Curr Genet (1992)21:319-324

Current Genetics 9 Springer-Verlag 1992

Isolation and characterization of additional genes influencing resistance to various mutagens in the yeast Saccharomyces

cerevisiae* Eckard Haase, J~rg Servos, and Martin Brendel Institut ffir Mikrobiologie der J. W. Goethe-Universit/it, Theodor-Stern-Kai 7, Haus 75, W-6000 Frankfurt/Main, Federal Republic of Germany Received December 20, 1991

Summary. Screening of a multi-copy vector-based yeast genomic library in haploid cells of wild-type Saccharomyces cerevisiae yielded transformants hyper-resistant to various chemical mutagens. Genetical analysis of the yeast insert DNAs revealed three genes SNG1, SNQ2, and SNQ3 that confer the phenotype hyper-resistance to MNNG, to 4-NQO and triaziquone, and to mutagens 4-NQO, MNNG, and triaziquone, respectively. Integration of the gene disruption-constructs into the haploid yeast genome yielded viable null-mutants with a mutagen-sensitive phenotype. Thus, copy number of these non-essential yeast genes determines the relative resistance to certain chemical mutagens, with zero copies yielding a phenotype of mutagen sensitivity and multiple copies one of mutagen hyper-resistance, respectively. Key words: Multi-copy plasmid - Hyper-resistance 4-NQO - M N N G Triaziquone Introduction The killing of yeast cells treated with mutagens such as 4-NQO, M N N G , HN2, and triaziquone, is predominantly the result of the D N A damage caused by these chemicals (Sugimura et al. 1965; Ruhland and Brendel 1979; Fleer and Brendel 1979; Brendel and Ruhland 1984; Kistler et al. 1986). The removal of mutagen-inflicted D N A lesions is mediated by a cellular D N A repair system. In the yeast Saceharomyces cerevisiae about 50 different genes have been shown to be involved in metabolic processes ultimately leading to repair of damaged D N A and hence to survival of the cell; according to the interaction of their defective mutant alleles when placed in double mutants, these genes have been ordered in three different epistasis groups that may represent three repair pathways (Game and Cox 1972; Brendel and Haynes 1973; see review by Friedberg 1988). * Dedicated to Professor Dr. R. W. Kaplan on the occasion of his 80th birthday Offprint requests to: M. Brendel

Many chemicals are frequently not carcinogenic or mutagenic per se but require metabolic activation to form reactive intermediates which themselves are able to combine with critical cellular components. For example 4-NQO (4-nitroquinoline-N-oxide) (Sugimura et al. 1965; Tada and Tada 1975), cyclophosphamide (Fleer etal. 1982), and M N N G (N-methyl-N'-nitro-N-nitrosoguanidine) (Sedgwick and Robins 1980) must undergo metabolic activation processes before becoming DNA damaging agents. If a multi-copy vector-contained, and hence over-expressed, yeast gene would negatively interfere with these processes this would alter a cell's response to the chemical, most probably making it more resistant. A higher mutagen resistance would also be expected if genes with a function in the de-activation of chemical mutagens were over-expressed in a transformant. Thus, while a yeast cell's relative resistance to mutagen treatment may largely depend on this ability to repair damaged nuclear DNA it may also, in the case of chemical mutagens, depend on extranuclear activities, i.e., on cellular processes such as transport and biotransformation that may lower the relative input of the active mutagen to the target DNA (Goldenberg and Begleiter 1984; Curt et al. 1984). In order to isolate yeast genes involved either in the repair of chemical mutagen-damaged DNA or in the cellular metabolism of chemical mutagens, we screened haploid wild-type yeast transformed with a multi-copy vector-contained genomic yeast gene library (Carlson and Botstein 1982) for the phenotype of mutagen hyper-resistance (HYR phenotype) following the experimental approach described by Ruhland et al. (1986). In previous communications we have shown that in S. cerevisiae the genes SNQ1, SNQ3, SFA1, and HNM1 are involved in controlling sensitivity to the chemical mutagens 4-NQO, formaldehyde, and nitrogen mustard, (HN2) , respectively (Mack et al. 1988; G6mpel-Klein et al. 1989; Haase and Brendel 1990; Hertle et al. 1991; Li et al. 1991). Here we report on the isolation and characterization of additional yeast genes which, when present in multiple copies, are able to confer the H Y R phenotype on S. cerevisiae.

