal functions specified by RAD50,52 and 57 are not essential for either the initial or terminal steps in meiosis, but are required for successful recombination. The rad6 strain may be recombination-defective, o r it may fail to progress past DNA replication in the overall sequence leading to formation and recovery of meiotic recombinants.

I N Saccharomyces cerevisiae, mutations in several genes controlling radiation sensitivity (rad mutants) block the successful completion of meiosis (Cox

and PARRY 1968; RESNICK 1969; RODARTE-RAMON 1972; GAME and MORTIMER 1974). The RAD genes required for meiosis chiefly are those whose mutant alleles confer cellular sensitivity to ionizing radiation (GAME and MORTIMER

Present address Department of Genetics, 345 Mulford Hall, University of California, Berkeley, California 94720. Present address: Department of Microbiology, University of Chicago, Chicago, Illinois 60637. Present address: Laboratory of Molecular Genetics, National Institute of Environmental Health Science, Research

Triangle Park, North Carolina 27709.

Genetics 94: 51-68 January, 1980.

52 J. c. GAME et al.

1974). Those rad mutants that confer sensitivity to UV radiation without substantially affecting sensitivity to ionizing radiation do not in general influence sporulation or spore viability (Cox and PARRY 1968; SNOW 1968). Since it is be- lieved that sensitization to the lethal effects of ionizing radiation in rad mutants results from defective DNA repair (NAKAI and MATSUMOTO 1967; GAME and MORTIMER 1974; Ho 1975; RESNICK and MARTIN 1976), those rad mutants defective in meiosis may identify genes whose function during meiosis and recombination is specifically concerned with aspects of DNA metabolism. We have therefore studied the defective meiosis that occurs in four X-ray sensitive mutants in yeast. These mutants are rad6-1 (Cox and PARRY 1968; GAME and Cox 1971), which is highly sensitive to X-rays and to UV radiation, and rad50-1, rad52-I and rad57-1 (GAME and MORTIMER 1974), which are highly sensitive to X-rays but only marginally sensitive to UV. Of these, rad6-1 has been reported to block sporulation (Cox and PARRY 1968), whereas rad50-1 and rad57-I have been reported to permit sporulation but to yield inviable spores (GAME and MORTIMER 1974). Rad52-I has been reported to reduce both sporulation and spore viability (RESNICK 1969; GAME and MORTIMER 1974).

We constructed diploid strains homozygous for each of these rad mutations in turn that also carried genetic markers for the study of recombination and haploidization. To characterize the meiotic defects in each strain, exponentially growing cultures were washed and transferred into liquid sporulation medium. At zero time, and at intervals throughout the sporulation cycle, samples were plated on selective growth media to interrupt meiosis and to monitor (1) viability, (2) the frequency of heteroallelic recombinants at the hisl locus, and (3) the frequency of colonies expressing recessive markers (can1 and ade2) for which the strains were heterozygous. At the same time intervals, we also monitored increases in mass, DNA content, propprtion of cells completing mitotic cell division and proportion of asci. Two radiabon-resistant strains heterozygous for a number of rad mutations were similarly studied. From these data and some other results, we have determined which of the above aspects of meiosis are defective in each of the four rad mutants studied.

MATERIALS AND METHODS

Yeast strains: The detailed genotype of each strain used in this study is presented in the text. The linkage relationships shown are those determined or cited by MORTIMER and HAW- THORNE (1975). Gene symbols are those cited by PLISCHKE, et al. (1976). Diploids homozygous for lesions in genes m d 6 , 50, 52, and 57 were constructed by mating haploids derived from the control strain, g538. Each diploid was also heteroallelic at the histidine (hisl) locus and heterozygous for adenine-2 (&a), canavanine resistance-l (canl) and other nutritional markers. Strain g53S was constructed from stocks obtained f r m the yeast culture collections of R. K. MORTIMER and S. FOGEL at Berkeley, California, and R. C. VON BORSTEL at Edmonton, Canada. The procedures used to sporulate g538, to dissect the tetrads and to score the nutritional markers and the mating type of each haploid spore clone have been described (MORTIMER and HAW- THORNE 1969). The presence of the hisl-1 or hisl-7 lietercallele was determined by a modifica- tion of the method described by HURST and FOGEL (1964). First spore clones were crossed with radiation-resistant (wild-type) tester strains carrying either the hisl-1 or hisl-7 allele. Next, the resulting diploids were monitored for heteroallelic recombination (prototroph formation),

rad GENES IN YEAST 53

which was enhanced with a sublethal dose of ultraviolet irradiation from a germicidal lamp. The rad mutation(s) carried by each haploid were identified by complementation tests performed with tester strains carrying single lesions in either rad 6, 50, 52, 54 or 57. The presence or absence of a rad mutation was judged by the sensitivity o r resistance of the diploid to either X-ray or gamma irradiation.

