Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6529-6533, December 1979

Genetics

Genetic control of enhanced mutability of mitochondrial DNA and y-ray sensitivity in Saccharomyces cerevisiae (mutators/DNA repair/gam mutants/rad mutants/antibiotic-resistant strains)

FRANQOISE FOURY

AND ANDRE GOFFEAU

Universite de Louvain, Laboratoire d'Enzymologie, Place Croix du Sud, 1, B-1348 Louvain-la-Neuve, Belgium

Communicated by Herschel L. Roman, September 20,1979

Five nuclear mutants enhancing the spontaABSTRACT neous mutation rate of mtDNA have been isolated in Saccharomyces cerevisiae. These mutators fall into five complementation groups and are located at five genetic loci different from rad5O to rad57 loci. Three mutants (gaml, gam2, and gam4), insensitive or weakly sensitive to y-rays, exhibit increased frequency of spontaneous production of mutants with large deletions of the mtDNA (p-) and of all tested mitochondrial drugresistant mutants. Two other mutants (gam3 and gam5), highly sensitive to 7-rays, increase only the mutation rate of particular alleles of the mtDNA. The mutant gam5 enhances only the production of p- and erythromycin-resistant clones. The mutant gam3 exhibits an enhanced rate of oligomycin-resistant clones as well as a collateral increase of nuclear mutability. The existence of gam3 and gamS mutants indicates that at least two common steps control both nuclear DNA repair and the mutability of particular alleles of the mtDNA. However, the general spontaneous mutability of the mtDNA includes at least three steps not involved in the repair of nuclear DNA, as revealed by the gami, gam2, and gam4 mutations.

In Saccharomyces cerevisiae, the rho- (p-) mutation characterized by large deletions and amplification of the mitochondrial genome (1) occurs spontaneously at a puzzling high rate, up to 3 X 10-3 per cell division (2). However, the frequency of the spontaneous mtDNA mutations conferring resistance to antibiotics is much lower (e.g., 10-8 to 10-7) and comparable to spontaneous nuclear mutations rates (3). As in the case of the yeast nuclear DNA, there must be complex mutagenic pathways that control the spontaneous mutability of yeast mitochondrial genome. Therefore, the isolation of mutator and antimutator strains exhibiting respectively increased or decreased spontaneous mutation rates of mitochondrial genes is of interest for better understanding of spontaneous mutagenesis and of possibly related processes such as repair and recombination of mtDNA. It has already been shown that the spontaneous production of p- strains can be increased by nuclear mutations (2, 4, 5), especially in the cdc8 and cdc2l genes which are required for replication of both nuclear DNA and mtDNA (6) and in uvsp mutants whose mtDNA is hypersensitive to UV light (7). More recently, it has been reported that nuclear genes can act as mutators of mitochondrial genes (8, 9). Fourteen mutants falling into two complementation groups were shown to exhibit increased spontaneous production of erythromycin-, oligomycin-, and spiramycin-resistant strains (9), even though they did not display mutator activity for nuclear genes. It is also known that dTMP auxotroph mutants deprived of the nucleotide behave as mutators for mitochondrial genes (10). Our approach to the spontaneous mutability of yeast mtDNA was different. Although it was shown years ago (11, 12) that no

