179

Mutation Research, 4 3 ( 1 9 7 7 ) 1 7 9 - - 2 0 4 © Elsevier/North-Holland Biomedical Press

PATHWAYS OF U L T R A V I O L E T MUTABILITY IN S A C C H A R O M Y C E S CERE VISIAE. III. GENETIC ANALYSIS AND PROPERTIES OF MUTANTS RESISTANT TO ULTRAVIOLET-INDUCED F O R W A R D MUTATION *

JEFFREY

F. L E M O N T T

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (U.S.A.) (Received October 6th, 1976) (Accepted December 14th, 1976)

Summary Non-allelic mutants of Saccharomyces cerevisiae with reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were assigned to seven distinct genetic loci, each with allele designations urnrl-1, umr2-1, . . . , umr7-1 to indicate UV mutation resistance. Each allele complemented revl-1, rev2-1, and rev3-1. None conferred a great deal of UV sensitivity. When assayed on yeast extract-peptone-dextrose complex growth agar, umrl, umr3, and umr7 (a mating type) were the most UV-sensitive, with a dose-reduction factor of approximately 1.2 at 10% survival. When assayed on synthetic agar lacking arginine, however, umr3 was the most UV-sensitive (dose-reduction factor of 1.5 at 10% survival). UV revertibility of his5-2, lysl-1, and ura4-1 was normal in strains carrying the single genes umr4, umr5, umr6, and umr7; umrl reduced revertibility of his5-2 and ura4-1 but n o t lysl-1 ; urnr2 reduced only ura4-1 revertibility; umr3 reduced UV reversion of all three test alleles. Five a/a homozygous umr diploids (except umrl and umr4) failed to sporulate. One of these, umr7, blocked normal secretion of alpha hormone in a segregants and could not conjugate with a strains. The phenotypes of umr mutants are consistent with the existence of branched UV mutation pathways of different specificity, some of which may function in the single RAD6-dependent error-prone pathway for repair of UV damage. Other possible pathways of

* Research sponsored by the U.S. Energy Research and Development A dmi ni s t ra t i on under contract with the Union Carbide Corporation. By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Governm e n t to retain a nonexclusive, royalty-free license in and to any copyright covering the article. Abbreviations: ARG, L-arginine; HIS, L-histidine; LYS, L-lysine; URA, uracil; LEU, L-leucine; MIN, minimal; SC, synthetic complete; CAN, L-canavanine; SDS, second-division segregation; PD, parental ditype; NPD, non-parental ditype; T, tetratype; YEPD, yeast e x t r a c t - - p e p t o n e -dextrose; UV, ultraviolet light.

180 action are discussed. It is also suggested that regulatory functions interacting with the mating-type locus or its gene products may play some role in UV mutagenesis or error-prone repair.