Growth, sporulation and analytical procedures: Complex growth medium (GNA) contained the following additions per liter : glucose 50g; Bacto-peptone (Difco) 5g; Bacto yeast extract log; Bacto beef extract 3g; adenine 20 mp; uracil 2Omg; 1-histidine 2Omg; 1-methionine 20mg; 1-tryptophan 40mg; 1-arginine 40mg; 1-phenylalanine 4 h g ; 1-tyrosine 0.lg; 1-lysine 0.12g; 1-isoleucine 0.12g; I-leucine 0.12g; 1-valine 0.16g; d-1 threonine 0.6g. The medium was solidified with 26g of agar. The preparatioa of acetate presporulation and sporulation media was previously described (ROTH and FOGEL 1971; KUENZI and ROTH 1974). To initiate meiosis, cells growing logarithmically in acetate presporulation medium were harvested, washed and resuspended in 1% potassium acetate sporulation medium (SM); this was considered zero time, t = 0 (KUENZI and ROTH 1974). At intervals, samples of various size were withdrawn from each meiotic culture to monitor: (1) completion of previously initiated mitotic cell division cycles; (2) viability; (3) increases in cellular mass; (4) DNA synthesis; (5) the appearance of histidine prototrophic recombinants; (6) the appearance of canavanine-resistant cells; (7) expression of the recessive adenine-2 mutation; and (8) sporulation. Completion of mitotic cells cycles in sporulation medium was assessed by monitoring the ability of gentle sonication to separate budded cells into two units, i.e.. a mother and daughter cell (ESPOSITO and ESPOSITO 1978). TO monitor viability culture samples were diluted, plated on solid complex medium and enumerated after three to five days. Viability plates were retained and re-examined after seven to ten days to score pink or red colonies, or sectors, expressing the recessive adenine-2 mutation. Mass increase was monitored by Iollowing absorbance changes at 600 nm (ROTH 1970). DNA synthesis was measured using a semi-micro diphenylamine procedure (ZAMB and ROTH 1977). The appearance of histidine prototrophs was followed by plating samples on histidine-free synthetic medium; HIS+ colonies were enumerated after five to seven days. The appearance of canavanine- resistant cells was determined by platings to synthetic medium supplemented with 30 pg per ml of canavanine sulfate (Sigma). In some experiments, samples were also plated on canavanine- containing, uracil-free synthetic medium to monitor the appearance of canavanine-resistant, uracil prototrophs. Canavanine-containing media was prepared without arginine. Sporulation percentages were determined by phase-contrast microscopy.

Incubations were carried out at 30" for all strains except g551, which was homozygous for the temperature-dependent lesion rad57-I. For 9551, we used a restrictive temperature of 23" and a permissive temperature of 34.5".

RESULTS

We have examined two radiation-resistant control strains, g538 and g550. The genotype of g538 is as follows:

g538: a CANIS + hom3-10 hid-7 4- lysl leu2 4- - - - - - -- -0 O, canl' ura3 + hid-1 trp2 + + ade2 ade4

rad&-1 + rad52-1 rad54-3 + + rad50-l + + rad57-1

Strain g550 has similar auxotrophic markers, but is heterozygous for only two rad loci, rad50 and rad54. Strain g538 was the parental diploid used to generate the haploid strains from which most of the homozygous rad mutant diploids were constructed. We chose related diploids for this study to minimize variation due to difierences in background genotype. As described below, normal meiosis oc-

54 J. e. GAME et al.

0 1 0 2 0 3 0 4 0 5 0 Time (hr)

FIGURE 1 .-Premeiotic DNA synthesis, recombination, sporulation, and viability in diploid g538. At zero time, a culture growing logarithmically in presporulation medium was harvested, washed and resuspended in sporulation medium to induce meiosis. At intervals, samples were removed to determine viability and monitor the progress of meiotic events. Upper panel: Viabil- ity (0) was determined by plating diluted samples on complex medium; viability is expressed as a percentage of surviving colonies relative to those present at zero time. Middle panel: Total cellular DNA ( ) was measured with a diphenylamine assay. The sporulation percentage (0) was evaluated microscopically. Lower panel: The appearance of histidine prototrophs ( W ) was evaluated by platings to histidine-free synthetic medium. The appearance of canavanine- resistant colonies (v) was followed by plating to synthetic medium containing canavanine sulfate. The appearance of canavanine-resistant uracil prototrophs (A) was scored on uracil- free synthetic medium containing canavanine sulfate. All incubations were at 30".


curs in g538 (Figure 1) and in g550, confirming that the rad mutations studied are recessive with respect to both their meiotic and radiation-sensitive phenotypes. Hence, the presence of additional heterozygous rad mutations in some of the rad mutant diploids is unlikely to have affecied the results.

Commitment to intragenic recombination in sporulation medium (SM) was measured at the his1 locus by plating on histidine-free medium. Viability and the appearance of red adenine-requiring colonies were measured by plating on complex (GNA) medium, and the appearance of canavanine-resistant (cand') colonies was monitored by plating both on CAN medium and on CAN medium without uracil (CAN-URA medium). Results for control strain g538 are given in Figure 1, together with data on DNA synthesis and sporulation. OD increased more than two-fold in SM (not known), indicating that the strain was able to metabolize the acetate medium, and less than 5% of cells remained budded following sonication after 13 hours in SM (compared with 32% at zero time) , indicating completion of mitotic cycles in most cells. It can be seen (Figure 1) that, after transfer to SM, there is an approximate doubling in DNA content over a period of 15 hours. Histidine prototrophs begin to appear after four hours, and rise to a maximum freuency of 3,400 per lo6 viable colony-forming units. Asci are first observed at 11 hours, and sporulation reaches a final value of 75%. Notice that histidine prototrophs were recovered from the meiotic cultures rela- tively early and that their accumulation coincided with DNA synthesis rather than with the appearance of mature spores. This agrees with previous studies in yeast that established that commitment to meiotic recombination occurs before commitment to the meiotic division; as a consequence histidine prototrophs recovered at early times are probably diploid, while those recovered later are probably haploid (SHERMAN and ROMAN 1963; ESPOSITO and ESPOSITO 1974a; SILVA- LOPEZ, ZAMB and ROTH 1975).

