Aug 8, 1988 - tional repair of damaged DNA in the germ line of organisms ... whether DNA damage promotes mating, as distinct from ..... theories of aging.
Vol. 171, No. 4
JOURNAL OF BACTERIOLOGY, Apr. 1989, p. 1893-1897
0021-9193/89/041893-05$02.00/0 Copyright © 1989, American Society for Microbiology
Sexual Reproduction as a Response to H202 Damage in Schizosaccharomyces pombe CAROL BERNSTEIN* AND VIRGINIA JOHNSt Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, Arizona 85724 Received 8 August 1988/Accepted 6 January 1989
Although sexual reproduction is widespread, its adaptive advantage over asexual reproduction is unclear. One major advantage of sex may be its promotion of recombinational repair of DNA damage during meiosis. This idea predicts that treatment of the asexual form of a facultatively sexual-asexual eucaryote with a DNA-damaging agent may cause it to enter the sexual cycle more freqtiently. Endogenous hydrogen peroxide is a major natural source of DNA damage. Thus, we treated vegetative cells of Schizosaccharomyces pombe with hydrogen peroxide to test if sexual reproduction increases. Among untreated stationary-phase S. pombe populations the sexual spores produced by meiosis represented about 1 % of the total cells. However, treatment of late-exponential-phase vegetative cells with hydrogen peroxide increased the percentage of meiotic spores in the stationary phase by 4- to 18-fold. Oxidative damage therefore induces sexual reproduction in a facultatively sexual organism, a result expected by the hypothesis that sex promotes DNA repair.
Most organisms devote considerable effort to sexual reproduction. The short-term costs of sex are large, however, and include the costs of males (19, 33), high recombinational load (29), and mating (4). To balance these costs the adaptive benefit of sex must be large. Genetic variation has traditionally been proposed to be the major benefit of sex, but there are difficulties in finding short-term benefits of variation (5, 21). It has been proposed that a major short-term benefit of sexual reproduction is its promotion of efficient recombinational repair of damaged DNA in the germ line of organisms (2, 5). In eucaryotes it is likely that recombinational repair occurs during meiosis, leading to the removal of potentially lethal damages from germ line DNA. One prediction of the repair hypothesis of sex is that a facultatively sexual-asexual organism may enter the sexual cycle more frequently when treated with a DNA-damaging agent. Mating (outcrossing) and recombination of DNA can be regarded as the two phenomena central to sexual reproduction. Only one previous study addressed the question of whether DNA damage promotes mating, as distinct from recombination. Bacteriophage T4 has been shown to undergo mixed infection, a form of mating, more frequently after the introduction of DNA damage (3). However, it is important to know if an organism with meiotic sex (a eucaryote) also mates more frequently upon the introduction of DNA damage. Thus, the eucaryotic yeast Schizosaccharomyces pombe was tested to determine whether sexual reproduction is increased in response to treatment with a DNA-damaging agent. An S. pombe wild-type population is a mixture of two mating types, since a cell of one mating type can give rise to a cell of the opposite mating type every two generations (17). A vegetative cell can enter the mitotic cell cycle and continue to grow vegetatively, or it can form an aggregate with a cell of the opposite mating type, fuse, undergo meiosis, and form sexual spores. In growth medium with 250 ,ug or more of NH4Cl per ml (adequate nitrogen), the frequency of sexual spores is less than 10-3 during growth. If nitrogen is
removed and incubation is continued for 24 h, about 20% of the cells form zygotic structures or sporulate (26). In the experiments to be described, hydrogen peroxide (H202) was used as the DNA-damaging agent. H202 is a by-product of oxidative cellular metabolism and has recently been proposed to be ah important natural cause of oxidative DNA damage in cells (1, 8). In human cells there may be considerably more than 1,000 oxidative DNA hits per cell per day because of H202 and its product, the OH radical, produced during normal metabolism. H202 can damage proteins (10) and lipids (31) as well as DNA. However, the major lethal effect of H202 appears to be on DNA. The damaged proteins seem to rapidly turn over (10). Lipid peroxidation is a chain reaction having initiation, propagation, and termination steps. Intermediates in all three steps are thought to cause DNA damage (31). That the primary attack of H202 is on DNA is further suggested by the fact that in Escherichia coli, mutants defective in the recA or xth pathways of DNA repair have 10 and 50 times as many lethal hits per unit H202 treatment, respectively, as have cells which are wild type for DNA repair (11, 34). Treatment of E. coli with external H202 generates single-strand breaks in intracellular DNA (12). Imlay and Linn (15) presented an extensive review of the mechanism by which H202 causes DNA damage in E. coli. They indicated that the Fenton reaction is the basis of H202 contribution to mutagenicity and cell death and pointed out that cellular DNA is a weak link in resistance to oxygen radicals. The yeast Saccharomyces cerevisiae, treated extracellularly with H202, shows increased levels of thymine glycol in its DNA (one type of H202-induced DNA damage) in proportion to the dose of H202 used (16). Thus, externally added H202 enters yeast cells and causes DNA damage. MATERIALS AND METHODS Wild-type S. pombe 968 cells were kindly provided by P. Nurse. Cells were prepared by growth in EMM2-M medium (22, as modified in 25) with NH4C1 at 5 mg/ml in an oscillating water bath at 25°C to about 1.5 x 107/ml. This is about 1.5 generations before the stationary phase is reached in this nitrogen-rich medium. At this concentration the diffusible sex factors that may be utilized by S. pombe to
* Corresponding author. t Present address: Office of Management Analysis and Research, University of Arizona, Tucson, AZ 85721.
