Pathways of ultraviolet mutability in Saccharomyces cerevisiae

d~lutation Research

319

Elsevier Publishing Company, Amsterdam Printed in The Netherlands

P A T H W A Y S OF U L T R A V I O L E T M U T A B I L I T Y IN S A C C H A R O M Y C E S CEREVISIAE

II. T H E E F F E C T OF rev GENES ON RECOMBINATION*

J E F F R E Y F. LEMONTT

Donner Laboratory, University of California, Berkeley, Cal@ (U.S.A .) (Received June 2nd, 1971) (Revision received August 24th, 1971)

SUMMARY

Recombination at both intergenic and intragenic levels has been investigated in diploid yeast strains each homozygous for one allele of three different rev loci that cause reduced UV mutability. Meiotic recombination between leuz and trp5 and between arg4-6 and arg4-z 7 was found to occur at control frequencies. The frequencies of UV- and X-ray-induced mitotic recombination, measured for both the ade2centromere and arg4-6-arg4-~7 regions, increased more sharply with radiation dose in rev/rev diploids than in wild type. Since rev mutations do not cause recombination deficiency, it is suggested that mitotic recombination as measured in this study, although induced by UV damage, is not correlated with UV mutagenesis.

INTRODUCTION

Genetic recombination appears to play an important role in UV-induced mutagenesis of E. coli ~. Induced nmtations are thought to be produced during a postreplication recombinational repair process that acts independently of excision repair 22. Strains carrying rec or exr genes, which cause varying degrees of both recombination deficiency and radiation (UV and X-ray) sensitivity, are defective in UV-induced mutationg,2°, 21, presumably because they are unable to carry out some mutagenic step in recombination repair 2.. Strains defective in excision repair are also UV-sensitive but, however, are neither recombination-deficient nor defective in UV mutability 3,19. In bacteriophage lambda, on the other hand, it has been suggested that no correlation exists between UV mutation and recombination 1°. * Part of a dissertation submitted for the Ph.D. degree, Supported by U.S. Public Health Service Fellowship number 5-PoI-GM4io59-o2. Facilities provided by U.S. Atomic Energy Commission. Present address: Division of Biology, National Research Council of Canada, Ottawa, Ontario [(IA OR6 (Canada).

Mutation Res., 13 (1971) 319-326

320

JEFFREY F. LEMONTT

If the mechanisms of both UV-induced mutagenesis and of recombination in yeast are similar to those operative in bacteria, then recombination-deficient yeast nmtants should be defective in UV mutability. Conversely, one might expect to find that some genes involved in UV mutation also regulate recombination. Mutants at three loci have been isolated in E. cerevisiae which are defective in UV-induced reversion and which also confer moderate UV and X-ray sensitivitv'%~L It has been suggested in the preceding papeU that such rcv genes block repair pathways that act independently of excision repair. If rev nmtants are found to be recombinationdeficient, it could be inferred that the mechanisn~ of UV-induced mutation in yeast inwslves recombinational events. S.'
Veast straiJzs. Diploids homozygous for rev mutations were constructed by crossing appropiiate rev segregants obtained from R E V / r e v crosses after sporulation and ascus dissections 4. To study intergenic recombination in both the ade2 centromere and the let~z IU55 regions n, strains XYI2() ( R E V / R E V ) , XYI5o (revI-,/revx-i), XYI27 (rev2-I/rev2-I) and XYI28 (rev3-z/rev3-I) were made to carry ade2-I/A l)E and le~tz-I2 [email protected]/LEU T R P . These diploids also carried ar~4-I7/arg4-z 7 and have been described previously. "". To study intragenic recombination the following heteroallelic diploids of the type aN4-6/arg4-± 7 rev/rev were obtained s by crossing roy ,~rg4-O and rev arg4-I 7 segregants: XYI82 ( R E I ' / R E I ' ) , XYI83 (r~vI-l/revI-I), XYI84 (ft'i,2-I/fC~!2-I) and XYI85 (rev3-I/rev3-i). Genetic symbols have been dcscril)ed ~2. E@erimeutal proced~¢res. Meiotic recombination was studied using random spore analysis 2. After sonic disruption of enzyme-treated 4 asci for 3 rain at 21 ke/see (IOO W Ultrasonic Disintegrator, Measuring and Scientific Ltd., London), suspensions containing both random spores and unsporulatcd diploids were diluted and plated onto YEPD. To measure intergenic recombination, the resulting clones were tested for adenine, leucine and tryptophan growth requirements. Parental and recombinant phenotypes were scored with respect to leuz and tr]55. Only adenine-deficient clones were considered in order to avoid including unsporulated diploids among the random spore sample. All lnedia including YEPD, C-AR (arginine), C-LE (leucine), C-AI) (adenine), C-TR (tlyptophala) and synthetic complete (C) have been described previously". Prototrophic haploid meiotic recombinants produced by arg 4 heteroallelic diphfids were selected as arginine-independent revertants growing on ('-AR after incubation of plates ~pread with between 3ooo and qooo ceils from the sonicated suspension (haploids and diploids). Heteroallelie diploids would also produce mitotic Mulatio~z I&s., 13 (~971 ) 3 i~)-326