p- strains were induced by ionizing radiations, we decided to explore whether a relationship existed between y-ray sensitivity and deficiencies in mtDNA repair. In most organisms, 7-rays produce a large range of damages repaired by specific pathways (13, 14), and the failure of inducing p- strains might originate from a similar sensitivity of the nuclear DNA and mtDNA to ionizing irradiations so that most of the p- strains would be accompanied by lethal nuclear mutations. Actually, we were able to isolate y-ray-sensitive mutants exhibiting either decreased (unpublished results) or increased spontaneous mutability of the mtDNA. In this communication, five mtDNA mutator strains are characterized which mark at least five distinct nuclear loci. Two of them are very sensitive to D-rays; the other three mutators are either insensitive or weakly sensitive. MATERIALS AND METHODS Media. The following media were used: YD medium (2% yeast extract, 2% glucose), YG medium (2% yeast extract, 2% glycerol), WO medium (0.67% yeast nitrogen base Difco, 3% glucose supplemented with amino acids when indicated), and Kac sporulation medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose). The antibiotic-containing media were composed of 1% yeast extract, 2% bactopeptone, 2% glycerol, and 50 mM potassium sodium phosphate at pH 6.2 and were supplemented with either 4 g of erythromycin per liter, 3 mg of oligomycin per liter, or 75 uM diuron. Solid media were supplemented with 2% agar. Strains. The mutants listed in Table 1 were all derived from S. cerevisiae D273-1OB/A1. A strain isogeneic to D273-10B in the opposite mating type was constructed and named NW384C. The rad mutants were a gift of the Yeast Genetic Stock Center (Berkeley, CA). Isolation of the Mutator Strains. Cells of D273-IOB/A1 were grown overnight and mutagenized with 2% ethyl methanesulfonate under conditions yielding 50% lethality (15). Among 40,000 survivors, 450 y-ray-sensitive mutants were isolated according to the method described by Game and Mortimer (16) for the selection of x-ray-sensitive mutants. The parental strain and the mutants were spread evenly on YD plates. After 2 days' incubation at 300C, they were replicated on antibiotic-containing media and incubated for 8 days. Mutator and antimutator strains were easily detected by the number of resistant colonies arising from the homogeneous lawn of sensitive strains. Determination of the Mutation Rates. The mutation rates Abbreviations: rho- (pj), strain with large deletions of the mtDNA; mtDNA leading to a specific respiratory deficiency; antR, antibioticresistant; ants, antibiotic-sensitive; ER, erythromycin-resistant; OR, oligomycin-resistant; DR, diuron-resistant; canR, canavanine-resistant; PD, parental ditype; NPD, nonparental ditype; T, tetratype.

mitr, mutant produced by a point mutation or a small deletion of the

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Proc. Natl. Acad. Sci. USA 76 (1979)

Genetics: Foury and Goffeau Table 1. Genotypes of the strains Nuclear genotype Name D273-1OB D273-1OB/Al

a

prototroph

a

met2

D273-1OB/auxl4 D273-1OB/auxl6

auxotroph a his

NW38-4C N123 CB11 F2-12C

F1-4B IR28 IR195 IR537 IR612 aIR195-lB

aIR195-19C

a

a

his

ahisl a adel MAL6 a adel adel gam5 met2 a gaml gam2 met2 a gam3 met2 gam4 met2 a gaml met2 his a gam2 met2 his a

a

D273-1OB, D273-lOB/Al, and CB11 were given by A. Tzagoloff (Columbia University, New York). N123 was given by E. Moustacchi (Orsay, France). F1-4B and F2-12C are meiotic segregants from the cross D273-1OB/Al X CB11. The mutator meiotic segregants aIR195-1B and aIR195-19C were from the cross IR195 X NW38-4C. NW38-4C is a strain isogeneic to D273-1OB in the opposite mating type obtained as follows. A mixture of spontaneous auxotrophs from D273-1OB (aux 14 and aux 16) was plated as a thick lawn on YD medium and incubated for 5 days before replication on glucose minimum medium. Among the colonies arising in the minimal medium, some were diploids formed by the spontaneous mutation of into a mating type of a haploid cell and subsequent mating of this mutant with a nonmutant haploid having a complementary auxotrophy. The diploids were spread on sporulation medium and the tetrads gave a clear 2:2 segregation of the mating type. The spore NW38-4C was his- and in the a mating type. Spore viability was >99o. a