1893
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20 40 60 80 0 20 40 60 80 Minutes 20% Ethanol Minutes 30% Ethanol FIG. 1. Killing of vegetative cells and spores by ethanol. Cells were suspended at 1.5 x 106/ml in EMM2-M medium with no NH4Cl and incubated at 25°C for 6 (0), 26 (0), 48 (l), 96 (a), and 144 (A) h. The vegetative cells and spores that had formed at each time point
were disaggregated with pronase at 50 xg/ml at 37°C for 1 h, treated for the times indicated with 30% ethanol (A) or 20% ethanol (B), diluted, and plated.
initiate conjugation (9) should be available. The cells were pelleted, washed once with 1% saline, and suspended in the medium appropriate for each experiment. RESULTS S. pombe cells form aggregates as they enter the stationary phase or are starved for nitrogen (9). Therefore, sonication (26) and pronase (7) treatments were tested as disaggregation methods to estimate cell number accurately in succeeding experiments. Cells prepared as described in Materials and Methods were suspended at 1.5 x 106/ml in EMM2-M medium with no NH4Cl and incubated for 24 h at 25TC to induce aggregation, mating, and spore formation. Aggregated cells were treated with sonication (50 W for 5, 10, or 15 s) or with pronase (50 ,ug/inl [CALBIOCHEM] at 37°C for up to 120 min). CFU increased a maximum of threefold with either treatment, and cells viewed with a microscope were seen to be disaggregated. Sonication under the conditions used, however, begah to kill CFU when carried out for more than 5 s. Pronase, on the other hand, released a maximum number of CFU at 40 min, and the number of CFU did not decrease with further treatment. Thus, subsequently, 60 min of pronase treatment was used for disaggregation. Ethanol at 30% has been reported to kill vegetative cells but not spores (14). To test the killing effect of ethanol, we initiated sporulation of vegetative cells by transfer of the population to medium without nitrogen, and samples taken at various times were observed with a light microscope at 400x under phase-contrast optics. Judged by their appearance in the microscope, at 6 h the population was entirely vegetative cells. At 26 h there were single vegetative cells, paired cells, aggregates of more than two cells, zygotes, and asci which contained spores. At 48 h there were a few zygotes and what seemed to be disintegrating asci and free spores. At 96 and 144 h there seemed to be only spores. The vegetative cells and/or spores in each of these samples were treated with either 30% ethanol (Fig. 1A) or 20% ethanol (Fig. 1B). Both vegetative cells and spores were killed by 30% ethanol, while 20% ethanol quickly killed vegetative
Minutes of H 202 Treatment FIG. 2. H202 inactivation of vegetative cells. Cells were suspended in a lOx volume of either EMM2-M medium with NH4Cl at 250 ,ug/ml (0) or tMM2-M medium with no NH4Cl (U). After 6 h of incubation in an oscillating water bath at 25°C, the cells were treated with freshly mixed 43 mM H202 and 10 ,uM CuSO4 for the times indicated. The cells were stirred vigorously, and a sample was removed and treated with catalase (Sigma Chemical Co.) at 0.018 mg of protein per ml to inactivate the H202. The cells were treated with pronase at 50 ,ug/ml at 37°C for 1 h, diluted, and plated. Panels A and B show the results of two separate experiments.