PATHWAYS OF U V MUTABILITY IN S.

cerevisiae. I I .

321

arg ÷ prototrophs but at a frequency about IOO times lower than in meiosis (see RESULTS). Numbers of diploids in the suspension were estimated by first diluting and plating about 5o cells/plate onto YEPD. C-AR replicas of the resulting Y E P D clones were exposed to IO kR of X-rays and incubated for 3 4 days. Diploids (heteroallelic) exhibited numerous X-ray-induced mitotic arg ÷ prototrophs growing on the replicas. This was possible since rev/rev diploids are not deticient in such recombination (see RESULTS). Haploids, however, containing either arg4-I7 or arg4-6 or rarely both, failed to exhibit prototrophs since the frequency of X-ray-induced reversion of either allele is several orders of magnitude lower than the frequency of X-ray-induced heteroallelic mitotic recombination. Thus, the frequency of meiotic prototrophs was based on the numbers of prototrophs per viable haploid spore plated. Induced mitotic recombination was examined in cultures grown for about 4 days in liquid Y E P D after inoculation with cells from a single colony isolate. Cells were harvested, washed twice with distilled water and then placed on ice at the proper titer. To measure intergenic recombination, cells were plated onto Y E P D and subsequently exposed to varying doses of either X-rays or UV, followed by incubation for about 5 days at 3 o°. Since a homozygous ade2 marker 11 causes the accumulation of an intracellular red pigment readily seen at the clonal level 16, mitotic recombination between ade2 and its centromere leading to ade2 homozygosis was scored by observing red-sectored clones among the survivors. Intragenic mitotic recombination was assayed as prototroph production on arginineless synthetic medium in diploids heteroallelic for arg4. Large numbers ( ~ IO6) of cells from washed 4-day cultures were plated onto C-AR and subsequently exposed to varying doses of UV or X-rays. Radiation doses used to induce intragenic recombination were small so that viability remained at about IOO}~ in all strains. The UV and X-ray sources have already been described 6. RESULTS AND DISCUSSION

Meiotic recombination. Tables I and I I show that homozygous rev genes exert no detectable effect on the production of meiotic recombinants either in theleuz-trp5 or in the arg4-6-arg4-r 7 regions. Studying other intergenic and intragenic systems, SNOW17 obtained similar results in 6 diploids each homozygous for different genes leading to UV sensitivity, including uvs 9. Thus, the repair of lethal UV damage, whether or not it leads to mutation production, probably occurs in pathways independent of steps leading to meiotic recombination. Induced mitotic recombination. Both UV and X-rays were employed to induce mitotic recombination. Nonreciprocal recombination (gene conversion) can lead to red-sectored clones especially at higher doses, but NAKAI AND MORT1MER14 found that most radiation-induced events leading to homozygosis were reciprocal mitotic crossovers. The effect of rev genes on radiation-induced mitotic segregation of ade2 is seen in Figs. I a and lb. Table I I I gives the survival levels. Although the curves are complex, greater frequencies of recombination are observed in homozygous rev diploids than in wild type when compared at equal radiation doses. MOUSTACCH113, SNOW17, and ZAKHAROVel al. 2a have observed similar effects on UV-induced intergenic mitotic recombination in diploids homozygous for uvs mutations. Heteroallelic mitotic recombination, however, occurs predominantly during a 2~lulation Res., 13 ( I 9 7 i ) 319 326

