Apr 12, 1973 - UV or ethidium bromide (EB) induction of cytoplasmic genetic lesions. The induction of the respiratory-incompetent "petite" colony mutation or ...
JOURNAL OF BACTERIOLOGY, Sept. 1973, p. 805-809 Copyright 0 1973 American Society for Microbiology
Vol. 115, No. 3
Printed in U.S.A.
Cytoplasmic "Petite" Induction in Recombination-Deficient Mutants of Saccharomyces cerevisiae ETHEL MOUSTACCHI Fondation Curie, Institut du Radium, Biologie, Bdtiment 110, Orsay (91), France
Received for publication 12 April 1973
As compared to the original wild type, the induction of the cytoplasmic "petite" mutation by ultraviolet light and by the intercalating dye, ethidium bromide, is reduced in two mutants (rec4 and rec5) of Saccharomyces cerevisiae. These mutants are blocked in X rays or ultraviolet light-induced intragenic recombination. It then appears that the products of nuclear genes necessary for the completion of nuclear intragenic recombination events are also involved in steps of the metabolic chain which leads to the mitochondrial mutation, p-.
Yeast mutants deficient in X rays and ultraviolet light (UV)-induced mitotic heteroallelic reversion were recently isolated from disomic yeast cells (14). These two mutations segregate as recessive genes; complementation and allelism tests demonstrate that they are independent (14). We have examined whether a block, due to a nuclear mutation in one of the steps required for exchanges of nuclear genetic material, would modify the response to UV or ethidium bromide (EB) induction of cytoplasmic genetic lesions. The induction of the respiratory-incompetent "petite" colony mutation or p- was studied. The mitochondrial deoxyribonucleic acid (DNA) of the p- mutants may be altered in base composition (2) or lost (10). A previous study of the UV induction of pin several nuclear repair mutants (9) already showed that some products coded by nuclear genes are required for the final expression of mitochondrial UV damage. We now demonstrate that the two recombination-deficient mutants tested for p- induction are significantly less mutable than the original wild type. MATERIALS AND METHODS Strains. The parental disomic strain of Sac-
The strains are disomic in order to test for heteroallelic recombination between arg4-2 and arg4-17. The rec loci are not on the disomic chromosome VIII. Disomic strains tend to lose their extra chromosome with repeated subculturing (R. K. Mortimer, personal communication). The disomic condition, which is necessary to assert the rec+ or rec- phenotype of the strains, was systematically monitored by crosses with arg4-2 and arg4-17 haploid tester strains. The trisomic strain resulting from a cross of either of these testers with the disomic strains should show recombination when irradiated with 5 krad. If one of the chromosomes is lost, one of the crosses will fail to show recombination. Media, growth conditions, irradiation procedure and dosimetry, detection of "petites." All of the above were as previously described (7). The EB mutagenesis was performed under nongrowing conditions according to the procedure described in (18).
RESULTS Effect of UV on survival and "petite" induction. Figure 1 shows the survival as a function of UV dose for strains 2C4 and 2C8 as compared to the wild type. In agreement with Rodarte-Ramon and Mortimer (14), the two recombination-deficient mutants are slightly more sensitive than the wild type, charomyces cerevisiae was Z140-51C, and the two the magnitude of the shoulder is decreased, and mutants exhibiting depressed frequencies of X rays the final slope is steeper. It should be mentioned and UV-induced reversion in the heteroallelic region, that in these strains the UV inactivation is arg4-2 +/+ arg4-17, on disomic chromosome VIII were strongly dependent upon the stage of growth of strains 2C4 and 2C8 (14). These mutations which the culture. The difference in UV sensitivity specifically affect mitotic intragenic recombination segregated as one recessive gene denoted rec5 between the wild type and the recombination(R. K. Mortimer, personal communication) and rec4 deficient strains was amplified in log-phase (14), respectively. These strains were kindly provided cells mainly due to an increase in resistance of the wild type. by R. K. Mortimer. i05
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UV- DOSE
FIG. 1. Survival after UV irradiation of the disomic strain Z140-51C (0) and the derived recombinationdeficient mutants 2C4 (A) and 2C8 (0). A mean of 1,000 colonies per dose was scored. Doses are given in ergs per square millimeter.