cells (6-h sample) but allowed the majority of spores to survive (26-, 48-, 96-, and 144-h samples). Spore maturity had some effect on resistance to ethanol. S. pombe was next treated with H202 to determine the range of H202 exposure that would result in low to moderate killing. Figure 2 shows the survival of vegetative cells following various exposures to 43 mM H202 (duplicate experiments are shown in panels A and B). Growth for 6 h in nitrogen-deficient medium allowed vegetative cells to become somewhat H202 resistant. In either adequate-nitrogen medium or nitrogen-deficient medium, H202 exposures for 5 to 30 min resulted in ca. 100% to ca. 2% survival. This is the equivalent of zero to four lethal hits per cell, an appropriate range for testing the effect of DNA damage on mating. As a further control, the frequency with which vegetative cells not treated with H202 can mate and form spores was determined under both nitrogen-deficient conditions (Fig. 3A) and adequate-nitrogen conditions (Fig. 3B). The vegetative cells used were those from which the zero-treatment survival points in Fig. 2A and B were determined. When cells were incubated in nitrogen-deficient medium meiosis was promoted (Fig. 3A). By 24 h there were equivalent numbers of ethanol-resistant spores and total CFU. The measurement of ethanol-resistant spores involves their release by snail enzyme (,3-glucuronidase) (14) from asci (which usually contain four spores). In measurements of total CFU each ascus gave rise to a single colony. These results imply that after 24 h in nitrogen-deficient medium about 25% of the CFU are asci and 75% are vegetative cells that can be killed by ethanol. When cells were incubated in medium with adequate nitrogen meiosis was not promoted (Fig. 3B). By 24 h the ratio of ethanol-resistant spores to viable CFU was about 10-. After incubation for 144 h (6 days) the ratio of spores to total CFU rose to about 2 x 10-2. For both nitrogendeficient conditions and adequate-nitrogen conditions, most spore formation took place while the populations were in the stationary phase.
1895
SEX IN RESPONSE TO H202 DAMAGE IN S. POMBE
VOL. 171, 1989
101A plus
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FIG. 3. Ratio of spores to CFU. Cells treated with catalaseinactivated H202 (zero-treatment controls in Fig. 2) were monitored after sham treatment to measure the ratio of spores to CFU. Incubation was continued in EMM2-M medium with no NH4Cl (A) or EMM2-M medium with NH4Cl at 250 jig/ml (B). The cells incubated in EMM2-M medium with no added H202 had a very small amount of nitrogen available from the catalase used to inactivate the H202 (3 jig of nitrogen per ml within the enzyme), an amount that should not have affected the rate of sporulation (26). The cells were incubated at 25°C for the total times shown (including 6 h of incubation before the sham treatment) and vortexed to break up any clumps, and six samples of the cell suspension were removed. In three samples the cells were disaggregated with pronase at 50 ,ug/ml at 37°C for 1 h, diluted, and plated for CFU. The other three samples were diluted 10-fold into 0.2% P-glucuronidase (Sigma) in distilled water and incubated at 32°C for 21 h to release the spores from asci (14). These three samples were then brought to 20% ethanol, incubated for 10 min at 32°C, and plated to measure spore numbers. The ratio of spores to CFU is plotted. The symbols (0 and *) represent data from two experiments.