Red colonies (including those with red sectors) in control strain g538 first ap- peared on the GNA plates from cells plated after four hours in SM. They are expected to arise from both recombination and haploidization. I n strain g538, they increased from 1.6% of all colonies at four hours to 11.5% at 15 hours, followed by a slower rise to 14.6% at 52.5 hours. Those from early time points are likely to be diploid recombinants arising from crossing over between the centromere and the ade2 locus, whereas those from later times are likely to arise from haploidization with o r without recombination. One-half of all live spore clones will carry the ade2 mutant allele, but the presence of the second adenine mutation, a d d , will block the formation of red pigment in half of these (ROMAN 1956). The fact that the final frequency of red colonies, ILEA%, is lower than 250/;, probably reflects the facts that only 75% of cells sporulated and that mat- ings of sister spores after germination may lead to white diploids in place of some red sectored colonies. Canavanine-resistant colonies first showed an increase in strain g538 among cells sampled after seven hours in SM, significantly later than the histidine prototrophs. In principle, canavanine-resistant colonies (like red colonies) could arise either by recombination between the c a d locus and its centromere followed by a mitotic division or by haploidization (with or without recombination) leading to segregation of the resistant and sensitive alleles at

56 J. c. GAME et al. meiosis. Several observations suggest that, under our conditions, canavanine- resistant colonies of the first type (mitotic recombinants) do not arise and that most if not all of the observed colonies on CAN plates arise via haploidization. These observations include the following: (1) The CAN plates were observed microscopically IO determine whether canavanine-sensitive cells were capable of going through at least one residual division before being blocked by canavanine. After several days' incubation, only 45 out of 110 cells examined on a CAN plate sampled at six hours were found to be budded. This is similar to the proportion of budded cells after a comparable period in sporulation medium prior to exposure to CAN, indicating that few if any cells go through the residual division needed for the expression of mitotic recombinants on CAN plates. ( 2 ) The expression of the recessive markers ura3 and leu2 was studied in canavanine- resistant colonies. Forty-one of 55 can1 colonies recovered from samples plated after 11 hours in sporulation medium were auxotrophic for uracil, leucine or both. Few such colonies should arise by recombination without haploidization, since ura3 and leu2 are closely centromere-linked. Some colonies that have undergone haploidization will nevertheless be prototrophic for leucine and uracil (in the latter case only if recombination has occurred between ura3 and canl ) , and these may account for the remaining 14. Clearly, at least most of the canlc colonies at 1 1 hours have undergone haploidization.

I[ seems clear, therefore, that plating on CAN medium monitors an event later in the meiotic cycle than commitment to recombination. Similar results have been obtained by SHERMAN and ROMAN (1963), who found that the frequency of canavanine-resistant colonies obtained correlated with the frequency of cells in sporulation medium that had undergone at least one nuclear division. Our results (Figure I ) , in which canavanine-resistant colonies first appear three hours after the histidine prototrophs but four hours before the first visible asci, are consistent with this.

Canavanine-resistant colonies that were prototrophic for uracil were scored by plating on CAN medium lacking uracil. They begin to appear at the same time as colonies on the CAN plates, and comprise more than one-third of the total c a d r colonies (Figure 1) . This confirms that meiotic recombination occurs nornially in the gene interval cad-ura3.

Viability in strain g538 in SM remains above 75% throughout the period of the experiment.

Strain g538, therefore, shows all the characteristics of a normal meiosis (SHER- MAN and ROMAN 1963; ESPOSITO and ESPOSITO 1976; SILVA-LOPEZ, ZAMB and ROTH 1975) with respect to kinetics of DNA synthesis, appearance of recombinants and canavanine-resistant colonies, sporulation and viability. This confirms that the rcrd mutations present in heterozygous condition in this strain are recessive and without consequence in meiosis.

Results for control strain g550 (not shown) were broadly similar to those for g538, except that sporulation occurred later and reached a lower final value (39%).

rad GENES IN YEAST

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FIGURE 2.-Premeiotic DNA synthesis, recombination, sporulation and viability in diploid g544, homozygous for the radb-I lesion. At zero time, a meiotic culture was established; a t intervals, samples were removed to determine viability and the progress of meiotic events as described in Figure 1. Top panel: viability (0). Bottom panel: DNA synthesis ( 0 ) ; sporulation percentage (0) ; histidine prototrophs (U) ; canavanine-resistant colonies (v).

rad6: Two diploids homozygous for rad6-I were studied. Results are presented in Figure 2 for g544, which had the following genotype:

g554: a C A N I S + o hom3-IQ fzisl-7 + + leu2 ade2 --- - _____- 01 can' ura3 + hid-I trp2 lysl + + + rad&-I + - ---

ade4 radb-1 iad57-1

The strain was highly sensitive to UV light and to X-rays. Confirmation that it was heteroallelic for his1 was obtained by exposing cells on histidine-free medium to a small dose of UV. A large increase in histidine prototrophs was observed, indicative of heteroallelic recombination.