Patterns of p- induction as a function of UV dose and survival are shown on Fig. 2. Complete and variegated colonies (solid lines) are scored as mutants, whereas only complete p - colonies (dotted lines) are scored as mutants. It is clear that, in both cases, strain 2C4 exhibits a reduced sensitivity to p- induction, manifested in a more pronounced sigmoidal shape of the curve and a smaller slope of the linear part of the curve as compared to that of the wild
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type. The same observations hold, but to a lesser extent, for strain 2C8. It should be noted that the spontaneous background of "petites" represents around 3% of the population and is similar for the three strains studied. The survival curves (Fig. 1) do not reveal any heterogeneity in the populations with respect to their radiosensitivity at least down to a level of 10-3 to 10-4 survival. Moreover, no substantial differences were found in the radiosensitivity to killing between isolated p- clones from Z140, 2C4 and 2C8, and the whole population of the corresponding strains. A differential enrichment in p- mutants particularly in the wild type is consequently excluded. "Petites" induction by EB. The transformation of nongrowing p+ cells to p- mutants as a function of time of storage with 1 ,ug of EB per ml are shown for the three strains in Fig. 3 with a semilog representation. It is clear that, in this case too, the two recombination-deficient mutants are less mutable than the wild type. Although the cultures were taken in stationary phase that is in a derepressed condition, the extrapolation numbers are in all cases low (around 2) and comparable for the three strains. The main difference concerns the slopes of the curves; the dose-modifying factor at a frequency of 50% of induction for strains 2C4 and 2C8 relative to the wild type were 1.8 and 2, respec-
tively. Reduced frequency of- p- is a consequence of the rec- mutation. It may be argued that the reduced cytoplasmic p- induction by UV and EB is not a consequence of the rec- mutation itself but is due to another nuclear mutation produced concomitantly by the mutagenizing treatment on the original wild type. To test this, 7 asci were analysed. All showed 2:2 segregation in association with rec-, indicating either that this property is closely linked to the rec gene or that there is a pleiotropic effect of the rec mutation. Two main arguments favor this second interpretation. First, as seen in Fig. 2 and 3, the induced p - frequencies are reduced in two independent mutants, 2C4 and 2C8, as compared to the original wild type. Secondly, rec+ revertants were obtained from the two rec mutants. These revertants, in which the disomic condition was monitored as described in the Materials and Methods section, demonstrated the pattern of p- induction characteristic of the wild type. It is difficult to accept the fact that the hypothetical mutation which would modify the response to p- induction by UV and EB was induced in two independent rec- clones and moreover, that it was reverted simultaneously with the rec- alleles.
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FIG. 2. UV induction of "petites" in Z140-51C (0), 2C4 (A), and 2C8 (0) strains. The spontaneous background was subtracted for each dose. The abscissa give the incident dose in ergs/mm2 in the left part of the graph and the percentage of survival in the right part. The curves with the dotted lines are obtained by scoring only the complete white colonies as "petites" and the full lines by scoring the complete white colonies plus the sectored white and red colonies as "petites."
