Mutation Research, 284 (1992) 111-123 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

111

MUT 00379

DNA damage induced mating type switching in Saccharomyces cerevisiae Robert H. Schiestl a and Ulrike Wintersberger b a Department of Molecular and Cellular Toxicology, Haruard University, School of Public Health, Boston, MA 02115, USA and b Department of Molecular Genetics, Institute for Tumor Biology and Cancer Research, University of Vienna, A-1090 Vienna, Austria (Accepted 30 March 1992)

Keywords: DNA damage; Mating type switching; Saccharomyces cerevisiae

Summary Haploid cells of the yeast Saccharomyces cerevisiae are able to undergo a differentiation-like process: they can switch their mating type between the a and the a state. The molecular mechanism of this interconversion of mating types is intrachromosomal gene conversion. It has been shown in a variety of studies that mating type switching in heterothallic strains can be induced by DNA damaging agents, and that different DNA damaging agents differ in the length of incubation after treatment required for induction. Because X-rays induce switching immediately after irradiation and because the DNA doublestrand break repair pathway is required for switching, the event initiating heterothallic mating type switching is likely to be a DNA double-strand break. Therefore the assay for heterothallic mating type switching may screen for the induction of DNA double-strand breaks. Several aspects indicating a relationship of mating type switching to mechanisms associated with carcinogenesis are discussed.

I. Introduction During the etiology of cancer, changes in the differentiation state of cells occur. In addition, genome rearrangements are found in tumor cells and may be associated with carcinogenesis (Marx, 1982; Klein, 1981; Pall, 1981; Cairns, 1981; Wintersberger, 1982; Haluska el al., 1987). Therefore, it seems to be an important question of whether changes in the differentiation state of cells may be caused by genome rearrangements induced by carcinogens. Mating type switching in the yeast

Correspondence: Dr. R.H. Schiestl, Department of Molecular and Cellular Toxicology, Harvard University, School of Public Health, 665 Huntington Ave, Boston, MA 02115, USA.

Saccharomyces cerevisiae gives rise to a change in the differentiation state of the cells by a genome rearrangement, the interconversion between the a and the a state. Therefore, it was desirable to develop a system to measure the frequency of heterothallic mating type switching, and to determine whether this process is inducible by carcinogenic agents. This review is concerned with the inducibility of heterothallic mating type switching by DNA damaging agents. The life cycle of yeast and the mechanism of mating type switching in homothallic and heterothallic strains is described briefly, but for further and more specific information the comprehensive reviews (Herskowitz and Oshima, 1981; Klar et al., 1984b; Nasmyth, 1982; Nasmyth

112 a n d Shore, 1987) a n d the extensive list o f references cited t h e r e i n should b e c o n s u l t e d .

1.1. Life cycle of yeast Cells o f the y e a s t Saccharomyces cerevisiae exist in t h r e e cell types. T h e r e a r e h a p l o i d cells showing an a or an a m a t i n g type a n d d i p l o i d cells showing an a / a n o n m a t i n g p h e n o t y p e . H a p l o i d cells a r e able to m a t e exclusively with h a p l o i d cells o f t h e o p p o s i t e m a t i n g type to f o r m d i p l o i d zygotes, b u t h a p l o i d ceils a r e u n a b l e to s p o r u l a t e . D i p l o i d a / a cells a r e able to u n d e r g o meiosis a n d s p o r u l a t i o n b u t a r e u n a b l e to mate. D u r i n g s p o r u l a t i o n o f d i p l o i d a / ~ cells, asci a r e p r o d u c e d which c o n t a i n four h a p l o i d spores. U p o n g e r m i n a t i o n , two of t h e s e s p o r e s will develop into cells of t h e a type, a n d two will de-

v e l o p into cells of the a type. H a p l o i d as well as d i p l o i d cells are able to divide mitotically. T h e cell type ( m a t i n g type) is d e t e r m i n e d by the M A T locus on c h r o m o s o m e I I I (see Fig. 1; for review see H e r s k o w i t z a n d O s h i m a , 1981). T h e M A T locus c o n t a i n s o n e of two alleles which a r e d i f f e r e n t in t h e i r D N A s e q u e n c e . T h e Ya a n d the Y a allele cause the cells to show an a m a t i n g type o r an a m a t i n g type respectively. In diploid cells s i m u l t a n e o u s e x p r e s s i o n o f b o t h alleles is r e q u i r e d to i n d u c e meiosis a n d s p o r u l a t i o n u p o n starvation. In a d d i t i o n , m a n y o t h e r g e n e s unl i n k e d to t h e m a t i n g type locus are r e q u i r e d for p h e n o t y p i c e x p r e s s i o n o f the m a t i n g types, for m a t i n g a n d for s p o r u l a t i o n ( M a c K a y a n d M a n ney, 1974a,b), s o m e of which are a specific, some a r e a specific a n d s o m e are d i p l o i d specific.