After the shift into sporulation medium, the rad6 diploid showed a rise in OD and a drop in the percentage of cells remaining budded after sonication (data not shown), indicating that most cells were able to metabolize the acetate and


to complete mitotic cycles. A round of DNA synthesis occurred after the shift (Figure 2) , indicating initiation of meiosis, but i t can be seen (Figure 2 ) that no sporulation and no increase in canavanine-resistant colonies occurred, and that the rise in histidine prototrophs was much smaller than in the control. The frequency of whose or partial red colonies on GNA plates rose from none at t = 0 to values between 0.7% and 2.3% at t = 6.5 hours and subsequent times (data not shown), which is also a much smaller increase than that seen in the control. Viability remained high in SM in the rad6 strain, as it did in the control. It is clear from these results that rd6-I effectively blocks meiotic recombination in sporulation medium and prevents cells from advancing beyond DNA synthesis in the meiotic cycle. However, on plating cells on nonselective medium, the first division could, in principle, be either mitotic o r meiotic. Were it meiotic, segregation of resistaiit colonies on CAN medium might not be observed because cells might not reach the point before plating at which a cell division could be completed subsequently in the presence of canavanine (see above). However, the low frequency of red colonies seen on the GNA plates makes it unlikely that more than a small percentage of cells undergo recombination or meiotic centromere disjunction after plating. In addition, we also scored colonies on some plates for the expression of other heterozygous markers in the strain, by replica- plating on to appropriate omission media. Of 391 colonies tested (from the 24 hour or 27.3 hour plates), none expressed a leucine or uracil requirement, one whole or partial colony required threonine, two required lysine, three required tryptophan and 15 required adenine. The absence of colonies expressing the centromere-linked markers leu2 and ura3 indicates that meiotic centromere dis- juiiction and haploidization do not occur, and the first division on GNA plates after 24 liaurs in SM is therefore mitotic rather than meiotic. The few auxotrophic colonies seen occur at a frequency far below that expected for meiotic recombination and mainly express markers such as ade4, which are remote from their cenlromere and which presumably arise by mitotic crossing over between the centromere and the locus concerned.

The small rise in histidine prototrophs that is seen in the rad6 diploid is about 9yA of that in the control, and a similar small rise in adenine (red) recombinants occurs. This could reflect a slight amount of recombination in the population as a whole, or it could arise if a small percentage of cells underwent a full round of meiotic recombination. If the latter were the case, a high frequency of recombination for other markers should be seen among the histidine prototrophs. Tests showed that the ade2 marker was expressed more frequently among histidine prototrophs than i t was on the GNA plates, but did not approach the frequency expected for meiotic recombination. In any event, recombinant cells do not complete meiosis, since no sporulation is seen.

The other iad6-2 strain studied, g547, was not heteroallelic for hid, but was heterozygous for canavanine resistance and for adea. It gave results similar to those for g544, except that a small increase in canavanine-resistant colonies occurred at late times, to a maximum of 3.7% of viable cells at 47 hours. This was probably due to the presence of some cells in which the rad&-I allele was partially suppressed. Of 41 canavanine-resistant colonies taken from t = 24 hour


plates, 25 were significantly more resistant to UV in spot-tests than was the original g547 strain. Twenty-seven of these 41 colonies also expressed the ura3 and/or the leu2 requirements, suggesting that they were products of centromere disjunction rather than mitotic recombinants. However, no sporulation was seen in strain g547. rad&-l is a noiisense allele that reverts by suppression quite frequently (GAME 1971; LAWRENCE et aZ. 1974), and the apparent occurrence of some aspects of meiosis in partially revertant cells provides additional evidence that the radiation sensitivity and the meiotic phenotype are conferred by the same lesion.

In summary, it seems clear that radb-I blocks meiotic recombination as well as haploidization and sporulation, but allows a round of DNA synthesis to occur in sporulation medium without loss of viability.

rad50: A diploid homozygous for rad50-I, g552, was constructed from g538 spore clones. It had the following genotype:

hom3-I0 hisl-I trp2 lysl leu2 add? --- + hisl-7 + + + 4- + O g552: a C A N I S - -

cy can17 U T

ade4 rad50-I rad54-3 + rad50-1 +

Tests confirmed that the strain was sensitive to X-rays and slightly sensitive to UV. The spontaneous frequency and rate of UV-induced histidine prototrophs confirmed that it was heteroallelic for hisl.

On transfer to sporulation medium, the rad50 diploid showed a somewhat smaller rise in OD than that seen in the control strain, but the percentage of cells completing mitotic cycles was similar to that in the control (data not shown). It can be seen from Figure 3 that a round of DNA synthesis occurs that starts later and is less complete than in the control strain g538 (Figure I ) , but may still represent a doubling of DNA content in a majority of cells. Similarly, the percentage of cells that sporulate is less than in the control g538, but reaches a final value of nearly 40%. However, the rad50 strain differs qualitatively from the cmtrol in showing no increase in histidine prototrophs or canavanine-resistant colonies and in showing a large drop in viability starting at the same time as, or slightly before, DNA synthesis (Figure 3 ) . In a separate experiment, red ade2 colonies were also found to show no increase with time in SM in the rad50 diploid.

It has been reported before (GAME and MORTIMER 1974) that no live asco- spores are produced in rad50 diploids, and the absence of canavanine-resistant or red colonies in our rad50 strain confirms this. The lack of histidine prototrophs or red coloiiies also indicates that the RAD50 gene is required for the formation of live recombinants. It is noteworthy that the lethality (or commitment to lethality) occm-s early in the rad50 diploid, implying that the lethal lesions arise or are committed to during DNA synthesis rather than during haploidization or any subsequent process. The absence of histidine prototrophs also indicates either a specific defect in recombination or a lethal lesion that OCCUTS early in the meiotic cycle, since such prototrophs arise in diploid wild-type cells independently

60

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10 20 30 40 50

Time (hrl

U) Q) .- E 5 0 0.5 1.0 + E 0 0 U) a w c c E .-

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FIGURE 3.-Premeiotic DNA synthesis, recombination, sporulation and viability in diploid g552, homozygous for the rad50-I mutation. At zero time, a meiotic culture was established; at intervals, samples were removed to determine viability and the progress of meiotic events as described in Figure 1. Top panel: viability (0). Bottom panel: DNA synthesis ( 0 ) ; sporulation percentage (U) ; histidine prototrophs (m) ; canavanine-resistant colonies (v) . of sporulation (see above). The lethality in rad50 cannot be explained solely by a defect in meiotic nuclear division.