DISCUSSION Two strains almost completely blocked in _\X\"\\ intragenic recombination are simultaneously ~--__~ \\ A > ^ ~ ---__ less susceptible to the induction of the cytoplasE _CS \ sX0 ~A~~~~mic p- mutation as compared to the original type. In other words, in addition to the \>4 wild (8), two new 2C\ 'rad" genes previously studied nuclear genes involved in repair processes ap\ 0\°"pear to interfere with the response to cytoplasmic damage. Since the rate of growth on nonfermentable sugars is the same for the three strains, and E \ sX since the respiratory adaptation follows the same kinetics, it is highly unlikely that the number of mitochondria per cell changed from the original wild type to the derived mutants. The E differences in sensitivity to induction by one < .single nuclear mutation represent, in our view, modifications in the events which follow the initial damage, that is, pyrimidine dimer forI mation in one case, as demonstrated by the I I I I large photoreactivation factor (around 3) obtime in hours served in this range of doses, and intercalation of the dye in the mitochondrial DNA in the FIG. 3. EB mutangenesis in Z140-51C (0), 2C4 other. The gene products of the recombination (A), and 2C8 (0) strains under nongrowing condi- loci tested appear to be implicated in the chain tions. The total number of cells (p+, sectored and P-) of metabolic events which will lead to the premains constant. In all cases the numbers of cells per ~0
X
milliliter have been normalized so that 1 = 2.2 x 106 mutation. cells per ml (initial number at time zero). The same Before one speculates on the nature of these representation as in Fig. 2 is adopted for complete events it may be recalled that the strains (dotted line) or complete plus sectored (solid line) examined here are specifically blocked in intragenic recombination but not in intergenic ex"petites".
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changes (13, 14). In other words, these genes participate in the events involved in conversion but not in crossing over. Similarities in response to liquid holding after UV treatment between intragenic recombination and p- induction were already suggested by previous work (8). The observations reported here point again to the fact that exchange events of the kind involved in intragenic recombination, that is, the replacement of an informational segment in one homolog with information contained in the corresponding section of a nonsister homolog, are required in the process of p- induction, some of the products necessary in this process being controlled by nuclear genes. It is generally accepted that the steps in the pathways of gene conversion and crossing over are different in yeast. Mutants affected in mitotic or meiotic crossing over are not yet identified in this organism. Consequently, the possibility has not been excluded that a mechanism which concerns homologous DNA molecules at the four-strand stage is not also required for the "petite" induction. All the varied molecular models for recombination, though they differ in the sequence of events, include breakage of DNA strands, formation of heteroduplex regions, DNA degradation, and resynthesis and rejoining of the ends. These events are expected to entail the participation of nucleases, polymerase(s), and ligase. Cells exhibiting altered recombinational capacities are supposed to lack one of these enzymatic activities. Exchanges of mitochondrial genetic markers are known to occur in wild type (16-2) and even in p- yeast cells (G. Michaelis and E. Petrochilo, personal communication). Observations of extracted mitochondrial DNA molecules by electron microscopy also suggest that such exchanges are frequent (4). It is generally accepted that the distortions introduced in the mitochondrial DNA molecules by the pyrimidine dimers after UV treatment or by a complex between EB and DNA are recognized and susceptible to enzymatic attack. Mitochondrial DNA degradation is indeed observed after EB treatment (3-12) and a loss of pyrimidine dimers is also suggested by the lack of photoreactivibility of p- induction after liquid holding of UV-treated yeast cells (8). This process of enzymatic attack may lead to deletions of mitochondrial DNA segments which indeed are described in the cases of some "petites" (6), or to a more or less random reassociation of broken segments of mitochondrial DNA molecules favored by the fact that at least two copies of