320

Materials and methods

Table 1. Yeast strains employed

Yeast strains, bacteria, bacteriophage, and plasmids. The genotypes

Strain

Genotype

MKPo

MATe, his3-A200, ura3-52, leu2-3, 112, trpl-A901, lys2-1, ade2-1, canl-lO0 MATe, ade2.199, trp5-b, ilvl-92 MATe, ade2-40, trp5-a, ilvl-92 MATe, ade2-40, trp5-a, ilvl-92 MATe, ura3-52, leu2-3, 112, trpl-289 MATa, his5-2, lysl-1, ade2-1, ura3-52, leu2-3, 112 MATa, his5-2, lysl-1, ade2-1, ura3-52, snml-i MATa, hisl, ade, ura3-52, leu2-3, 112, tup7, rev2-1 MATc~, his3/4, ura3-52, [eu2-3, 112, radl8-2 MATe, his3, lys2-1, ade2-1, ura3-52, leu2-3, 112, trpl-289, rad2-20 MATe, his3, ade2-1, ura3-52, leu2-3, 112, trpl-A901, tup7, radl-1 Mate, his3-A200, ade2-1, ura3-52, leu2-3, 112, trpl-289, rad52 MATe, ura3-52, leu2-3, 112, trpl-289, rad4-4 MATe, ura3-52, leu2-3, 112, rad6-1 MATe, ura3-52, leu2-3, 112, trpl-289, rad3-x MATe, ade2-tOl, lysl-1, lys2-801, arg4-17, ura3-52, leu2A-1, trplA-1

of the yeast strains used in this study are shown in Table 1. Strain JTO-3A was a gift from 17.K. Zimmmermann. The two multi-copy yeast-Escherichia coli shuttle vectors Yep24, carrying the URA3, gene, and YEpl3, carrying the LEU2 gene, have been described previously (Botstein et al. 1979; Broach et al. 1979). A YEp24-based yeast genomic library (Carlson and Botstein 1982) was used for the cloning of yeast genes conferring hyper-resistance. The bacteria, plasmids and bacteriophages for TnlO mutagenesis were kindly provided by N. Kleckner.

Media and growth conditions. Standard growth medium for yeast was YEPD as described by Fleer and Brendel (1979). Prototrophic yeast transformants were screened on the appropriately supplemented synthetic media (SynCo), as described by Ruhland and Brendel (1979). LaeZ gene fusions were detected as blue-colored colonies on buffered M63 medium prepared according to Clifton et al. (1978) with 40 ~tg/ml X-Gal (5-bromo-4-chloro-3-undolyl-//D-galactopyranoside) added. E. eoli was grown in LB medium as described by Miller (1972), with antibiotics supplemented according to procedures given in Huisman et al. (1987). Determination of HYR (wild-type) phenotype. Mutagen resistance was determined by replica-plating of transformants onto ice-cold Synco-ura medium (Ruhland and Brendel 1979) supplemented with either 4-NQO, MNNG or triaziquone, and verification of the HYR phenotype via the agar diffusion test as described by Ruhland et al. (1981). Survival curves were calculated as described by Ruhland and Brendel (1979). The cells were pre-grown in liquid Synco-ura medium at 30 ~ on a gyrotary shaker to stationary growth phase, washed three times with phosphate buffer (0.067 tool/l, pH 7.0) and treated with 4-NQO or MNNG as described by Ruhland and Brendel (1979). Preparation of DNA and transformation procedures. Plasmid DNA was isolated from E. coli by alkaline lysis (Birnboim and Doly 1979). Purification, restriction, ligation, and analysis on agarose gels of plasmid DNA were performed as described by Maniatis et al. (1982). Electroeluted DNA was further purified on Elutip columns (Schleicher and Schfill and Dassel, FRG). E. coli was transformed according to Dagert and Ehrlich (1979), and yeast according to the method of Ito et al. (1983) with modifications as described by Rodriguez and Tait (1983). Yeast plasmid DNA was isolated according to methods described by Rodriguez and Tait (1983) or by Holm et al. (1986). Subeloning of the HYR genes. DNA of the plasmids pEH7, pEH16 and pEH22 were partially digested with Sau3A and the resulting fragments were separated by preparative agarose gel electrophoresis (Maniatis et al. 1982). Fragments sized between 2 and 6 kb were isolated by electro-elution and ligated into YEp24, which had been cut by BamHI and treated with alkaline phosphatase. The mixture was transformed into E. eoli and transformants that were sensitive to tetracycline and resistant to ampicillin were selected. Some 300 clones were washed off the agar plates and grown for 3 h in liquid culture (LB medium + ampicillin). Isolated plasmids were used to transform wild-type yeast strain EH3714-2B and transformants were tested for the HYR phenotype. Plasmid DNA was prepared from transformants that demonstrated the appropriate HYR phenotype and after re-transformation and propagation in E. coli was used for further restriction analysis. TnlO transposon mutagenesis and mapping of SNQ2. E. coli strain NK5830 containing the transposase plasmid pNK629 was transformed with a monomer of the plasmid pEH22 containing the SNQ2 gene and with pEH1605 containing the SNQ3 gene. Transposition of TnlO-lacZ-URA3-kanR (Huisman et al. 1987) - from now on abbreviated Tnl0-LUK- into the plasmids pEH22 and pEH1605 was carried out by the "lambda-hop" procedure as described by Way et al. (1984). Transformants of E. coli resistant to kanamycin