The central experimental point was then addressed. Cell populations treated with H202 were examined to see if their frequencies of sexual spore formation increased over the spontaneous levels. The cells were those used to assay survival after H202 treatment for 7.5 and 15.0 min (Fig. 2A) and 8.0, 12.0 and 28.0 min (Fig. 2B). The cells were incubated in either nitrogen-deficient medium (Fig. 4A) or adequate-nitrogen medium (Fig. 4B). The ratios of spores to CFU were determined after 24, 48, 96, and 144 h of incubation. Upon incubation in nitrogen-deficient medium, which by itself causes mating and sporulation, the frequency of spores in the H202-treated samples was about the same as that in the samples not treated with H202 for each time of incubation (Fig. 4A). Upon incubation in adequate-nitrogen medium, however, the H202-treated cells underwent mating and spore formation at a 4- to 18-fold higher rate than did cells incubated in adequate-nitrogen medium but not exposed to H202 (Fig. 4B). The actual factors by which the frequencies of spores in the H202-treated populations exceeded the frequencies of spores in the untreated populations are given in Table 1. The average factor of increase was 8.4-fold. There was no systematic trend with time of H202 treatment and no systematic trend in the factor of increase in spore
nnnl . nvj.vvv
120
formation beyond 48 h of incubation of the damaged
cells. It might be possible that vegetative cells growing in adequate nitrogen, when treated with H202, could become induced to resistance to ethanol. Such an occurrence would artificially increase the apparent spore number in adequatenitrogen cultures, as measured here. To test for this possibility, we measured asci and spores when cells were grown
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FIG. 4. Ratio of spores to CFU after treatment of cells with H202 and incubation for the times shown. The cells were sampled from the experiments shown in Fig. 2. The cells used were those treated with H202 for 7.5 min (0) or 15 min (O) (Fig. 2A) and for 8 min (0), 12 min (U), or 28 min (A) (Fig. 2B). (A) Cells incubated in EMM2-M medium with no added H202. (B) Cells incubated in EMM2-M medium with 250 ,ug of NH4Cl per ml. The incubation times shown were measured from the time cells were suspended (6 h before H202 treatment). The dashed lines show the spore/CFU ratios that occurred when inactivated H202 was used (Fig. 3).
in both nitrogen-deficient and adequate-nitrogen media for the experiment shown as filled symbols in Fig. 4. To measure asci, we treated three samples of each culture with ethanol without releasing spores with snail enzyme (P-glucuronidase). This procedure should kill susceptible free vegetative cells but not asci which contain spores. Spores were measured after both snail enzyme treatment and ethanol treatment as described in the legend to Fig. 3. If a significant TABLE 1. Factors of increase in spore formation in populations of S. pombe cells treated with H202'
Expt
of H202 Length treatment (min)
Length of(h) incubation
Factor of increase in spores
1
7.5
48 96 144
5.6 7.7 4.3
15.0
48 96 144
17.6 4.2 4.0
8.0
48 96 144
8.2 7.3 7.1
12.0
48 96 144
13.0 13.4 7.7
28.0
48 96 144
6.0 15.5 5.1
2
The populations were those inactivated for 7.5 and 15.0 min in Fig. 2A (experiment 1) and 8.0, 12.0, and 28.0 min in Fig. 2B (experiment 2). At each time of incubation, the frequency of spores in the H202-treated population was divided by the frequency of spores in the untreated population to yield the
factor of increase in spore formation. The comparisons are between the open symbols in Fig. 4B and the squares in Fig. 3B (experiment 1) and between the filled symbols in Fig. 4B and the circles in Fig. 3B (experiment 2).
1896
BERNSTEIN AND JOHNS
J. BACTERIOL.
TABLE 2. Comparison of the number of spores per milliliter observed and the maximum number predicted by the model of vegetatively dead cells mating and forming viable spores'a Length of Expt
LefH2
(reamen) (min)
Predicted maximum
Observed
cells/ml
sore/m spores/ml
spores/ml
0.00 X 106 1.88 x 106 2.82 x 106
0.08 x 106 0.39 x 106 0.32 x 106
106
0.28 x 106
106 106 106
1.36 x 106 1.84 x 106 1.63 x 106
No. of
H202killed
1
0 7.5 15
00.0 X 106 0.94 x 106 1.41 x 106
2
0 8 12 28
0.00 0.67 0.97 3.51
x x x x
106 106 106 106
0.00 X 1.34 x 1.94 x 7.02 x
Osre
a The number of H202-killed cells was the initial number of CFU per milliliter minus the final number of CFU per milliliter at each time of H202 treatment as measured in Fig. 2A (experiment 1) or in Fig. 2B (experiment 2). The predicted maximum numbers of spores per milliliter were estimated by assuming that two vegetatively dead cells mate and generate four live spores. The observed numbers of spores per milliliter were from the 144-h populations in Fig. 4B.