322

JEFFREY F. LEMONTT


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PATHWAYS OF U V MUTABILITY IN S . cerevisiae.

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T A B L E II I N T R A G I ~ N I C M E I O T I C R E C O M B I N A T I O N 13ETXVEEN

Diploid

Viable cells per plate × rO~2

rev genotype

arg4-6

AND arg.l-I 7 IN

Haploid ascosporaI clones ( % )

REV/RE V

Viable haploids per plate × ZO-z

AND

rev/rev

Average number prototrophs per plate

DIPLOIDS

Frequency of prototrophs per viable haploid spore X ZO "-~

REV/REV revi-r/revl-z rev2-I/rev2, r rev3-I ~re'v3~

XYI82 XYI83 XYt84 X Y 185

38.4 56,7 30.0 82.5

53.2 3Lo 53.0 47 .0

2o.4 I7.6 15. 9 38.8

19.o 22.7 ]6.8 48.8

:t_ _f: ~ ~

3,6 a 3-3 2.8 4.4

0.93 ~.29 [.o6 1.z6

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a D e n o t e s s t a n d a r d error of six plate c o u n t s . 10

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Fig. L I n t e r g e n i c m i t o t i c r e c o m b i n a t i o n b e t w e e n ade2 a n d its c e n t r o m e r e i n d u c e d b y UV (Ia) or X - r a y s (Ib) in R E V / R E V a n d rev/rev diploids.

nonreciprocal process in which prototrophs are produced by gene conversion of one allele~L Coconversions in meiosis would occur more frequently than single-site events and do not produce prototrophs 1. If mitotic conversions are also mostly coconversions of these alleles, then the observed prototrophs represent a minority population of single-site conversions among all conversions. The effect of rev genes on such radiationinduced gene conversion is shown in Figs. 2a and 2b. Exposure to either radiation produces greater prototroph frequencies in rev/rev diploids than in wild type. The Mutation Res., I3 (1971) 319-326

324 TABLE

J E F F R E Y F. LEMONTT Ill

SURVIVAL

AT R A D I A T I O N

1~]~['" AND

r~,1)/r(,t) DIPLOIDS

Radiat~o~z dose

DOSES USED

TO I N D U C E

INI"I-'RGEN1C MITOTIC R E C O M B I N A T I O N

IN

lelil'/

S u r v i v a l ('~,) Rt:. L'/I?I:~ l"