JTO-3A EH3879-5C EH3867-1B EH3255-6A EH3714-2B EH3709-2A EH3666-3A EH3644-10A EH3719-3C EH3696-2B EH3752-1A EH3745-5A EH3763-1C EH3759-1D EH3871-2B

and ampicillin were selected and DNA of the pooled transformants was isolated by alkaline lysis (Maniatis et al. 1982). After re-transformation of the lambda-resistant E. coli strain NK8017 plasmid DNA was prepared from some blue coloured transformants (X-Gal). A yeast wild-type strain was transformed and tested for its HYR phenotype. Isolates not exhibiting the HYR phenotype were further analysed by restriction mapping.

Disruption and gene transplacement of HYR genes. Yeast genes contained in plasmids pEH7, pEH1605, and pEH22 which confer the mutagen HYR phenotype were disrupted by the insertion of TnlOLUK. Additionally, gene SNG1 contained in pEH705 was disrupted by insertion of the LEU2 gene in the BglII site (see Fig. 3). Linearized constructs were employed for gene transplacement studies in haploid wild-type according to the protocol of Rothstein (1983). Uracil or leucine prototrophic transformants exhibited an enhanced sensitivity to the appropriate mutagens. Gene transplacemerit was verified by genetical analysis.

Results T h e three m u t a g e n s 4 - N Q O , M N N G , a n d t r i a z i q u o n e , differing in their specificity o f D N A i n t e r a c t i o n (cf. Brendel a n d R u h l a n d 1984) a n d hence l e a d i n g to different D N A lesions, were e m p l o y e d for screening o f a p p r o x i m a t e l y 6 000 y e a s t t r a n s f o r m a n t s for the m u t a g e n h y p e r resistance p h e n o t y p e ( H Y R p h e n o t y p e ) . We f o u n d 39 p r i m a r y H Y R isolates (Table 2) t h a t were f u r t h e r screened for their H Y R p h e n o t y p e in the a g a r d i f f u s i o n test ( R u h l a n d et al. 1986); nine isolates, h a r b o u r i n g inform a t i o n for 14 H Y R p h e n o t y p e s t o w a r d s the three m u t a gens were finally d e t e r m i n e d (Table 3). T h e c o r r e l a t i o n o f v e c t o r - c o n t a i n e d y e a s t insert D N A w i t h the e x p r e s s i o n o f the H Y R p h e n o t y p e c o u l d be verified b y p l a s m i d - l o s s e x p e r i m e n t s in all o f them. T h r e e o f these y e a s t insert D N A s , p E H 7 , p E H 1 6 , a n d p E H 2 2 , c o n t a i n e d in the respective p l a s m i d s o f y e a s t t r a n s f o r m a n t s i7, i16, a n d i22,

321 _1

lOG

[

I

l

(~

Table 4. Complementation of mutagen sensitivity by HYR-conferring plasmids in DNA repair mutants Mutant

'5.> ,> ~c

0

I

I

I

1 2 3 4NQO (tumol/t)