number of vegetative cells were resistant to killing by ethanol, then the ratio of spores to asci would really be spores plus resistant vegetative cells to asci plus resistant vegetative cells and would be less than four. If the resistant vegetative cell number were large enough to account for the average 8.4-fold increase in apparent spore number, the ratio of spores to asci would be (1.0 + 7.4)/(0.25 + 7.4) or 1.1. At 24 and 48 h, for both nitrogen-deficient and adequatenitrogen cultures, the measured ratio of spores to asci was somewhat above four. Thus, there was no evidence for ethanol-resistant vegetative cells. Repair processes in vegetative cells apparently deal with most of the individual DNA damages caused by H202. In humans about 1,000 oxidized thymine residues are removed by repair per cell per day (28). However, no particular repair mechanism is 100% efficient. When DNA is overloaded with damage special repair processes are expected to be induced. If meiotic recombinational repair evolved to cope with damages in DNA (see above), then in facultatively sexualasexual organisms an overload of DNA damage would be expected to call forth sexual reproduction. A population of cells treated with H202 in adequate-nitrogen medium should be a mixture of cells with an overload of damage (vegetatively dead cells) and cells which cope with the damage (viable CFU). The data were evaluated to see if the number of spores formed was consistent with the expectation that only vegetatively dead cells would mate and form viable spores. The number of spores formed was either somewhat less than or about equal to that expected by this hypothesis (except for 0 min of H202 treatment, in which H202 killing was not a factor) (Table 2). Some vegetatively dead cells may also have been unable to mate, so this pattern was expected. Thus, our data are consistent with the idea that if an S. pombe cell is able to grow vegetatively, it does so. However, if it has DNA damage which prevents vegetative growth, it may mate, undergo recombinational repair, and generate viable spore progeny. DISCUSSION Previous studies of S. pombe have indicated that nitrogen starvation or glucose limitation induces sexual reproduction (9, 20). Heterozygosity at the mating type locus is also required, indicating that the products of the mat] and mat2
genes must be present (9). In addition, as discussed by Watanabe et al. (32), genes steX and pacl as well as the positive regulators encoded by mei2 and mei3, the negative regulator encoded by rani or pati, and cyclic AMP are involved. Further, genes rasi and byri are needed (13, 24). Thus, the pathway for turning on the sexual process is beginning to be elucidated. It is not clear how H202 treatment induces sexual reproduction. In Salmonella typhimurium H202 treatment induces 30 proteins (including 2 catalases and 2 subunits of alkyl hydroperoxide reductase) (23). Of the 30 proteins, 5 are also induced by heat shock, and other small subsets are induced by other stresses such as nalidixic acid, ethanol, or cumene hydroperoxide treatments. Five adenylated nucleotides (AppppA, ApppGpp, AppppG, ApppG, and ApppA) are produced in response to heat, ethanol, and oxidative stress in S. typhimurium and E. coli (18). These adenylated nucleotides were proposed as possible "alarmones," and mechanisms were suggested for how a variety of stresses could all act to turn them on. Ubiquitin, a 76-residue polypeptide (that shows extraordinary sequence conservation among eucaryotes), increases rapidly in S. cerevisiae cells in response either to treatment with DNA-damaging agents or to the cells being suspended under starvation conditions in sporulation medium (30). This result indicates a response common to both DNA-damaging agents and starvation. It is possible that starvation itself causes DNA damage. In any case, our data (Fig. 4B and Table 1) show that treatment of S. pombe with the DNA-damaging agent H202, like starvation, increases sexual reproduction. The data in Table 2 were consistent with the idea that a vegetative cell with an overload of DNA damage and hence vegetatively dead may enter the sexual pathway, take advantage of meiotic recombinational repair, and be revived. However, spores are probably resistant to many physiological stresses, just as they were more resistant to ethanol than were vegetative cells (Fig. 