E x p r e s s e d locus

Repressed locus

Repressed locus

A W

W

X

Yo~ Z l Z 2

W

HML~

X

Yet Z l Z 2

MATa

X

Ya Z1 HMRa

JI E x p r e s s e d locus

W

X

Ya Z l Z 2

MATa Fig. 1. The structures of the three mating type cassettes on chromosome III of Saccharomyces cerevisiae. Boxes with the same patterns contain homologous sequences. The wavy lines symbolize transcription of the MAT locus. HML and HMR are not expressed and contain silent copies of Ya and Ya respectively. All three loci are flanked by homologous sequences X and Z1. HML and MAT in addition to X and Z1 are flanked by W and Z2. Mating type switching results from replacement of the Ya sequence at MATo~ by the Ya sequence of HMRa or it results from replacement of Ya at MATa by the Ya sequence of HMLa, by an intrachromosomal gene conversion mechanism. The mating type is determined by whether the Ya or Ya sequence is expressed at MAT.

113

Therefore, the genes expressed at MAT play a regulatory role controlling a cascade of other functions, which ultimately are the effectors for phenotypic expression of the cell types (Herskowitz, 1984). The genes coding for mating type specific functions are under control of the MAT locus. In MATa cells, a specific genes are constitutively expressed, and in MATa cells, a specific genes are repressed and a specific genes are induced (e.g. Nasmyth, 1982). A mutation in MATa resuits in release of repression of a specific genes and lack of induction of a specific genes, therefore, it results in a cell showing an a phenotype (e.g., Herskowitz and Oshima, 1981).

1.2. Homothallism Homothallic cells contain the HO gene, which was originally called D for diploidization by Winge and Roberts (1949). Homothallic cells can change their mating type from a to a and vice versa, as often as every cell division. Two divisions after germination of a single spore, two of the four ceils are likely to have changed their mating type and are able to mate with the other two cells. Once the diploid state is reached after mating, no more interconversion takes place (Hicks and Herskowitz, 1977). Thus homothallic mating type switching is a very efficient mechanism to minimize the time required for a single haploid spore to reach diploidy. 1.3. Mechanism of mating type switching In addition to the HO gene, two more loci are needed for homothallic mating type switching. These alleles are HMLa on the left arm and HMRa on the right arm of chromosome III (see Fig. 1) containing silent, nonexpressed alleles of MATa and MATa respectively. For switching from MATa to MATa an intact HMLa allele is required, and for switching from MATa to MATa an intact HMRa allele is required. These alleles contain two different unique sequences Ya at HMRa and Ya at HMLa. In addition, constant sequences X and Z1, which are homologous at the two silent loci as well as at the MAT locus flank the Y sequences (Fig. 1). The result of mating type interconversion is the replacement of

Ya for Ya and vice versa at MAT. The mechanism for homothallic mating type switching has been identified to be intrachromosomal gene conversion. During a switch from a to a, HMLa serves as donor and MATa is the recipient, and during a switch from a to a, HMRa is the donor and MATa is the recipient. The original DNA at MAT is replaced and lost during the conversion process (Rine et al., 1981). The switching process in homothallic strains is initiated by the action of the H O function. The product of the HO gene is a site specific endonuclease, H O endo, which produces a double strand break at the junction between the Y and the Z sequence at MAT (Kostriken et al., 1983; Kostriken and Heffron, 1984). The same junction exists at the silent loci at HML and HMR, but cutting at these silent loci by H O endo is efficiently repressed by the products of at least four additional genes, the same genes which cause transcriptional repression of the silent loci (Klar et al., 1981). Therefore H O endo cuts exclusively at MAT. HO is expressed and switching is initiated in the late G 1 phase of haploid cells (Nasmyth, 1983) after commitment to a new cell cycle, so that after cell division two cells of the opposite mating type emerge. Following the initial cut at the YZ junction at MAT an intrachromosomal conversion event between MAT and one of the silent loci takes place. For the conversion event on the Y side of the DNA break, the entire Y sequence has to be degraded until homology is found within the X region to initiate invasion and recombination. Thus there is a gradient of coconversion events across the X region (McGill et al., 1989) which is in agreement with the involvement of the double-strand gap repair mechanism suggested by Szostak et al. (1983) for mating type switching. Heterothallic strains can change their mating type only about once per 106 cells (Hawthorne, 1963; Schiestl and Wintersberger, 1982). The switching rates of several heterothallic strains were more accurately determined by employing a special adaptation of the fluctuation assay of Luria and Delbriick (1943). The rates varied for different strains within an order of magnitude, amounting to one to ten switching events per 107 cell doublings (Klein and Wintersberger, 1988,