The RAD50 gene is clearly not required for sporulation, as judged by the appearance of visible asci.

rad52: A diploid strain, g553, homozygous for rad52 was constructed with the following genotype:

hisl-1 lysl leu2 ade2 3- rad52-1 a c a d c ura3 hom3-10 hid-7 + + + ade4 rad52-1 ---- g553: a CANlS + + _ _

+ rad50-1 + rad6-1 + rad57-1

The strain was highly sensitive to X rays. I t showed a much lower spontaneous frequency of histidine prototrophs than the control strain and there was little or no increase in prototrophs after W treatment. To confirm that it was hetero-


allelic at hisl, a tetraploid strain was selected on appropriate medium from a mixture of g553 and a diploid that was wild-type for RAD52 and HIS1 and homozygous for mating type. Spore clones from this tetraploid were monitored for radiation sensitivity and behavior on histidine-free medium. Several radiation-resistant his1 spore colonies were found to give high levels of histidine prototrophs after a small dose of UV radiation, suggesting that they had inherited hisl heteroalleles from g553. As a further test, a g553 derivative auxotrophic for adenine was isolated after treatment with UV light. The requirement for adenine allowed his1 triploids to be selected from mixtures of this strain with each of the his1 tester-strains. Triploids formed with a hid-1 and a hid-7 tester each gave many histidine prototrophs after a small dose of UV light, indicating the presence of heteroalleles in g553.

On transfer to sporulation medium, the rad52 diploid showed an increase in OD arid a completion of mitotic cell cycles similar to that seen in the rad50 strain (data not shown). Other aspects of the sporulation process are shown in Figure 4,

. .... - ,... , FIGURE 4.-Premeiotic DNA synthesis, recombination, sporulation and viability in diploid

g553, homozygous for the rud52-I lesion. At zero time, a meiotic culture was established; at intervals, samples were removed to determine viability and the progress of meiotic events as described in Figure 1. Top panel: viability (0). Bottom panel: DNA synthesis (e); sporulation percentage (0) ; histidine prototrophs (H) ; canavanine-resistant colonies (v).

62 J. c. GAME et al. y" and are also broadly similar to those of the rad50 diploid (Figure 3). There is a nearly two-fold increase in DNA content, but a complete absence of histidine prototrophs and canavanine-resistant colonies. In a separate experiment (not shown), red ade2 colonies also showed no increase in frequency with time in sporulation medium. Some sporulation occurs (Figure 4), but it reaches a lower percentage (1 7%) than in the control or the rad50 strains. This is consistent with previous findings (GAME and MORTIMER 1974). There is a substantial early drop in viability, coinciding with the time of DNA synthesis and resembling that seen in the rad50 strain.

I t is noteworthy that the rad52 strain shows little or no heteroallelic recombination at the his1 locus under any conditions tested, suggesting that the RAD52 gene product may be involved in the generation of live recombinants by all mechanisms in both mitosis and meiosis. RESNICK (1975) has previously shown that rad52-I blocks induction of recombinants by ionizing radiation at the arg4 locus, but he found some recombinations at arg4 after UV radiation.

rad57: A diploid strain, g551, was constructed which was homozygous for rad57-I. Its genotype was as follows:

hom3-10 hid-7 4- rad57-I + h a - I trp2 rad57-I 0-

g551: a CANIS + - -___--- (\I c a d ' ura3

The strain resembled other rad57-1 strains in being sensitive to ionizing radiation at 23", but not (or only marginally) at 34" (GAME and MORTIMER, unpub- lished observations). The presence of the his1 heteroallele; was confirmed by induction o€ histidine prototrophs with UV light. Because of the temperature- dependent radiation sensitivity, meiotic experiments were done at 23 O and plates were incubated at this temperature. In addition, a preliminary experiment was done using 34.5" as the temperature of the sporulation medium and for subsequent incubation of plates. On transfer to sporulation medium at 23", the rad57 diploid resembled the control strain in its rise in OD and completion of mitotic cell cycles (data not shown). Other results for the strain at 23" are shown in Figure 5, and resemble those for the rad50 strain (Figure 3). Sporulation reaches a final value of almost 40%, and there is a substantial drop in viability approxi- mately coinciding with the time of DNA synthesis. The almost complete absence of canavanine-resistant colonies, even when sporulation is completed, indicates that viable spores are not produced, and similarly the very low frequency of histidine prototrophs compared to the control strain (Figure 1) implicates the RAD57 gene product in generating live recombinants. The strain was not heterozygous for ade2; hence, red colonies could not be scored.