I

4

[, 0

4-NQO

MNNG

Triaziquone

UV

HN2

S WT S S

S S WT HYR

S WT S S

S S S S

S S S S

rad2 YEp24 rad2 pEH22 rad2 pEH16 rad2 pEH7

S HYR S S

S S S HYR

S HYR S S

S S S S

S S S S

rad3 YEp24 rad3 pEH22 rad3 pEHI6 rad3 pEH7

S WT S S

S S S HYR

S HYR S S

S S S S

S S S S

rad4 Yep24 rad4 pEH22 rad4 pEH16 rad4 pEH7

S HYR WT S

S S WT HYR

S HYR S S

S S S S

S S S S

snml snml snml snml

WT HYR HYR WT

WT WT WT HYR

S S S S

WT WT WT WT

S S S S

rad5 YEp24 rad5 pEH22 rad5 pEH16 rad5 pEH7

S S S S

S S S WT

S S S S

S S S S

S S S S

tad6 YEp24 rad6 pEH22 rad6 pEH16 rad6 pEH7

S HYR S S

S S HYR HYR

S HYR S S

S S S S

S S S S

rad18 YEp24 rad18 pEH22 rad18 pEH16 rad18 pEH7

S S S S

S S S S

S S S S

S S S S

S S S S

rad52 YEp24 rad52 pEH22 rad52 pEH16 rad52 pEH7

S WT S S

WT WT HYR HYR

S HYR WT S

WT WT WT WT

S S S S

radl radl rad! radl

20 40 60 NNNG (/umol/I }

80

Fig. 1 A, B. Survival of haploid yeast cells transformed with plasmids YEp24 (open circles), pEH7 (filled squares), pEHI6 (filled triangles), and pEH22 (filled triangles, pointed downwards) to 4-NQO (A) or MNNG (B)

Table 2. Selection of mutagen hyper-resistant transformants Concentration of mutagen (mM)

Number of isolates"

No. with verified HYR phenotype b

4-NQO (4.0) MNNG (5.0) Triaziquone (4.3)

17 10 12

5 6 3

Ura-prototrophic transformants were replica-plated onto ice-cold SynCo-ura agar plates freshly supplemented with the appropriate mutagen and incubated at 30~ for 3 days b Verification ofmutagen HYR phenotype in the agar diffusion test (Ruhland et al. 1981); 200 gL of mutagen solutions were applied with concentrations of 4-NQO (0.1 mM), MNNG (6.7 mM), and triaziquone (0.86 mM) a

Table 3. Phenotypes of the 16 HYR isolates Pattern of HYR

Name of isolate a

Only 4-NQO Only MNNG 4-NQO and MNNG 4-NQO and triaziquone 4-NQO, MNNG, and triaziquone

i24 i5, i7, i14 i26, i38 i22, i25 i16

Mutagen

YEp24 pEH22 pEH16 pEH7

YEp24 pEH22 pEH16 pEH7

a Isolates in bold script were used for further studies

DNA repair-deficient mutants were transformed with multi-copycontained HYR genes and tested for their sensitivity towards mutagens with varying specificity in their interaction with DNA. Sensitivity/resistance was determined via the agar diffusion test with all chemicals and with a spot test when applying UV-light. S, WT, and HYR are qualitative "markers" and describe the sensitivity, wild type-like, and the hyper-resistance phenotype, respectively

were c h a r a c t e r i z e d in m o r e d e t a i l a n d will be d e s c r i b e d in this c o m m u n i c a t i o n . R e - t r a n s f o r m a t i o n o f w i l d - t y p e y e a s t w i t h E. coil-amplified p E H 7 , p E H I 6 , a n d p E H 2 2 resulted in the e x p e c t e d H Y R p h e n o t y p e (Table 3). T h e survival kinetics o f such H Y R t r a n s f o r m a n t s after m u t a gen e x p o s u r e a r e s h o w n in Fig. 1 A a n d B. T h e largest increase in 4 - N Q O resistance as c o m p a r e d to the wildt y p e is seen w i t h m u l t i - c o p y p E H 2 2 ( S N Q 2 ) , w h e r e a s m u l t i - c o p y p E H 1 6 ( S N Q 3 ) confers o n l y a small, b u t sig-

nificant, H Y R to 4 - N Q O (Fig. 1 A ) a n d H Y R to M N N G (Fig. 1 B) p h e n o t y p e . M u l t i - c o p y p l a s m i d p E H 7 ( S N G 1 ) confers o n l y the p h e n o t y p e H Y R to M N N G , b u t to a l a r g e r extent t h a n p E H 1 6 ( S N Q 3 ) (Fig. 1 B). I n o r d e r to test the p u t a t i v e i n v o l v e m e n t in D N A rep a i r , the H Y R p h e n o t y p e - c o n f e r r i n g y e a s t insert D N A s were used for c o m p l e m e n t a t i o n studies o f m u t a g e n sensitivity in a n u m b e r o f selected D N A r e p a i r m u t a n t s (Table 4). F i v e m u t a n t s defective in excision r e p a i r