1). Thus, it is possible that any one of many stresses could have produced some kind of alarmone that would cause entry into the meiotic pathway, leading to the physiologically protective spore state. Our evidence does not eliminate the possibility that entry into the sexual cycle after H202 treatment may be a generalized response to a stress. On the other hand, it is possible that entry into the sexual cycle served to revive vegetatively dead cells which had died because of DNA damage. If this is the case, what kind of recombinational repair would account for the approximately 100% survival of meiospores seen in some cases (Table 2)? In the case of X-ray inactivation of vegetative eucaryotic cells, lethal damages are thought to be largely caused by double-strand breaks (6). As briefly summarized by Imlay and Linn (15), X-ray damage is thought to occur mainly through oxide radicals, just like those produced by H202. Thus, X-ray lethality and H202 lethality are both likely to be caused by double-strand breaks. Since double-strand breaks destroy information on both strands of a chromosome, they can not be repaired by excision repair or other vegetative repair process depending on one chromosome. However, meiotic recombinational repair, involving two parental chromosomes, can repair double-strand breaks. Orr-Weaver and Szostak (27) have published a model for meiotic recombination which starts with a double-strand gapped chromosome that is paired with a homologous chromosome intact in that region. In the simplest version of their double-strand break repair model, a 3' end from the side of the gap in one strand invades the homologous duplex. A D-loop is displaced from
VOL. 171, 1989 the intact duplex and is enlarged by single-strand synthesis from the invading 3' end. The displaced D-loop of the intact duplex becomes available for single-strand copying by the
gapped chromosome. Two Holliday junctions are formed by these two rounds of single-strand synthesis. When these junctions are resolved by cutting, two intact regions are produced. Thus, as long as the two original damaged chromosomes do not have their damages in overlapping regions, recombinational repair can produce two entire intact chromosomes by repeated operation of the double-strand break repair model at damaged sites. These two intact chromosomes can replicate to produce four meiospores. The idea that a major selective advantage of sexual reproduction is its promotion of DNA repair was based on a broad range of observations in viruses, bacteria, fungi, ciliates, insects, plants, and mammals (2, 5). One obvious expectation is that DNA damage will induce mating in a facultatively sexual-asexual organism. We have shown that oxidative damage, in addition to the previously known signal of nitrogen or glucose starvation, induces sexual reproduction in S. pombe, possibly reflecting a response to an overload of a frequent natural type of DNA damage. ACKNOWLEDGMENTS This study was supported by Public Health Service grant GM27219-08 from the National Institutes of Health and by BRSG grant 2S07 RR05675. We thank H. Bernstein, R. E. Michod, S. T. Abedon, J. Spizizen, and P. Hyman for valuable discussions. LITERATURE CITED 1. Ames, B. N., R. L. Saul, E. Schwiers, R. Adelman, and R. Cathcart. 1985. Oxidative DNA damage as related to cancer and aging: assay of thymine glycol, thymidine glycol, and hydroxymethyluracil in buman and rat urine, p. 137-144. In R. S. Sohal, L. S. Birnbaum, and R. G. Cutler (ed.), Molecular biology of aging: gene stability and gene expression. Raven Press, Publishers, New York. 2. Bernstein, C. 1979. Why are babies young? Meiosis may prevent aging of the germ line. Perspect. Biol. Med. 22:539-544. 3. Bernstein, C. 1987. Damage in DNA of an infecting phage T4 shifts reproduction from asexual to sexual allowing rescue of its genes. Genet. Res. 49:183-189. 4. Bernstein, H., H. C. Byerly, F. A. Hopf, and R. E. Michod. 1985. Sex and the emergence of species. J. Theor. Biol. 117:665-690. 5. Bernstein, H., F. A. Hopf, and R. E. Michod. 1987. The molecular basis of the evolution of sex. Adv. Genet. 24:323-370. 6. Bryant, P. E. 1985. Enzymatic restriction of mammalian cell DNA: evidence for double-strand breaks as potentially lethal lesions. Int. J. Radiat. Biol. 48:55-60. 7. Calleja, G. B. 1974. On the nature of the forces involved in the sex directed flocculation of a fission yeast. Can. J. Microbiol. 