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for description of the fluctuation assay see section 2.2). Heterothallic strains switch inefficiently because they contain the ho allele of the HO gene, and therefore lack any detectable HO endo activity (Kostriken et al., 1983). Nevertheless, heterothallic mating type switching, like homothallic switching, leads to replacement of the mating type information at the MAT locus by the information from a silent locus (Schiestl and Wintersberger, 1982). Heterothallic switching probably depends on an accidental DNA break at MAT, because the ho allele behaves like a true HO null mutant allele (Schiestl, 1986). Almost all natural isolates of S. cerevisiae undergo efficient switching (Klar et al., 1984b). The heterothallic laboratory strains, presently in use, originated from a few natural haploid isolates showing a stable mating type (Lindegren and Lindegren, 1943), presumably mutants in the HO gene.

2. DNA damage induced switching Two assays were developed to measure the frequency of heterothallic mating type switching (ho switching), and to determine whether ho switching may be inducible by DNA damaging agents.

2.1. Conventional plating assay 2.1.1. Assay to measure the frequency of ho switching Schiestl and Wintersberger (1982) developed a system for accurately measuring the frequency of mating type switching, which was used for most of the studies carried out. Two haploid heterothallic strains with complementing markers were used. After one cell switched its mating type, it was allowed to mate with a cell with complementing markers, and the diploid cell was identified after growth on selective medium. Switches from a to a were assayed, since the reverse, a to a switches could be caused by mutation, deletion or transient phenotypic changes in addition to bona fide ho switching (McCusker and Haber, 1981; Schiestl, 1986; Repnevskaia et al., 1989; see also section 1.1). To limit switching to the 'switching strain', the 'tester strain' contained only a infor-

mation at all three loci, HML, MAT and HMR, and thus could not change its mating type. The growth medium had to serve two requirements. Switching and mating can only take place on medium in which the haploids grow very well, but for selection of the diploids, a medium selecting against the haploids is needed. Several experiments were carried out to optimize the assay (Schiestl and Wintersberger, 1982), and addition of 5% (3% in the study of Luggen-Holscher and Kiefer, 1988) of the normal concentration of full medium to the selective medium finally proved to be appropriate. In the standardized assay 1 x 10 s cells of the 'tester strain' and 0.5-1 x 10 7 cells (4 X 10 7 in the study of Luggen-Holscher and Kiefer, 1988) of the switching strain were used per petri dish. To avoid contamination with diploids, the culture of cells of the switching strain was started with a fresh colony for every experiment. Stationary phase cells of the switching strain were treated with various DNA damaging agents, and thereafter they were incubated in full medium until 10% of the cells showed buds. This was done to counteract the effect of cell cycle arrest in response to DNA damage (Brunborg and Williamson, 1978; Kupiec and Simchen, 1985; Wintersberger and Karwan, 1987; Schiestl et al., 1989c). The cells of the switching strain were mixed with ceils of the tester strain, plated onto the above selective medium, and the arising diploid colonies were counted after several days of incubation at 30°C. In a special procedure, ho switching was determined after different periods of posttreatment incubation in growth medium (Schiestl, 1989b). Because wild type cells which have changed their mating type will, in a logarithmically growing culture, mate with their siblings to produce diploids and thus will escape detection, a semisterile strain had to be used for experiments involving proliferation of the switching strain after mutagen treatment (Klein and Wintersberger, 1988). Strain VY5 was used; it harbors the ste2-2 mutation, which confers a sterile phenotype to a but not to a cells (MacKay and Manney, 1974a). As soon as an a sterile cell switches to a, it is able to mate, but it cannot mate with its siblings. It can only mate with the 'tester strain' to form a prototrophic diploid colony.

115 TABLE 1 DNA DAMAGE INDUCED HETEROTHALLIC MATING TYPE SWITCHING Agent

Highest effect. dose

Surviving switching cells rel. to contr, in %

Prototrophic colonies/ 106 survivors (fold increase) Control

Treated

X-Ray

100 Gy 60 Gy 30 J / m 2 150 J / m 2 0.20 mM 3.0 p.g/ml 2 mg/ml 3% 0.5 mg/ml 100 mM

24 45 17 45 6.6 13 64 84 10 100

0.55 3 0.42 1 0.43 0.30 0.47 0.50 (1) -

26 (48) 41 (14) 18.40 (44) 41.5 (42) 32.10 (54) 14.10 (47) 5.40 (11) 1.7 (3.3) (7) 5 × rate increase