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mitochondrial DNA molecules per mitochondrion are present (17). The protection and the reversibility of EB pinduction by heat treatment (11) or by drugs such as choloramphenicol or antimycin A (5) may be due to inactivation or inhibition of one of the enzymes required in such a process. A genetic defect in one of the presumptive steps proposed could similarly account for the decrease in the proportion of p- cells recovered after treatment in the present experiments. A precedent for such a decrease in mutability related to deficiency in repair capacity is already well documented in recombination-deficient bacteria (18). It should be emphasized that the yeast recmutants tested retain other repair capacities: they are able to perform intergenic recombination and probably excision-resynthesis repair (unpublished data). Consequently, since these mutants still produce after treatment a significant fraction of p- mutants, it is likely that other processes are also involved in the induction of p- mutants. ACKNOWLEDGMENTS I thank R. Davies (John Innes Institute) for critical reading of the manuscript and S. Enteric for excellent technical assistance. This investigation was supported in part by the Fondation pour la Recherche Medicale Francaise and the Commissariat a l'Energie Atomique, (Saclay). LITERATURE CITED 1. Bernardi, G., F. Carnevali. A. Nicolaieff, G. Piperno, and G. Tecce. 1968. Separation and characteristic of a satellite DNA from a yeast cytoplasmic "petite" mutant. J. Mol. Biol. 37:493-506. 2. Coen, D., J. Deutsch, P. Netter, E. Petrochilo, and P. Slonimski. 1970. Mitochondrial genetics. I. Methodology and phenomenology. Symp. Soc. Exp. Biol. 24:449496. 3. Goldring, E. S., L. I. Grossman, D. Krupnick, D. R. Cryer, and J. Marmur. 1970. The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J. Mol. Biol. 52:323-335. 4. Hudson, B., and J. Vinograd. 1967. Catenated circular DNA molecules in HeLa cells mitochondria. Nature (London) 216:647-652. 5. Mahler, H. R., and P. S. Perlman. 1972. Mutangenesis by ethidium bromide and mitochondrial membrane. J. Supramol. Struct. 1:105-124. 6. Michaelis, G., S. Douglas, M. J. Tsai, K. Burchiel, and A. S. Criddle. 1972. In vitro transcription of mitochondrial deoxyribonucleic acid from yeast. Biochemistry
11:2026-2036. 7. Moustacchi, E. 1969. Cytoplasmic and nuclear genetic events induced by UV light in strains of Saccharomyces cerevisiae with different UV sensitivities. Mutat. Res. 7:171-185. 8. Moustacchi, E., and S. Enteric. 1970. Differential "liquid holding recovery" for the lethal effect and cytoplasmic "petite" induction by UV light in Saccharomyces
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cerevisiae. Mol. Gen. Genet. 109:69-83. 9. Moustacchi, E. 1972. Evidence for nucleus independent steps in control of repair of mitochondrial damage. I. UV induction of the cytoplasmic "petite" mutation in UV-sensitive nuclear mutants of Saccharomyces cerevisiae. Mol. Gen. Genet. 114:50-58. 10. Nagley, P., and A. W. Linnane. 1970. Mitochondrial DNA deficient petite mutants of yeast. Biochem. Biophys. Res. Commun. 39:989-996. 11. Perlman, P. S., and H. R. Mahler. 1971. A premutational state induced in yeast by ethidium bromide. Biochem. Biophys. Res. Commun. 44:261-267. 12. Perlman, P. S., and H. R. Mahler. 1971. Molecular consequences of ethidium bromide mutagenesis. Nature N. Biol. 231:12-16. 13. Rodarte-Ramon, U. S. 1972. Radiation induced recombination in Saccharomyces: isolation and genetic study of recombination deficient mutants. Radiat. Res. 49:133-147.
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tion induced recombination in Saccharomyces: the genetic control of recombination in mitosis and meiosis. Radiat. Res. 49:148-151. Slonimski, P. P., G. Perrodin, and J. H. Croft. 1968. Ethidium bromide induced mutation of yeast mitochondria: complete transformation of cells into respiratory deficient nonchromosomal "petites." Biochem. Biophys. Res. Commun. 30:232-239. Thomas, D. Y., and D. Wilkie. 1969. Recombination of mitochondrial drug-resistance factors in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 30:368-372. Williamson, D. H. 1970. The effect of environmental and genetic factors on the replication of mitochondrial DNA in yeast. Symp. Soc. Exp. Biol. 24:247-276. Witkin, E. 1969. Mutability towards ultraviolet light of recombination-deficient strains of Escherichia coli. Mutat. Res. 8:9-14.