Introduction

Induced mutagenesis is believed to require functional enzymatic pathways and may be modified genetically and physiologically (for recent review see Clarke and Shankel [1]). The lex and recA loci in Escherichia coli confer considerable resistance to mutation induced by UV, ionizing radiation, and certain chemical mutagens by blocking error-prone pathways of DNA repair [17,34, 41]. UV-induced mutagenesis in the eukaryotic microorganism Saccharomyces cerevisiae may be blocked genetically by a variety of unlinked loci (revl, rev3, tad5, rad6, tad8, rad9, and rad18), which significantly increase cellular sensitivity to UV and ionizing radiation in both single and multiple mutants [20,21, 26]. Double-mutant haploids carrying tad6 and either revl, rev3, rad9, or tad18 are not significantly more UV-sensitive than a rad6 strain [20]. Moreover, rad6 and rev3 block UV mutability in every mutation system studied, whereas the other genes reduce UV reversion of certain alleles but not of others [20,26]. These findings support the existence of a single error-prone pathway for repair of UV damage in yeast. Envisioned are steps required for all UV mutation (controlled by RAD6 and REV3), with specific branches (controlled by REV1, RAD9, RAD18, and possibly RAD5 and RAD8) responsible for different modes of mutational alteration. The basis for the specificity is unknown but could possibly include (1) the kinds of UV lesions in DNA (pyrimidine dimers are responsible for much but n o t all UV mutagenesis in yeast [35], (2) the class of base-pair change (e.g. transition, transversion, or frameshift), (3) position within the gene, (4) nucleotide sequence in the region of the mutational site. Data obtained by Lawrence and Christensen [19,21] involving REVl-independent (RAD6, REV3-dependent) UV reversions of four cycl alleles suggest that the specificity of this branch of the error-prone pathway may depend upon an interaction between the repair enzyme and a specific nucleotide sequence rather than upon a simple preference for position within the gene, type of base-pair change, or particular DNA triplet altered. Since UV is known to induce a variety of base alterations in Saccharomyces [39], it should be possible to identify new genes controlling other specific modes of UV mutagenesis if the selection procedure is based upon forward mutability rather than UV reversion of a particular ochre allele [23] or UV sensitivity in general [2]. If there exist repair-independent pathways of UV mutagenesis, or if there are certain branches of the tad6 pathway which contribute to a small fraction of the cell's total repair potential, this procedure might uncover such mutation-resistant strains with little or no increased UV sensitivity. RAD6-dependent forward mutations to can1 (canavanine resistance) are induced with high frequency by UV in a RAD ARG CAN1 wild-type haploid

181

[22]. Grenson et al. [13] were the first to demonstrate that mutants resistant to the toxic effects of the arginine analogue canavanine arose by forward mutation at a single recessive gene (can1) responsible for an arginine-specific permease activity. More recently, Whelan [40] has confirmed this finding and moreover failed to detect allelic complementation among nearly 2000 different dihybrids, suggesting that arginine permease is functional as a single polypeptide. Fine-structure mapping of the most widely spaced alleles indicated that the permease could have a molecular weight of the order of 260,000 daltons [40]. The isolation of strains with reduced UV mutability to can1 has been described previously [26]. Included in the original set were putative isolates which initially exhibited reduced, but not totally defective, mutability. In this paper properties are presented of seven mutants carrying alleles of seven different genes termed umr (for "ultraviolet mutation resistant"). Materials and methods Strains Considerable attention was paid to developing strains with similar genetic backgrounds in order to achieve meaningful comparison between umr and wildtype UMR. Table I gives the genotype of all haploid strains initially employed in this study. XY222-1A, XY222-1C, XY220-6C, and XY225-6A are haploid meiotic segregants obtained from crosses described previously [25] and have genetic backgrounds similar to the commonly used $288C (a wild type). Strains were derived from those in the Berkeley collection carrying ochresuppressible alleles his5-2, lysl-1, ura4-1, and leu2-1 in addition to the chromosome V marker ura3. All other strains were further derived from these by crosses, as discussed in Results. Genetic symbols are described by Plischke et al. [33]. Media The following media were the same as described previously [23,25] : YEPD, MIN, SC, SC-HIS, SC-LYS, SC-URA, SC-ARG, presporulation, sporulation. Sporulation was also obtained on an agar medium containing (per liter) 3 g

TABLE I GENOTYPES OF HAPLOIDSTRAINS USED IN GENETICANALYSISOF u m r MUTANTS Strain

Genotype

XY222-1A XY222-1C XY220-6C XY225-6A XY392-11C XY410-31D XY491-4B

a REVARG HIS LYS URA LEUMET c~ r e v l - 1 a r g 4 - 1 7 c~ rev2-1 a r g 4 - 1 7 c~ rev3-1 arg4-1 7 c~ u r a 3 h i s 5 - 2 lys 1-1 a ura4-1 l y s l - 1 leu2-1 c~ ura4-1 l y s l - 1 leu2-1 m e t 8 - 1 t r p l - 1

a All s t r a i n s a r e C A N 1 .

a TRP

182 potassium acetate, 0.22 g raffinose • 5H20, and 20 g agar. To prepare SC-ARG + CAN, L-canavanine sulfate was added to SC-ARG at 40 mg/1. MIN + ARG contained ARG at the usual 20 mg/1. UV irradiation