UV MNNG 4-NQO NaNO 2 EMS FdUMP Na-butyrate

Reference

a,b,c d b,e d b,e e e c,f g h

Shown is the number of diploid cells arising from mating with haploid switching cells after treatment with different doses of the respective mutagens, calculated per number of surviving cells of the switching strain. The two data points with X-ray and UV irradiation originate from different laboratories and different strains were used which may be the reason for differences in survival levels. Chemicals (CAS Registry No.): MNNG, N-methyl-N'-nitro-N-nitrosoguanidine (70-25-7); 4-NQO, 4-nitroquinoline-N-oxide (56-575); NaNO2, nitrous acid (7782-77-6); EMS, ethyl methanesulfonate (62-50-0); FdUMP, fluorodeoxyuridylate; Na-butyrate (107-92-6). References: (a) Schiestl and Wintersberger, 1982; (b) Wintersberger and Schiestl, 1982; (c) Schiestl, 1983; (d) Luggen-Holscher and Kiefer, 1988; (e) Schiestl and Winlersberger, 1983; (f) Schiestl, 1989b; (g) Kunz et al., 1985; (h) Klein and Wintersberger, 1988.

2.1.2. DNA damage induced ho switching Table 1 summarizes the results obtained with the assay for ho switching for different DNA damaging agents. X-Rays directly induce doubleand single-strand breaks in DNA (for reviews see Ward, 1990; Frankenberg-Schwager and Frankenberg, 1990). Irradiation with UV light causes a variety of photoproducts in DNA, of which pyrimidine dimers and the pyrimidinepyrimidine (6-4) lesions are thought to be the most biologically significant ones (for review see Rubin, 1988). The repair of these photoproducts creates single-strand gaps. Irradiation with either X-rays or UV light induces recombination in yeast (for review see Kunz and Haynes, 1981). Both irradiations were highly effective in inducing ho switching (Table 1). Luggen-Holscher and Kiefer (personal communication) used particle beams, in addition to UV and X-rays (Luggen-Holscher and Kiefer, 1988), to test for their effect on ho switching. They found that 23Su, 4°Ar and 4He beams enhanced the ho switching frequency about 10-fold at the highest dose used. However, UV irradiation and X-rays were 2-fold more effective than

the particle beams for enhancing the frequency of ho switching (Luggen-Holscher and Kiefer, personal communication). MNNG is an alkylating agent (Lawley, 1968) and a powerful carcinogen. 4-NQO induces GC to AT transitions and GC to TA transversions in yeast (Prakash et al., 1974) and is also a strong carcinogen. Nitrous acid is a direct mutagen by deamination of cytosine and adenine leading to base substitutions. EMS is an alkylating agent and is widely used as a mutagen and carcinogen (for review see Sega, 1984). An interesting observation is that after a single exposure to EMS, lethal fixation of the DNA damage may occur as late as during the third posttreatment generation (Klein et al., 1990). All four mutagens are known to induce mitotic recombination in yeast (for review see Zimmermann et al., 1984) and they enhance the frequency of ho switching severalfold. MNNG and 4-NQO were most effective (about 50-fold), nitrous acid was less, and EMS was least effective (Table 1). Deoxyribonucleotide pool imbalance entails a variety of genetic consequences (reviewed by Kunz, 1982). FdUMP inhibits synthesis of dTMP

116

in yeast, and thus disturbs the nucleotide pool. FdUMP is cytotoxic and highly recombinogenic, but does not induce nuclear mutations (Kunz et al., 1980). The frequency of ho switching is 7-fold enhanced after treatment of cells with FdUMP (Table 1, Kunz et al., 1985). Since butyrate has been tested with the fluctuation assay the experiments with butyrate will be explained in section 2.2.3 dealing with the fluctuation assay. To test whether the ability to mate with cells of the prior identical mating type actually was a result of a change in the mating type of the switching strain, caused by a bona fide switching event, the obtained diploid colonies were routinely checked for their sporulation ability (Schiestl and Wintersberger, 1982, 1983; Kunz et al., 1985; Luggen-Holscher and Kiefer, 1988). Expression of the information at both loci MATa and MATa is required for sporulation. Therefore, in a sporulating diploid MATa has to be expressed de novo, since it was not expressed in any of the haploid cells before switching. Therefore, the ability of the resulting diploids to sporulate is a first indication that one cell of the switching strain has switched its mating type prior to mating with, instead of merely fusing with one cell of the tester strain. Almost all colonies obtained from DNA damage induced switching events were able to sporulate (Schiestl and Wintersberger, 1982, 1983; Kunz et al., 1985; Luggen-Holscher and Kiefer, 1988). The apparent increase in the number of diploid colonies after treatment with DNA damaging agents could still be caused by other mechanisms in addition to bona fide mating type switching (i.e. the replacement of the Ya information at MAT by Yc~). Other causes could be reciprocal recombination leading to a ring chromosome and to expression of HMLa (Strathern et al., 1979; Haber et al., 1980), or rescue by mating of a spontaneously switched cell which has encountered a lethal mutation (which would give rise to an apparent increase of switching events if calculated per number of surviving colonies). These possibilities can be sorted out by genetic (tetrad) analysis of the diploid clones and by Southern blotting. Tetrad analysis and Southern blotting indicated that almost all diploids obtained after