The rad57 culture incubated in SM at 34.5" was sampled for histidine prototrophs, canavanine-resistant colonies, sporulation and viability. Results (Figure 6) differed sharply from those at 23". At 34.5", histidine prototrophs rose from an initial value of 23 per 106 colony-forming units (CFU) to a final value of 5,100 per lo6 CFU at 50 hours. This is a value higher than the maximum seen in ihe control strain at 30" (3,NO per 10 CFUs, Figure 1) and clearly indicates lhat rad57-1 does not block meiotic conversion at its permissive temperature. A

rad GENES IN YEAST

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b In-

0 IO 20 s Time (hr)

FIGURE 5.-Eff ects of a restrictive temperature on premeiotic DNA synthesis, recombination, sporulation and viability in diploid g551, homozygous for the rad57-I mutation. At zero time, a culture growing logarithmically in presporulation medium at 30", was harvested and washed. The culture was resuspended and continuously incubated in sporulation medium at 23" (the restrictive temperature). At intervals, samples were removed to determine viability and the progress of meiotic events as described in Figure 1. All incubation were at 23". Top panel: viability (0). Bottom panel: DNA synthesis ( ) ; sporulation percentage (U) ; histidine prototrophs (S) ; canavanine-resistant colonies (v).

large increase in canavanine-resistant colonies is also apparent in Figure 6, rising to a value of 50% of CFUs at 50 hours. On CAN-URA medium, the number of colonies was about one third that on CAN medium, suggesting that recombination in the canl-ura3 interval occurs at a frequency similar to that in the control strain at 30". However, the rad57 diploid at 34.5" differs from the control stain at 30" in showing a large loss of viability (Figure 6). This drop in viability may indicate that rad57-I is still defective in some aspect of meiosis, even at its permissive temperature; however, it may be an unrelated consequence of using 34.5", since many wild-type strains do not give good spore viability at comparable temperatures. The fact that the percent canavanine-resistant CFUs (50%) was finally higher than the percent sporulation (36.5%) may indicate a higher


Time (hr) FIGURE &-Effects of a permissive temperature on premeiotic DPU'A synthesis, recombination,

sporulation and viability in diploid g.551. At zero time, a culture growing logarithmically in presporulation medium, at 30", was harvested and washed. The culture was resuspended and continuously incubated in sporulation medium at 34.5" (the permissive temperature). At intervals, samples were removed to determine viability and the progress of meiotic events as described in Figure 1. All incubations were at 34.5". Upper panel: viability (0). Middle panel: DNA synthesis ( ) ; sporulation percentage (U). Lower panel: histidine prototrophs ( W) ; canavanine-resistant colonies (v) ; canavanine-resistant uracil prototrophs (A).


loss of viability in cells that did not sporulate than in asci. This could also account for the histidine prototroph frequency reaching a higher final number per CFU than in the control strain,

DISCUSSION

We have studied the meiotic defects that occur in yeast strains carrying mutations in the rad6, rad50, rad52 and rad57 loci. We find that all four of these mutants do undergo a round of premeiotic DNA synthesis, but none of them are able to produce viable haploid spores or meiotic recombinants. The meiotic defect in rad64 differs from that in rad50,52 and 57. In rad6-2, sporulation is blocked and recombination does not occur, but cells maintain viability and undergo an apparently normal mitotic division when plated on vegetative growth medium. In rad50,52 and 57, sporulation does occur but the spores are inviable and a large drop in viability is seen in cells from the meiotic culture plated on growth medium.

The loss of viability seen in rad50,52 and 57 strains appears to commence early in the meiotic cycle, concomitantly with DNA synthesis. I t is not clear if lethal damage actually occurs at this time, or if the apparent early drop in viability represents commitment to a subsequent lethal event. In molecular terms, an ab- normal meiotic DNA synthesis would be a possible explanation for early lethality. Alternatively, a defect in the recombination process, or in the resolution of recombinant structures, could also give an apparent early drop in viability, since commitment to lethality would occur as a consequence of commitment to recombination. This is concomitant with the onset of DNA synthesis (see Figure 1 ; also SILVA-LOPEZ, ROTH and ZAMB 1976). Work in progress with a rad57-1 strain in which cells are replated at the permissive temperature after incubation in sporulation medium at the restrictive temperature, and vice versa, may indicate when the lethal damage actually occurs. I t is noteworthy that rad6, which differs from rad50, 52 and 57 in its meiotic phenotype, also differs with respect io radiation-repair and mutagenesis. It is much more sensitive to UV and has been placed in a different UV “epistasis group” (see Cox and GAME 1974). This group includes the mutant rs2 (AVERBECK et al. 1970), which resembles rad6-1 in blocking sporulation (F. ECKARDT, personal communication). However, no simple correlation between repair defects and meiosis occurs, since several other rad mutants in the same group as rad6 (error-prone repair, see LAWRENCE and CHRISTENSEN 1976) do not block meiosis. Similarly, mutants in at least one rad loms, rad 54, have an apparently normal meiosis despite being espistatic to rad52 with respect to X-ray sensitivity. (GAME and MORTIMER 1974; GAME, unpub- lished observation). Conversely, the con1 mutant isolated by ROTH and FOGEL ( R ~ T H and FOGEL 1971; FOGEL and ROTH 1974) resembles rad6-2 with respect to meiotic defects, but is not substantially UV sensitive. The situation regarding RAD6 is further complicated by the isolation by DOUTHWRIGHT-FASSE, CHRIS- TENSEN and LAWRENCE of a rad6 allele (rad6-4) that shows normal sporulation and spore viability. Such sporulation could be explained if the RAD6 gene codes for two or more separate activities, o r if it codes for a single gene product with

66 s. c. GAME et al.

two or more functions that are differently affected in different alleles (LAW- RENCE, personal communication).