revz-1/rew-r

rev2 1/r~'v2-1

rcv3-I /rcv3-I

I oo 96 63

77 72 1,2

6() 5 .8 o.75

~oo 4.7 0.74

98 85 49

92 86 04

loo 9i ~7

t~ t" (erg/mm ~) 265 53 ° 795 X - r a y (kl?) 3,75 7.5 ° 15.°

97 94 85

45.0

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data points for X - r a y induction, known to be linear with dose in wild type ~, were fitted to straight lines using the least squares method. The slopes and standard errors of regression lines for X Y I 8 2 , X Y I 8 4 , X Y I 8 5 and X Y I 8 3 are 4,7 i 1.2, 7-3 d_ 1.5, 11. 5 ± 0. 9 and 14. 5 :[: 0.3 prototrophs per IO~ smvivors per R, respectively. The curves of UV induction exhibit the same order of enhancement among r e v / r e v strains. That is, the curves for r e v ± - z or r e v 3 - I homozygotes are steeper than that for the r e v 2 - z homozygote. Homozygous r e v z - i or re~,,3-I alleles also reduce UV reversion of a r g 4 - I 7 to a greater degree 6 than does r e v 2 - i . Studying UV-induced heteroallelic reversion of h i s l , SNOW~7 also found enhanced piototroph frequencies in strains carrying homozygous u v s mutations, including uvs9, compared to wild type at equal UV doses. If, with increasing proximity of the two heteroalleles, coconversions are produced mitotically at the expense of single-site conversions as in meiosis 1, then the enhanced recombination of r e v / r e v (or u v s / u v s ) diploids could be due to an increased proportion of single-site events. This could occur if rev (or re,s) genes reduce the length of the converted interval. To investigate the relation between UV-induced nmtation and recombination in yeast, three f r y loci have been tested for their effect on both meiotic and radiationinduced mitotic recombination at both intergenic and intragenic levels. If UV nmtations were produced by a mechanism similar to that found in E . coli, genes conferring defective UV nmtability ought to cause v m y i n g degrees of recombination deficiency like e x r and rec loci. Since it was found that rc~,' genes do not block recombination in the diploid systems studied, it can be a~gucd that UV-induced mutations are not produced by recombination events alone, if at all. W h a t then accounts for the enhanced frequencies of radiation-induced mitotic recombination observed in diploids homozygous for either rev or uvs genes ? It cannot sin',ply be due to a causative relation between nmtation induction and recombination, since u v s 9 strains, which are not blocked in UV mutationT, 1~, also exhibit these enhanced frequencies. The simplest interpretation is that mitotic recombination is not involved in the mutagenic steps associated with R E V pathways, but nevertheless is stimulated by agents such as UV that also induce lethal D N A damage. UV-sensitive strains (re~J o r w,,s) then would exhibit greater frequencies of [W-induced mitotic recombination at equal UV doses because they repair, by any mechanismL fewer recombination-promoting potentially lethal lesions than the wild type. .,llutatio~i Res., 13 (t97~) 3x9-326

PATHWAYS OF U V MUTABIIATY IN S . cerevisiae. II. 80

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Fig. 2. Intragenic mitotic recoulbination between arg 4- 6 and arg4-z 7 induced by UV (2a) or X-rays (2b) in R E I / ' / R E I 7 and rev/rev diploids.

In summary, pathways of gene-controlled UV mutability in yeast may be characterized as follows: A small number of R E V genes, at least three, act in pathways that share common enzymatic steps with the repair of lethal UV and X-ray DNA 2l]utation Res., 13 (1971) 319-326

326

JEFFREY F. LEMONTT

damage 6. These pathways are thought to operate later than the reaction controlled t)3, UVS9, itself likely responsible for excision repair of UV-induced pyrimidine dimers. Mitotic recombination as measured in this study, although induced by UV damage, is not correlated with UV nmtagenesis. ACKNOWLEDGMENTS