treatment with DNA damaging agents derived from bona fide mating type switching events (Schiestl and Wintersberger, 1982, 1983; Kunz et al., 1985). In addition, rescue by mating accounted for less than 10% of the events and occurred only at the highest doses of the agents, and therefore cannot explain the increases in switching events obtained (Schiestl and Wintersberger, 1982). In addition, if the switching frequency was calculated per number of plated cells, instead of per number of surviving cells, substantial increases were still found for all agents. 2.1.3. Induced and spontaneous ho switching in DNA repair deficient mutants In an attempt to determine the genetic control of ho switching, mutants from each of the three DNA repair epistasis groups of yeast (doublestrand break repair, excision repair and postreplication repair) were tested for their spontaneous and radiation induced switching frequencies. For detailed information about the DNA repair pathways in yeast, the following relevant reviews may be consulted: Haynes and Kunz (1982), Friedberg (1988), Rubin (1988). The RAD52 gene is involved in double-strand break repair (Ho, 1975; Resnick and Martin, 1976), meiosis and homothallic mating type switching (Malone and Esposito, 1980; Weiffenbach and Haber, 1981). Other recombination events are RAD52 independent (see references cited in Schiestl, 1986). Most of the mitotic interchromosomal (Malone and Esposito, 1980; Prakash et al., 1980) and intrachromosomal recombination events (Jackson and Fink, 1981; Klein, 1988; Schiestl and Prakash, 1988) require the RAD52 function, but some fraction of recombination events is RAD52 independent. On the other hand, ho switching is absolutely dependent on the RAD52 function. In fact, the spontaneous frequency of switching shows at least a 103-fold decrease in rad52 mutants, and the X-ray and UV induced frequencies show at least a 104-fold decrease (Schiestl, 1986). RAD3 is involved in excision repair (Cox and Game, 1974; Prakash, 1977). The rad3-2 mutation has almost no influence on spontaneous and X-ray induced ho switching, but it reduced induction by UV light (Schiestl, 1986). Reduced UV

117 induction has been previously reported for mitotic gene conversions in the rad3 mutant and it has been suggested to be due to unrepaired dimers hindering the conversion process rather than to R A D 3 being directly involved in induced recombination (Kern and Zimmermann, 1978). It may be equally possible that incisions at closely overlapping lesions, on opposite strands, may result in a double-strand break in the wild-type strain. The incision and the resulting doublestrand breaks would not be formed in a rad3 mutant. More recently, it has been shown that R A D 3 is a D N A helicase (Sung et al., 1987). Thus it may be tempting to speculate that local unwinding during excision repair may facilitate recombination at the site of damage. RAD18 belongs to the postreplication repair epistasis group (for review see Haynes and Kunz, 1981) and mutants in that gene show 10-70-fold elevated levels of spontaneous mitotic recombination (Boram and Roman, 1976; Schiestl et al., 1990 and references cited therein). On the other hand, the frequency of ho switching was only marginally, and not significantly, elevated in rad18 mutants (Schiestl et al., 1990).

2.1.4. Kinetics of induction during cell proliferation Different D N A damaging agents differ greatly in their kinetics of induction during incubation after treatment (Schiestl, 1989b). Irradiation with X-rays resulted in increase of the frequency immediately after exposure and no further increase was seen during incubation after treatment (Fig. 2). Nitrous acid and 4-NQO, on the other hand, did not cause any increase in frequency immediately after treatment, but required incubation of treated cells in growth medium to produce an enhanced frequency of heterothallic mating type switching (Fig. 2). U V irradiation (Fig. 2) and EMS (Schiestl, 1989b) result in induction to certain levels immediately after treatment, but further induction was seen during posttreatment incubation (Fig. 2). These results may indicate that certain kinds of D N A damage require repair or replication to be converted into recombinagenic lesions inducing ho switching. Alternatively, a D N A repair system may be induced, including