Shortly after plating, cells were irradiated directly on agar, one plate at a time, in the UV source; this source consisted of three 8-W germicidal lamps (General Electric G8T5, 90% intensity at 254 nm) positioned over a circular aperature 8 cm in diameter. The UV energy fluence rate at the agar surface was 2.8 J/m2/sec as determined by a calibrated Jagger meter [16]. A fast mechanical shutter device was activated by a preset time clock. To assure a constant lamp o u t p u t over a n u m b e r of hours, a thermostatically regulated fan maintained a constant t em pe r at ur e (~30°C) in the confined air space surrounding the lamps. Use of General Electric " g o l d " fluorescent lamps prevented photoreactivation during and after UV exposure until plates could be incubated continuously in the dark. u m r X rev c o m p l e r n e n t a t i o n

tests

Mating, zygote isolation, sporulation, ascus dissection by micromanipulation, and tetrad analysis were routinely carried o u t as described previously [15,23, 31]. The urnr-rev c o m p l e m e n t a t i o n tests were p e r f o r m e d according to the m e t h o d briefly described earlier [26]. Each u m r isolate was crossed t o each of the 3 r e v strains listed in Table I. Following zygote isolation by micromanipulation, vegetative progeny of hybrids were plated on YEPD ( ~ 1 0 0 cells/plate) and exposed to UV ( ~ 3 5 J/m2). Surviving colonies were replica-plated to MIN and MIN + ARG. Strains unable to grow on MIN but able to grow on MIN + ARG were considered to have undergone mitotic segregation to arg4-1 7 homozygosis. These were isolated and subsequently tested for UV revertibility of a r g 4 - 1 7 by comparing UV-irradiated with unirradiated SC-ARG replicas. Failure to observe UV revertants was taken as evidence for rev homozygosis [23]. Detection of the umr phenotype

Haploids from crosses were routinely tested for UV-induced forward mutation to canavanine resistance ( C A N 1 ~ c a n 1 ) . Cultures on YEPD master plates were replica-plated to two or more SC-ARG + CAN plates; one served as the unirradiated control for spontaneous mutation, and the other(s) received the necessary UV fluence to distinguish between urnr and U M R (28 or 56 J/m2). U M R segregants exhibited numerous can1 m u t a n t colonies growing from the replicas (often more than 50), while few or none arose w i t h o u t UV. Segregants of some putative u m r isolates with considerable effect on m ut at i on had few or no can1 colonies on either plate, whereas other isolates with tess effect exhibited induced mutants but with a net increase less than that of wild type. This semiquantitative procedure reflects only the m u t a n t frequency in terms of m u t a n t s p e r cell p l a t e d . A l l e l i s m t e s t s a m o n g urnr s t r a i n s

Complementation

tests could n o t be perform ed between u m r

mutants

183 because the wild-type function, UV mutation to recessive can1, is n o t expressed in the diploid. UV revertibility could not be used, as it had been with rev X rev complementation tests [23], because the effects of u m r on UV reversion were n o t known at this point nor could they be assumed. Indeed, a number of mutants were found eventually to have normal UV revertibility. All u m r X u m r hybrids were immediately sporulated on both types of sporulation agar and subjected to tetrad analysis. Haploid spore clones were tested for UV forward mutation. The parents were considered to be allelic if they failed to produce tetrads having U M R spores or nonallelic if wild types were present as NPD or T asci. M e a s u r e m e n t o f survival and m u t a t i o n f r e q u e n c y

In each independent experiment, cells from single-colony isolates were seeded into liquid YEPD and shaken vigorously at 30°C for 3 days to stationary phase. Cultures were washed three times with distilled water and resuspenced at high titer (~ 109 cells/ml). Cells were diluted in distilled water, plated in replicate on all relevant media, and finally exposed to UV. The surviving fraction after irradiation was assayed in the usual manner by plating appropriate numbers of cells to provide statistically reliable colony counts. To determine whether or not survival could be influenced by the kind of plating medium, cells were plated on both the complex YEPD agar and the synthetic SC-ARG agar. Forward mutations to canavanine resistance are recessive and occur only at the can1 locus [13,40]. Cells from u m r and U M R strains were plated on SC-