I wish to thank Dr. ROBERT K. MORTIMERfor his advice and support during the course of this study. I{F;FF;I{ENCES 1 FOGEL, S., D. D. HURST AND R, K. MORTIMER, Gene conversion in unselected t e t r a d s fronl nlultipoint crosses, Seco~zd Stadler Symposium, May i97o. 2 GILMORF., R. A., Super-suppressors in Saceharo~nyees cerevisiae, Genetics, 56 0907) 641-658. 3 HILL, l{. F., Ultraviolet induced lethality and reversion to p r o t o t r o p h y in Escherichia coli strains with normal and reduced dark repair ability, Photochem. Photobiol., 4 (1965) 563-.568. 4 JOHNSTON, J. R., AND R. K. MORTIMER, Use of snail digestive juice in isolation of yeast spore tetrads, .]. Bacteriol., 78 (I959} 292. 5 LEMONTT, J. F., GenetiecontrolofmutationindnctioninSaccharomycescerevisiae(Ph.D. Thesis), Uniw, rsity q[CaHfornia, Lawrence Radiation Laboratory Report UCLRL-2o2 z5, Sept. 197 o. 6 LEMONTT, J. F., M u t a n t s of yeast defective in m u t a t i o n induced by ultraviolet light, Genetics, 68 (1971 ) 21 337 LEMONTT, J. F., P a t h w a y s of ultraviolet nautability in Saccharomyces ccrevisiae, I. Some properties of double m u t a n t s involving uvs 9 and rev, 11~rutation Res., 13 (19711 311-317. 8 MANNEY, T. R., AND ]1. K. MORTIMER, Allelic m a p p i n g in yeast by X-ray-induced mitotic reversion, Science, 143 (19641 581-582. 9 MIURA, A., AND J. TOMIZAWA, Studies on radiation-sensitive m u t a n t s of E. coli, 11[. Partici p a t i o n of Rec s y s t e m in induction of m u t a t i o n by ultraviolet irradiation, ~lIol. Gen. Gen~t., lO 3 11968 ) I--IO. IO M1URA, A., AND J. TOMIZAWA, Mutation and recombination of bacteriophage lanlbda; Effect of ultraviolet radiation, Proc. Natl. Acad. Sci. (U.S.), 67 {197o } 1722 1726. i f MORTIMER, R. 11., AND D. C. HA\VTHORNE, Genetic m a p p i n g in Saccharonlyces, Gem'ties, 53 (t966) 165-173 • 12 ~{ORTIMER, ]~. K., F. ~HERMAN AND t~.. C. VON BORSTEL, Proposal for a new system of genetic nomenclature for use in yeast genetics research, Microbial (;enetics Bzdh'ti~L (1969) No. 3 I , Yeast Genetics Supplement. 13 M/)USTACCHI, ]']., Cytoplasmic and nuclear genetic eveuts induced by UV light in strains of Saccharomyces cerevisiae with different U V sensitivities, Mutalion Res., 7 (1969) 171 185. 14 NAKAI, S., AND 1{. K. MORTIMER, Studies o[ the genetic mechanism of radiation-induced nlitotic segregation in yeast, Mol. Gen. Genet., lO3 (I969) 329 338. 15 RESNICK, M. A., I n d u c t i o n of m u t a t i o n s in Saccharomyees cerevisiae by ultraviolet light, Mzttation Res., 7 11969) 315-332. 16 ROMAN, H., A s y s t e m selective for m u t a t i o n s affecting the synthesis ol adenine in yeast, Compt. Rend. Tray. Lab. Carlsberg, 26 (1956) 299 314 • 17 SNow, R., ReconIbination in ultraviolet-sensitive strains of Saccharomyces ccrevisiae, 3iutatio~ Res., 6 11968) 4o9-418. 18 WlLDENBER/~, J., The relation of mitotic recombination to D N A replication in yeast pedigrees, Ge~u,tics, 66 (197 o) 29I-3O 4. I9 \VITKIN, E. M., Radiation-induced m u t a t i o n s and their repair, Science, 152 (I966) 1345-1353. 20 WITK1N, 1:. M., Mutation-proof and m u t a t i o n - p r o n e modes of survival in derivatives of E. c~li B differing in sensitivity to ultraviolet light, Brookhave~ Syrup. Biol., 2o 119671 17 55. 21 WrrKIN, 1'2. M., The umtat)ility t o w a r d ultraviolet light of recombination-deficient strains of E. coli, Mutation Res., 8 119691 9-14. 22 WITKIN, F. M., U\:-induced m u t a t i o n and D N A repair, Ann. Rev. Genet., 3 (19691 525--552. 23 ZAKHAROV, 1. A., T. N. KOZlNA AND I. V. I"EDOROVA, Effects des m u t a t i o n s vers la sensibilitd au r a y o n n e n l e n t ultraviolet chez la levure, 3lz~tatio~ Rt,s., 9 (I97 o) 31-30 -

Mutation Res., 13 (I97~) 319-326