25

20

O

15

o ,-.i

I

0

1

2

No. of population doublings Fig. 2. The effect of incubation in complete medium after treatment of cells with DNA damaging agents on the frequency of heterothallic mating type switching is shown (as described in Schiestl, 1989). Samples were taken after emergence of 10% buds (0 generations), and after 1 and 2 population doublings in complete medium before cells were added to the 'tester strain' to test for heterothallic mating type switching. The number of diploid colonies arising from matings of haploid cells per 10 6 surviving cells of strain VY5 are shown. The doses of the DNA damaging agents were adjusted to give approximately 25% survivors. The symbols indicate: • control; [] nitrous acid, 3.5 mg/ml; • X-irradiation, 10 krad; o 4-NQO, 1.7 mg/ml; • UV irradiation, 35 J/m e. DNAases, which may enhance throughout the entire genome.

recombination

2.2. Fluctuation assay 2.2.1. Limitations of the conventional plating assay As described in section 2.1.4, enhanced frequencies of mating type switching were observed,

118

if cells induced for switching were plated (together with the cells of the tester strain) immediately after treatment with some DNA damaging agents. But, after proliferation of the exposed cells a further increase (to different degrees) of the fraction of switched cells was observed (Schiestl, 1989b). This could mean that the state of enhanced mating type interconversion frequency may continue over several generations after treatment, a presumption worthy of further experimental examination. Unfortunately, the conventional test system of directly plating cells from large populations on selective medium immediately after treatment has several limitations. Above all it compares frequencies and not rates of genetic events occurring in treated and untreated populations. These events are counted as colony forming ceils on selective medium, without information of whether the genetic change took place directly in the treated cell, or in a descendant arisen by residual divisions of nonswitched (and in general nonmutated) ceils, the number of cells, of which the genetically changed ones comprise a 'fraction', is unknown. Furthermore, direct plating disregards the influence of D N A damaging agents on the progression of cells through the cell cycle (Wintersberger and Karwan, 1987). Our 'conventional' switching assay only considered the lag until the emergence of the first buds after treatment. Also, residual divisions of lethally damaged cells and the appearance of inviable cells during later generations (Klein et al., 1989, 1990) escape observation. The assignment of the appearance of a genetic variant to a certain generation of the growing population would, however, be of great interest, e.g. in connection with speculations about the mechanisms of radiation and chemical carcinogenesis (Wintersberger and Klein, 1988).

2.2.2. Rationale for the fluctuation assay What one actually wants to determine is the kinetics of the changing rate of a genetic event (i.e. the number of events/cell division) during the proliferation of an induced population. In other words, one would like to reveal the shape and the height of the 'wave of induction' migrating through the growing population. To this aim a method for mathematically evaluating experimen-

tal data, obtained from fluctuation assays, in addition to those already available, had to be worked out. The principle of the fluctuation assay is that a large cell population is divided into many small independently growing subpopulations so that rare probabilistic events like mutations, gene conversions, etc., occur at different frequencies in the subpopulations. The mode of distribution of the events over the subpopulations (after their undisturbed proliferation without selection over several generations) is the basis for the calculation of the rates of the genetic events. Whereas for the determination of the expectedly constant rates of spontaneous mating type switching the Pc, method and the tables of Lea and Coulson (1949) could successfully be used (Klein and Wintersberger, 1988), rates varying with time had to be estimated by a newly developed computer simulation program (Klein, 1987). The program works such that the computer assumes certain kinetics for the variation of the rate over several generations of growth, and that it reports the distributions of the events (in our case the numbers of the switched cells) over the subpopulations which would be likely to result from the assumed rate kinetics. Next the computer searches for the best fit of one of its proposals for distributions to the real experimental data. In this way the most probable rate kinetics explaining an experimental distribution can be determined (Wintersberger and Klein, in preparation). It should be stressed that this test system attributes genetic events to certain posttreatment generations of the total surviving fraction of the treated cell population, i.e. it determines whether the events had happened before or after the first, second, third, and so on, doubling of the total number of the surviving cells. The system of course offers no information of whether or not the particular cell in which a genetic event actually took place was a zero-, first-, second- (and so on) generation cell, because the cell might be just one which has not divided, or it might descend from a cell family which has divided more or less often since the treatment than the bulk population. Naturally, the system is subject to the dilemma intrinsic to all attempts of getting exact information about randomly occurring rare events: While looking at high enough numbers of individuals to obtain

119

statistically relevant data the information about the fate of the individual is lost. The test system can also not discriminate between two possible reasons for the continuation of the enhanced switching rate, namely a preservation of DNA lesions over several rounds of D N A replication or the persistence of an induced state of an enhanced interconversion rate as indicated in section 2.1.4.