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F i g . 1. U V s u r v i v a l ( . . . . . . ) o f t h e w i l d t y p e X Y 5 0 5 - 1 8 C o n Y E P D (m) o r S C - A R G (u). U V m u t a b i l i t y ( ) i n X Y 5 0 5 - 1 8 C : F o r w a r d m u t a t i o n s t o can1 ( o ) a n d r e v e r s i o n s o f hisS-2 (o), l y s l - 1 (~), a n d urn4-1 (A). E a c h p o i n t r e p r e s e n t s t h e m e a n o f t h r e e i n d e p e n d e n t e x p e r i m e n t s . E r r o r b a r s i n d i c a t e s t a n d a r d e r r o r s o f t h e m e a n . E r r o r i n t e r v a l s less t h a n t h e size o f a s y m b o l are n o t d r a w n . M e a n s p o n t a n e o u s f r e q u e n c i e s are p l o t t e d a t z e r o U V f l u e n c e .

184 A

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fraction on SC-ARG. Frequencies of induced mutation in each independent experiment were calculated by subtracting the spontaneous frequencies from those obtained after UV exposure. Three independent experiments were performed for each strain except for cases in which results with closely related strains were combined. Values for surviving fraction and induced mutation frequency were averaged and are plotted in Figs. 1--10. The standard error of the mean (between experiments) was calculated and is indicated by an error bar

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F i g . 2 - - 7 . A. U V s u r v i v a l o f Y E P D (m) o r S C - A R G (~) o f t h e u r n r s t r a i n ( . . . . . . ), c o m p a r e d w i t h w i l d type XY505-18C (...... ). U V m u t a b i l i t y t o c a n 1 ( ) o f u m r (o) a n d w i l d - t y p e ( e ) s t r a i n s . B. U V r e v e r s i o n o f h i s 5 - 2 , l y s l 1, a ~ d u r a 4 - 1 i n u m r (o) a n d w i l d - t y p e ( e ) s t r a i n s . I n b o t h A a n d B, e a c h p o i n t represents the mean of three ihdependent experiments. Error bars indicate standard errors of the mean. E r r o r i n t e r v a l s less t h a n t h e size o f ~ s y m b o l are n o t d r a w n . M e a n s p o n t a n e o u s f r e q u e n c i e s are p l o t t e d a t zero UV fluence.

187

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All u m r and U M R strains carried the UV-revertible, ochre-suppressible his5-2, l y s l - 1 , and ura4-1 alleles, and two strains carried the leu2-1 ochre as well. These alleles are all suppressible by the strong, dominant ochre-specific SUP2, S U P 3 . . . . , S U P 8 , and S U P 1 1 nonsense suppressor loci comprising classes I and

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Fig. 9. A . U V s u r v i v a l o n Y E P D (m) o r S C - A R G (u) o f t h e a u m r 7 - 1 s t r a i n X Y 5 0 7 - 7 A ( . . . . . . ), c o m p a r e d with wild type XY505-18C (...... ). U V m u t a b i l i t y t o c a n 1 o f w i l d - t y p e (~) a n d u r n r (©, 1.7 X 1 0 7 c e l l s p e r p l a t e ; o, 1.7 X 1 0 8 c e l l s p e r p l a t e ) s t r a i n s . B. U V r e v e r s i o n o f h i s 5 - 2 , l y s l - 1 , a n d u r a 4 - 1 i n w i l d - t y p e (~) a n d u r n r ( o , 1.7 × 1 0 7 c e l l s / p l a t e ; o, 1.7 X 1 0 8 c e l l s / p l a t e ) s t r a i n s . P o i n t s , e r r o r b a r s , a n d s p o n t a n e o u s freq u e n c i e s are as d e s c r i b e d i n F i g s . 2 - - 7 .

188 A

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