20:797-803. 8. Cathcart, R., E. Schwiers, R. L. Saul, and B. N. Ames. 1984. Thymine glycol in human and rat urine: a possible assay for oxidative DNA damage. Proc. Natl. Acad. Sci. USA 81:56335637. 9. Crandall, R., R. Egel, and V. L. MacKay. 1977. Physiology of mating in three yeasts. Adv. Microb. Physiol. 15:307-398. 10. Davies, K. J. A., and A. L. Goldberg. 1987. Proteins damaged by oxygen radicals are rapidly degraded in extracts of red blood cells. J. Biol. Chem. 262:8227-8234. 11. Demple, B., J. Halbrook, and S. Linn. 1983. Escherichia coli xth mutants are hypersensitive to hydrogen peroxide. J. Bacteriol. 153:1079-1082. 12. Demple, B., A. Johnson, and D. Fung. 1986. Exonuclease III and endonuclease IV remove 3' blocks from DNA synthesis primers
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in H202-damaged Escherichia coli. Proc. Natl. Acad. Sci. USA 83:7731-7735. 13. Fukui, Y., T. Kozasa, Y. Kaziro, T. Takeda, and M. Yamamoto. 1986. Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. Cell 44:329-336. 14. Gutz, H., H. Heslot, U. Leupold, and N. Loprieno. 1974. Schizosaccharomyces pombe, p. 395-446. In R. C. King (ed.), Handbook of genetics, vol. 1. Plenum Publishing Corp., New York. 15. Imlay, J., and S. Linn. 1988. DNA damage and oxygen radical toxicity. Science 240:1302-1309. 16. Kaneko, M., S. A. Leadon, and T. Ito. 1988. Relationship between the induction of mitotic gene conversion and the formation of thymine glycols in yeast S. cerevisiae treated with hydrogen peroxide. Mutat. Res. 207:17-22. 17. Klar, A. J. S. 1987. Differentiated parental DNA strands confer developmental asymmetry on daughter cells in fission yeast. Nature (London) 326:466-470. 18. Lee, P. C., B. R. Bochner, and B. N. Ames. 1983. AppppA, heat shock stress, and cell oxidation. Proc. Natl. Acad. Sci. USA 80:7496-7500. 19. Maynard-Smith, J. 1978. The evolution of sex, p. 2-3. Cambridge University Press, Cambridge. 20. McDonald, I. J., G. B. Calleja, and B. J. Johnson. 1982. Conjugation in chemostat cultures of Schizosaccharomyces pombe. J. Gen. Microbiol. 128:1981-1987. 21. Michod, R. E., and B. R. Levin. 1988. The evolution of sex: an examination of current ideas, p. 1-4. Sinauer Associates, Sunderland, Mass. 22. Mitchison, J. M. 1970. Physiological and cytological methods for Schizosaccharomyces pombe, p. 131-165. In D. M. Prescott (ed.), Methods in cell physiology, vol. 4. Academic Press, Inc., New York. 23. Morgen, R. W., M. F. Christman, F. S. Jacobson, G. Storz, and B. N. Ames. 1986. Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proc. Natl. Acad. Sci. USA 83:8059-8063. 24. Nadin-Davis, S. A., and A. Nasim. 1988. A gene which encodes a predicted protein kinase can restore some functions of the ras gene in fission yeast. EMBO J. 7:985-993. 25. Nurse, P. 1975. Genetic control of cell size at cell division in yeast. Nature (London) 256:547-551. 26. Nurse, P. 1985. Mutants of the fission yeast Schizosaccharomyces pombe which alter the shift between cell proliferation and sporulation. Mol. Gen. Genet. 198:497-502. 27. Orr-Weaver, T. L., and J. W. Szostak. 1985. Fungal recombination. Microbiol. Rev. 49:33-58. 28. Saul, R. L., P. Gee, and B. N. Ames. 1987. Free radicals, DNA damage, and aging, p. 113-129. In H. R. Warner, R. N. Butler, R. L. Sprott, and E. L. Schneider (ed.), Modern biological theories of aging. Raven Press, Publishers, New York. 29. Shields, W. M. 1982. Philopatry, inbreeding and the evolution of sex. State University of New York Press, Albany. 30. Treger, J. M., K. A. Heichman, and K. McEntee. 1988. Expression of the yeast UBI4 gene increases in response to DNAdamaging agents and in meiosis. Mol. Cell. Biol. 8:1132-1136. 31. Vaca, C. E., J. Wilhelm, and M. Harms-Ringdahl. 1988. Interaction of lipid peroxidation products with DNA. A review. Mutat. Res. 195:137-149. 32. Watanabe, Y., Y. lino, K. Furuhata, C. Shimoda, and M. Yamamoto. 1988. The S. pombe mei2 gene encoding a crucial molecule for commitment to meiosis is under regulation of cAMP. EMBO J. 7:761-767. 33. Williams, G. C. 1975. Sex and evolution. Princeton University Press, Princeton, N.J. 34. Yonei, S., R. Yokota, and Y. Sato. 1987. The distinct role of catalase and DNA repair systems in protection against hydrogen peroxide in Escherichia coli. Biochem. Biophys. Res. Commun. 143:638-644.