2.2.3. Results obtained with the fluctuation assay With all necessary precautions in mind it could be shown that in X-irradiated populations the mating type interconversion rate was enhanced in a dose dependent mode for the first two posttreatment generations of the surviving cells. During the following two generations (i.e. up to the fourth generation after treatment) a rate slightly higher than for untreated populations was observed. The kinetics of enhanced switching rates for populations treated with UV light was found different from that of the X-irradiated cells. The highest rates were obtained for generation two to four after treatment, then for the higher UV dose tested the induced rates stayed less but significantly enhanced up to the sixth generation before the spontaneous value was reached again (Table

2). A more detailed description of the results is in preparation (Wintersberger and Klein). It should be noted that rates obtained with the fluctuation assay cannot directly be compared with frequencies obtained with the direct plating system. Interestingly, the presence of 0.1 M sodium butyrate (which triggers differentiation in certain mammalian cells in vitro, but which, according to current knowledge, does not produce DNA damage) increases the 'spontaneous' switching rate 5-fold (Klein and Wintersberger, 1988).

3. DNA damaging agents induced illegitimate mating Inge-Vechtomov et al. (1985) used the phenotypic change from mating type a to a as test system for genetic activity in yeast. They showed that /3-propiolactone, 1,2,7,8-diepoxyoctane, acridine mustard ICR-170, ethyl methanesulfonate, and 2-aminofluorene were positive. Since loss of the MATa function leads to an a mating type (for reviews see Herskowitz and Oshima, 1981; Nasmyth, 1982), a to a switches can in addition to bona fide ho switching be caused by mutation, deletion or transient phenotypic changes (McCusker and Haber, 1981; Schiestl,

TABLE 2 SWITCHING RATES OF HETEROTHALLIC POPULATIONS OF S. cerevisiae PROLIFERATING AFTER A SINGLE T R E A T M E N T WITH X-RAY OR UV LIGHT Irradiation

Mean rates ( x 107) a estimated for different periods of population growth b (1-2

X-ray (Gy(% CFC)) c 0(90) 5(92) 20(74) 40(49) 90(27) 160(17)

1.7+0.8 2.4+1.1 6.3+2.0 8.6+1.0 15.7+3.4 28.0+6.0

UV ( J / m 2 ( % CFC)) c 0(90) 20(65) 35(34)

1.5+0.4 3.7+2.6 12.6+6.4

2-4

4-6

6-8

1.7+0.8 2.0+0.6 4.6+1.1 3.7+1.4 4.3+0.7 6.8+1.8

1.7+0.8 1.7+0.4 1.0+0.6 1.1+0.6 1.7+0.4 3.1+0.6

n.d.

1.5+0,4 8.5+2,1 24.4+4.0

1.0+0.3 1.6+0.8 5.6+1.2

1.0+0.3 1.1+0.3 2.6+0.4

The rate is defined as conversions/number of cell divisions. b The periods are given in population doublings, e.g. 0 - 2 denotes the period from the start of population growth up to the 2nd doubling, 2-4 the period between the 2nd and 4th doubling, etc. c % colony forming cells directly after treatment.

120

1986; Repnevskaia et al, 1989). In fact the majority of the diploids obtained in the study of IngeVechtomov et al. (1985) did not show any genetic change and it has been suggested that the development of diploids is due to transient 'mutagenic' changes which have not been fixed yet (Repnevskaia et al, 1989). Therefore it seems to be difficult in our view to sort out transient phenotypic changes in the cells inducing cell fusion, for instance changes at the membrane or cell wall caused by the treatment, which would not be the result of a genotoxic effect. 4. Discussion

Two assays have been developed to measure the frequency or the rate of heterothallic mating type switching (ho switching). With these assays it has been shown in a variety of studies that ho switching is inducible by DNA damaging agents. Furthermore, heterothallic mating type switching (Schiestl, 1986), like homothallic switching (Malone and Esposito, 1980; Weiffenbach and Haber, 1981), is completely dependent on the function of the RAD52 gene. The RAD52 gene is involved in recombinational repair (for review see Kunz and Haynes, 1981), which is the major mechanism of repair of double-strand breaks and gaps in yeast (Ho, 1975; Resnick and Martin, 1976). It has been proposed that the absolute dependence of ho switching on the RAD52 function may indicate that the mechanism of ho switching proceeds via a double-strand break intermediate (Schiestl, 1986). Similarly, homothallic switching, a process known to involve the double-strand gap repair mechanism of yeast (Szostak et al, 1983), is also absolutely dependent on RAD52 (Klar et al, 1984a). Even though we do not have any direct physical evidence that ho switching is induced by double-strand breaks, the above genetic result, together with other results (see below), would imply that the assay for ho switching measures the frequency of doublestrand breaks produced by the D N A damaging agents. These breaks may occur directly under the influence of a DNA damaging agent, in the course of the repair of some DNA lesion, or because of the induction of a general recombination mechanism.