The relationship between meiotic defects and mitotic recombination in rad mutants is also complex. UV-induced mitotic heteroallelic recombination in radb-1 occurs at a rate per survivor higher than in wild-type (HUNNABLE and Cox 1971 ), in contrast to meiotic recombination, which is absent. However, it is not clear whether radl-l blocks the actual process of recombination itself or simply fails to commit to recombination in sporulation medium. RAD52, which blocks live prototroph formation in meiosis, also blocks ionizing radiation- induced mitotic heteroallelic recombination ( RESNICK 1975; NAKAI, personal communication; see also this study) and lowers the rate of spontaneous mitotic prototroph formation at the his1 locus (this study). However, rad50 and rad57, which resemble rad52 in meiotic phenotype, do show UV-induced and spontaneous mitotic prototroph formation (this study; see also HUNNABLE and COS 1971). Hence, if these rad mutants do directly affect meiotic recombination (rather than killing meiotic cells in some other way) , then the processes of meiotic and mitotic heteroallelic recombination probably share at least one step (mediated by the RAD52 gene product), but differ in others (mediated by the RAD50 and RAD57 gene products).

Radiation-sensitive mutants that block meiosis have been isolated in a number of other eukaryotes (see BAKER et a1 1976 for a review), suggesting that genes whose products function both in repair and meiosis are of general occurrence. In the smut fungus, Ustilago maydis, mutations in the recl locus are defective in meiosis, as well as conferring cellular sensitivity to UV and ionizing radiation. Another mutation, rec2, which has been placed in a separate repair pathway, reduces the viability of meiotic products (HOLLIDAY 1974; HOLLIDAY et al. 1976). Hence, the situation in U. maydis may resemble that in yeast. In Drosophila. several mutants with defective female meiosis also show increased sensitivity to UV, X rays and chemical mutagens. Females carrying mutant alleles at the mei-41 loc~is are largely or completely infertile, and this locus also controls sensitivity to X rays, UV, MMS, acetylaminofluorene and nitrogen mustard (BOYD et al. 1976; BAKER et a2. 1976). The mei-9 locus, which is largely defective in meiotic recombination (BAKER and CARPENTER 1972), has been shown to be defective in repair replication of DNA after UV and X ray treatment (NGUYEN aiid BOYD 1977). It functions in a different UV-repair pathway from mei-41 (BAKER et al. 1976). Mutants in at least three other loci isolated on the basis of MMS-sensitivity Lmus(lj lO5, mus(1 j109 and mus(2 j l 1 0 ] have also been found to confer partial or complete female infertility (SMITH 1976).

The ability of yeast cells to revert to vegetative growth on appropriate medium after starting the meiotic cycel permits marc detailed characterization of mutant phenotypes than is possible with many organisms. Further information concern- ing the role of the R A D genes in meiosis may come from work with double mu- tank involving rad genes and spo genes. The latter comprise a well-characterized set of sporulation mutants that are not radiation sensitive (ESPOSITO and ESPO- SITO 1978), and a study of rad-spo double mutant phenotypes may indicate more


precisely the phase of the meiotic cycle at which the RAD gene products act. Further information as to whether RAD gene products act sequentially or independently in meiosis may come from studies with double rad-mut-ant strains, which may allow epistatic relationships or other interactions between rad mutations to be revealed.

This work was supported by a grant (GM19330) from the Public Health Service. We thank the DNA Operant Recombination Group in Absentia (DORGA) for assistance.

LITERATURE CITED

AVERBECK, D., W. LASKOWSKI, F. ECKARDT and E. LEHMANN-BRAUNS, 1970 Four radiation sensitive mutants of Saccharomyces. M d . Gen. Genet. 107: 117-127.

BAKER, B. S., J. B. BOYD, ADELAIDE T. C. CARPENTER, M. M. GREEN, T. D. NGUYEN, P. RIPOLL and P. D. SMITH, 1976 Genetic controls of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster. Proc. Natl. Acad. Sci. U S . 73: 4140-414-4.

BAKER, B. S. and A. T. C. CARPENTER, 1972 Genetic analysis of sex chromosomal meiotic mutants in Drosophila melanogaster. Genetics 71 : 255-286.

BAKER, B. S., A. T. C. CARPENTER, M. S. ESPOSITO. R. E. ESPOSITO and L. SANDLER, 1976 The genetic control of meiosis. Ann. Rev. Genet. 10: 53-134.

BOYD, J. B., M. D. GOLINO, T. D. NGUYEN and M. M. GREEN, 1976 Isolation and characterization of X-linked mutants of Drosophila melanogaster which are sensitive to mutagens. Genetics 84: 485-506.

Cox, B. and J. C. GAME, 1974 Cox, B. S. and J. M. P~RRY, 1968

ESPOSITO, R. E. and M. S. ESPOSITO, 1974a

ESPOSITO, M. S. and R. E. ESPOSITO, 1974b

Repair systems in Saccharomyces. Mutation Res. 26: 257-264. The isolation, genetics, and survival characteristics of ultra-

Genetic recombination and commitment to meiosis violet light-sensitive mutants in yeast. Mutation Res. 6: 37-55.

in Saccharomyces. Proc. Natl. Acad. Sci. U.S. 71 :3172-3176. Genes controlling meiosis and spore formation in

yeast. Genetics 78: 215-225. - , 1978 Aspects of the genetic control of meiosis and ascospore development inferred from the study of spo (sporulation-deficient) mutants of Saccharomyces cereuisiae. Biol. Cellulaire 33 : 93-102.

Mutations affecting meiotic gene conversion in yeast. Mol. Gen. Genet. 130: 189-201.