y-Ray-Irradiation. The cells were irradiated on YD plates at 30 0C in a panoramic irradiator with a 6OCo source (Mecanique et Conception Industrielle Modernes, Clamart, France) at a dose rate of 48 krad (480 grays)/hr. Nomenclature. The original y-ray-sensitive mutants were designated by the prefix IR followed by a number. The same nomenclature was used for the mutator meiotic segregants issued from crosses between original mutants and the isogeneic parental strain NW38-4C. In addition, the a or a mating type and the name of the spore were indicated. The mutator loci were designated by the three letters gam followed by a number specific for each locus. The wild-type allele is designated as gam+. RESULTS A total of 40,000 clones were examined, and 450 were found to be sensitive to y-rays. Of these 450 y-ray-sensitive mutants, 4 had increased spontaneous mutation rates of mitochondrial mutants. Subsequent quantitative tests revealed that the IR612 mutant and possibly the IR195 mutant were not significantly more sensitive to 7-rays. 7-Ray Survival Curves. Fig. 1 shows that, in our irradiation conditions, survival of the wild-type strain D273-10B/Al was complex with a shoulder between 25 and 100 krad followed by a slower decrease for higher doses. The strains IR28 and IR537 were highly sensitive to 7-rays. The survival of the strain IR195 was significantly decreased only in the second phase of killing. The strain IR612, even though initially isolated as 7-raysensitive, did not show increased lethality in subsequent experiments. In none of the mutants was the yield of y-rayinduced p- mutations enhanced. Mutator Activity. Mitochondrial inheritance of antibiotic resistance can be defined by three criteria: (i) mitotic segregation of antR versus ants traits in the diploid progeny of a cross between ants and antRp+ strains; (ii) lack of meiotic segregation of antR versus ants traits in the tetrads issued from the sporulation of a pure diploid progeny; (iii) lack of mitotic segregation of antR trait in the diploid progeny issued from a cross between an antR strain and a p0 which is devoid of mtDNA. In all strains, the frequency of antR mutants encoded in the mtDNA was determined according to the above criteria. All ER and oR independent clones were found to be mitochondrially inherited. However, an appreciable number of DR clones exhibited other inheritance patterns and only the mitochondrial mutation rates are given in Table 2. The mutants could be arranged into several categories according to their mutator activities for mtDNA and nuclear DNA. The first group was represented by the mutants IR612 and IR195. The frequency of spontaneous glycerol-negative strains was 40-100 times higher than in the parental strains. It

for induction of forward and reverse nuclear mutations were determined by the method of Luria and Delbruck (17) by using the equation r = aNt lnCaNt in which r is the average number of mutants in the cultures, Nt is the number of cells at time t, a is the mutation rate per time unit, and C is the number of independent cultures. The same equation was used to determine the mutation rates of mitochondrial genes. In this case, however, because the multiple copies of mtDNA within cell may be subjected to intracellular selection, the mutation rate per cell must be considered as an apparent rate and not as the actual number of mutations altering the mitochondrial DNA. Tetrad Analysis. Prototrophic selection of the diploids was carried out as described (18). Sporulation of the diploids and dissection of the asci were performed as described by Mortimer and Hawthorne (19). Segregation of the 'y-ray-sensitivity trait was analyzed as described by Game and Mortimer (16). Segregation of the mutator properties was determined by the average frequency of antibiotic-resistant colonies produced in three to five independent cultures for each spore of complete 100 tetrads. Mitochondrial Inheritence of Antibiotic Resistant (ant.R) 100 Mutants. Erythromycin-resistant (ER), oligomycin-resistant -IR612 D273-10B/Al ~ 0D7-10 (OR), and diuron-resistant (DR) clones were picked up randomly o 1OB/Al subcloned. The and secondary from 10-25 independent cultures 1.0 > antR clones were crossed with a p+ants parental strain and a p0 R3 aIR195-1B were selected on glumutant devoid of mtDNA. The diploids ~~~~~0.1 cose minimal medium, grown for about 20 generations, and spread for single colonies. After 2 days, the colonies were replicated on glycerol and appropriate antibiotic-containing media. About 100 resistant or sensitive diploid colonies were scored. For each cross, ants and antR diploids were spread on sporulakrad krad tion medium and a few tetrads resulting from the sporulation FIG. 1. The cells were irradiated on YD plates at a dose rate of of a pure diploid progeny were analyzed. 48 krad/hr. ~~~1A

Genetics:

Foury and Goffeau

Proc. Natl. Acad. Sci. USA 76 (1979)

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Table 2. Spontaneous mutation rates of mutator strains Mutation rate per cell division X 107 Strains % pER OR DR his met2 canR Genotype Parental strain gam+ 0.3 0.8 0.1 0.1 0.1 1.7 0.9 IR612 gam4 31.0 26.7 1.8 5.6 0.1 1.8 1.0 IR195 gamlgam2 22.0 15.9 2.8 2.4 0.1 1.7 0.6 aIR195-LB gaml 32.0 11.6 0.8 0.1 0.1 2.4 ND aIR195-19C gam2 7.1 3.7 0.5 1.5 0.1 1.3 ND IR28 gam5 9.0 3.3 0.1