The study comparing different DNA damaging agents for their differences in the kinetics of induced ho switching during incubation after treatment, showed that irradiation with X-rays led to the full level of increase immediately after irradiation (Schiestl, 1989b). Nitrous acid and 4NQO, on the other hand, did not cause any increase in frequency immediately after treatment, but required posttreatment incubation to produce an increased frequency of heterothallic mating type switching. UV irradiation and EMS resulted in induction to certain levels immediately after treatment, but further induction was seen during posttreatment incubation. Experiments with the fluctuation assay also indicated that induction by UV becomes effective during later generations than induction by X-rays (Table 2). Since X-rays cause double-strand breaks directly (for reviews see Ward, 1990; FrankenbergSchwager and Frankenberg, 1990), this may be a further indication that ho switching is inducible by double-strand breaks. Other DNA damaging agents may produce lesions leading to doublestrand breaks during DNA repair or replication. If it is assumed that some kinds of DNA lesions may survive a round of DNA replication (see, e.g., Klein et al., 1990), it is imaginable that repair induced DNA breaks may also occur after one or even several divisions of treated cells. Mutations in radl8 show a hyperrecombination phenotype for interchromosomal recombination (Boram and Roman, 1976; Schiestl et al., 1990 and references cited therein). On the other hand, the frequencies of intrachromosomal recombination including ho switching is only marginally, and not significantly, elevated in the radl8 mutant (Schiestl et al., 1990). It has been suggested, that these results indicate a difference in the mechanism of interchromosomal and intrachromosomal recombination (Schiestl et al., 1990). These results also support the idea that the assay for ho switching may identify a spectrum of DNA damaging agents other than the commonly used recombination tests with yeast (reviewed by Zimmermann et al, 1984), and therefore may represent a valuable addition to the repertoire of recombination assays. For instance, another assay also selecting for intrachromosomal recombination events, the DEE assay, has been shown to be

121

inducible by nonmutagenic carcinogens (Schiestl, 1989a; Schiestl et al., 1989a,b). These are agents which cause cancer, but do not induce genetic activity in a variety of short term tests including tests for interchromosomal recombination in yeast. The fluctuation assay can be used for the determination of switching rates constant as well as varying with time. In addition other interesting questions can be studied. The growth conditions of the subpopulations may be altered, and tested for their influence on spontaneous as well as induced switching rates. The presence of sodium butyrate (which triggers differentiation in certain mammalian cells in vitro, but which, according to current knowledge, does not produce DNA damage) increases the spontaneous switching rate (Klein and Wintersberger, 1988). Substances suspected to modulate the effects of DNA damaging agents can be tested for their influence on the rate kinetics by adding them to the growth media. Because population sizes are evaluated before and after the proliferation of the subpopulations, the results are not blurred by possible growth inhibiting effects of modulators. Thus, antagonistic and synergistic effects of drugs can be diagnosed. The fluctuation assay together with the computer simulation unveils whether a drug, which in the plating assay diminished the number of induced mutants, convertants, and so on, did so by just inhibiting the residual growth on the selective plates, or by actually decreasing the rates of these events. In several respects the mechanism of ho switching relates to events which are associated with carcinogenesis. Phenotypic mating type switching is a process giving rise to a change in the differentiation state. Changes in the differentiation state of cells also occur during the etiology of cancer. On the molecular level, ho switching is a genome rearrangement giving rise to an alteration in gene expression by a change in the position of a regulatory gene within the genome. Genome rearrangements are also found in tumor cells, and may be associated with carcinogenesis (Marx, 1982; Klein, 1981; Pall, 1981; Cairns, 1981; Wintersberger, 1982; Haluska et al., 1987). Even though mating type switching is part of the normal life cycle of yeast, whereas genome rear-

rangements associated with cancer are most certainly not part of the normal cycle of eukaryotic somatic cells, the yeast system could detect agents which initiate these rearrangements, and it is conceivable that the mechanisms may be similar, i.e. due to induction by a double-strand break. Therefore it seems justified to further study the mechanism, genetic control and inducibility of heterothallic mating type switching.

Acknowledgements Thanks are due to Tom Petes for encouragement and support and R. Daniel Gietz for discussion. U.W. was supported by grants from the Jubil~iumsstiftung der (~sterreichischen Nationalbank (Grants 2108, 2993). R.H.S was supported by funds from the Department of Molecular and Cellular Toxicology, Harvard University, School of Public Health.

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