FOGEL, S. and R. ROTH, 1974

GAME, J. C., 1971 GAME, J. C. and B. S. Cox, 1971

GAME, J. C. and R. K. MORTIMER, 1974

Ho, K. S. Y., 1975

HOLLIDAY, R., 1974

HOLLIDAY, R., R. E. HALLIWELL, M. W. EVANS and V. ROWEIL, 1976

Radiation-sensitive mutants in yeast. D. Phil. Thesis, Oxford University. Allelism tests of mutants affecting sensitivity to radiation

A genetic study of X-ray sensitive mutants in yeast.

Induction of DNA double-strand breaks by X-rays in a radiosensitive strain of the yeast Saccharomyces cereuisiae. Mutation Res. 30: 327-334.

Ustilago maydis. Vol. 1, pp. 575-595. In: Handbook of Genetics. Edited by R. C. KING. Plenum Press, New York.

Genetic characterization of rec-I, a mutant o f Ustilago may& defective in repair and recombination. Genet. Res., Camb. 27: 413-453.

The genetic control of dark recombination in yeast. Mutation Res. 13: 297-309.

in yeast and a proposed nomenclature. Mutation Res. 12: 328-331.

Mutation Res. 24: 281-292.

HUNNABLE, E. G. and B. S. Cox, 1971

68 HURST, D, D. and S. FOGEL, 1% Mitotic recombination and heteroallelic repair in Saccharo-

myces cereuisiae. Genetics 50: 436458. KUENZI, M. T. and R. ROTH, 1974 Timing of mitochondrial DNA synthesis during meiosis in

Saccharomyces cerevisiae. Exptl. Cell Res. 85: 377-382. LAWRENCE, C. W. and R. CHRISTENSEN, 1976 UV mutagenesis in radiation-sensitive strains of

yeast. Genetics 82: 207-232. LAWRENCE, C. W., J. STEWART, F. SHERMAN and R. CHRISTENSEN, 1974 Specificity and fre-

quency of ultraviolet-induced reversion of an iso-l -cytochrome c ochre mutant in radiation- sensitive strains of yeast. J. Mol. Biol. 85: 137-162.

MORTIMER, R. K. and D. C. HAWTHORNE, 1%9 Yeast Genetics. pp. 385460. In: The Yeasts, Vol. 1. Edited by A. H. ROSE and J. S. HARRISON. Academic Press, New York.

MORTIMER, R. K. and D. C. HAWTHORNE, 1975 Genetic mapping in yeast. Vol. 11, pp. 221-233. In: Methods in Cell Biology. Edited by D. M. PRESCOTT. Academic Press, New York.

NAKAI, S. and S. MATSUMOTO, 1967 Two types of radiation-sensitive mutants in yeast. Muta- tion Res. 4: 129-136.

NGUYEN, T. D. and J. B. BOYD, 1977 The meiotic-9 (mei-9) mutants of Drosophila melanogaster are deficient in repair replication of DNA. Molec. Gen. Genet. 158: 141-147.

PLISCHKE, M. E., R. C. VON BORSTEL, R. K. MORTIMER and W. E. COHN, 1976 Genetic markers and associated gene products in S. cereuisiae. pp. 767-832. In: Handbook of Biochemistry and Molecular Biobgy, 3rd ed. Nucleic Acids, Vol. 2. Edited by G. E. FASMAN. Chemical Rub- ber Co. Press, Cleveland, Ohio.

Genetic control of radiation sensitivity in Saccharomyces cereuisiae. Genetics 62: 519-531.

The repair of double-strand breaks in chromosomal DNA of yeast. pp. 549-556. In: Moleculm Mechanisms for Repair of DNA. Part B. Edited by P. HANAWALT, and R. B. SETLOW. Plenum Press, New York.

RESNICK, M. A. and P. MARTIN, 1976 The repair of double-stranded breaks in the nuclear DNA 3f Saccharomyces cerevisiae and its genetic control. Molec. Gen. Genet. 143: 119-129.

RODARTE-RAMON, U. S., 1972 Radiation-induced recombination in Saccharomyces: The genetic control of recombination in mitosis and meiosis. Radiat. Res. 49: 148-154.

ROMAN, H., 1956 Studies of gene mutations in Saccharomyces. Cold Spring Harbor Symp. Quant. Biol. 21: 175-185.

ROTH, R., 1970 Carbohydrate accumulation during the sporulation of yeast. J. Bacteriol. 101 : 53- 57.

ROTH, R. and S. FOGEL, 1971 A system selective for yeast mutants deficient in meiotic recombination. Molec. Gen. Genet. 112: 295-305.

SHERMAN, F. and H. ROMAN, 1963 Evidence for two types of allelic recombination in yeast. Genetics 48: 255-261.

SILV4-LOPEZ, E., T. J. ZAMB and R. ROTH, 1975 Role of premeiotic replication in gene conversion. Nature 253: 212-214.

SMITH, P. D., 1976 Mutagen sensitivity of Drosophila melanogaster. 111. X-linked loci gov- erning sensitivity to methyl methanesulfonate. Molec. Gen. Genet. 149: 73-85.

SNOW, R., 1968 Recombination in ultraviolet-sensitive strains of Saccharomyces cereuisiae. Mutation Res. 6: 409-418.

ZAMB, T. J. and R. ROTH, 1977 Role of mitotic replication genes in chromosome duplication during meiosis. Proc. Natl. Acad. Sci. U.S. 74: 3951-3955.

Corresponding editor: R. E. ESPOSITO

J. e. GAME et al.

RESNICK, M. A., 1969

RESNICK, MICHAEL A., 1975

Documents

THE ROLE OF RADIATION (rad) GENES IN MEIOTIC ...rad GENES IN YEAST 55 curs in g538 (Figure 1) and in g550, confirming that the rad mutations studied are recessive with respect to both