322 ( R A D 3 pathway), three with a defective gene in the R A D 6 pathway, and one with a defect in the recombinational repair (RAD52 pathway), were transformed with

the originally characterized HYR-conferring plasmids pEH7, pEH16, and pEH22, as well as with the vector YEp24, and then subjected to treatments with 4-NQO, MNNG, triaziquone, UV254nm,and HN2. The non-yeast insert DNA-containing vector, YEp24, did not change the mutagen sensitivity of any of the transformed D N A repair mutants. A complementation-like response, i.e., increased resistance to some chemical mutagens, was found in some transformants; however, their unaltered sensitivity to mutagens like UV254,m and HN2, clearly revealed this phenomenon (wild-type like resistance to 4-NQO, MNNG, and triaziquone) not to be due to true complementation of the defective D N A repair gene. In some cases the complementation-like response of the multi-copy plasmids even led to a H Y R phenotype (mutagen resistance higher than the wild-type), indicating that processes not pertaining to the originally defective repair gene may lead to the novel phenotype. Since both pEH 16 and pEH22 influence cellular sensitivity to 4-NQO we compared sensitivity and mutability of transformants to a strain transformed with insertDNA free YEp24, Figure 2 shows the survival and the mutability of haploid yeast transformed with either pEH16 or pEH22 after 4-NQO treatment, pEH22 has clearly a more pronounced effect than pEH16 on both survival and forward mutagenesis after 4-NQO treatment. Though less pronounced, the effects on pEH16transformed cells are still significantly different from a wild-type transformed with yeast insert DNA-free YEp24 (Fig. 2). The H Y R plasmids were cloned in E. coli and their respective yeast insert DNAs were characterized by restriction mapping (Fig. 3). All three insert DNAs differ from one another, i.e., most probably contain different yeast genes. The size of the three putative HYR-conferring genes was narrowed down by two techniques: pEH7 and pEH16 were subcloned after partial S a u 3 A digestion and pEH22 was inactivated by Tnl0-LUK mutagenesis. The subcloned D N A fragments still able to confer the respective H Y R phenotypes are shown below the original yeast insert DNAs (Fig. 3). The putative genes on the subcloned HYR-conferring insert DNAs were either disrupted by transposon mutagenesis or by insertion of the LEU2 yeast gene and the resulting plasmids employed for a gene transplaeement experiment, according to the protocol of Rothstein (1983). In all cases we obtained viable haploid disruptants with the new phenotype of more or less pronounced mutagen sensitivity. Therefore, the newly found nonessential yeast genes were named S N G I (sensitive to MNNG), contained in pEH7, S N Q 2 (sensitive to 4-NQO), contained in pEH22, and SNQ3 (sensitive to 4-NQO), contained in pEH16. Correct integration of the disrupted gene into the genorne of the mutagen-sensitive transformants was proven by tetrad analysis of sporulated diploids constructed by mating each putative integrant with a haploid wild-type (Table 5). Abnormal segregation of the disruption marker genes URA3 or LEU2 revealed

i

105

u

i

i

i

boo

x

104

)

2

/

o

N

\

lO3

e 0

i 10

i 20

310

L

L ~0

J0

20

4NO0 I umol/I )

Fig. 2. Influence of HYR to 4-N()O-conferring plasmids on sensitivity and mutability of 4-NQO-treated haploid yeast cells. Symbols as in Fig. 1

pEH 7 BIS

E

Lt

~

I)

B/S

.o '

L '

P '

xb~

Lxb '. :

P P

~ ~d

' '

:'i)il

URA3 2pro

pEH 705

E I

u I

d etco I'::: "' ::

~ E ii

URA3 2pro

pEH 16 WS

E

u

,

I

~

Ltxb Ii '

XbLO ;;:

C I

G~e

CE

t"~V I' )1

III

URA3

~E ( ( 2pro

pEH 1605 B/S

t',

Xb B/SCE

...... J

im

i

|

|

URA3 2prn

pEH 16L8 B/S L'~ KonR URA3 LocZ

E CXb

L

I~

III

I

I

I

pEH 22 I

xbE II

B~

X

B~

913 6 185

Fig. 3. Physicalmap of the three originally isolated HYR-conferring plasmids and the subclonesderived from them. Solid bar, yeast insert DNA; open bar and thin line, vector DNA; open bar in pEH16L8, and numbered arrows with open andfilled heads in pEH22, transposon TnlO-LUK. Abbreviations for cleavage sites: B, BamHI; C, ClaI; E, EcoRI; G, BglII; L, Sail; Ne, Neol; S, Sau3A; U, PvulI; X, XhoI; Xb, XbaI

323 Verification by tetrad analysis of correct integration of disrupted genes SNQ2, SNQ3, and SNG1

Table 5.

Genetic marker

Segregation of alleles 4:0

3:1

2:2

(1) Diploid derived from cross EH3714-2B (snq2::TnlO-LUK) x EH3867-1B HIS5 LYS1 LEU2 ILV2 TRP1 URA3 SNQ2

0 0 0 0 0 4 0

0 0 0 0 0 3 0

9 9 9 9 9 2 9

(2) Diploid derived from cross EH3714-2B (snq3 : : TNI0-LUK) x EH3879-5C SNQ3 HIS5 LYS1 URA3 LEU2 TRP5 ILV2

0 0 0 1 0 0 0

0 0 0 3 0 0 0

6 6 6 2 6 6 6

(3) Diploid derived from cross EH3871-2B (sngl " :LEU2) • JT0-3A LEU2 ADE2 ILV1 URA3

3

9

2

0 0 0

0 0 0

14 14 14

Suppression of disruption-generated mutagen sensitivities amongst three SNQ genes

Table 6.

Mutant genotype (plasmid)

Phenotype

snql : LEU2 (Yep24) snqI : LEU2 (pEH16SNQ3) snql : LEU2 (PEH22SNQ2)

Sensitive Resistant Resistant Sensitive Resistant Resistant Sensitive Resistant Resistant

snq2 : : TnI0-LUK (YEpl3) snq2 : : Tnl0-LUK (pAR172SNQ1) snq2 : : Tnt0-LUK (pJSIO9ISNQ3) snq3 : : TnlO-LUK (YEpI3) snq3 : : TnlO-LUK (pAR172SNQ1) snq3 : : Tnl0-LUK (pEH5122SNQ2)

Yeast mutants containing the disrupted loci SNQ1, SNQ2, and SNQ3 were transformed with multi-copy vector YEp24, and/or with YEp24 containing one of the SNQ genes. Sensitivityto 4-NQO was checked by the agar diffusion test

their correct integration at the genomic locus of the resistance gene. Together with one already isolated H Y R to 4-NQO conferring yeast gene, called S N Q 1 (Mack et al. 1988; G6mpe!-Klein etal. 1989), the two genes S N Q 2 and S N Q 3 reveal a similar sensitivity to 4-NQO upon disruption of the gene, Table 6 shows that one multi-copy vector-contained S N Q gene can more or less suppress the 4-NQO sensitivity conferred by either one of the two other disrupted S N Q loci.

Discussion

Screening of a multi-copy yeast D N A gene bank with mutagens like M N N G , 4-NQO and triaziquone for mutagen resistance-conferring genes resulted in the isolation of 39 transformants with the H Y R phenotype and finally, in the characterization of three different plasmids, pEH7, pEH16 and pEH22, containing the yeast genes S N G 1 , S N Q 3 , and S N Q 2 , respectively. Not all mutagens can be successfully employed for such a task: When HN2 was used as selective agent for H Y R transformants, all H Y R isolates proved resistant to HN2 because of a recessive chromosomal mutation in the H N M 1 gene (Haase and Brendel 1990), which was subsequently found to also affect the choline transport in yeast and to be allelic to C T R (Li et al. 1991), the gene coding for choline permease (Nikawa et al. 1990). We do not assume that the three HYR-conferring genes, S N G I , S N Q 2 , and S N Q 3 , are involved in D N A repair, although some of the nine repair-deficient mutants transformed with a multi-copy vector containing one of the genes showed complementation-like responses (Table 4). However, complementation was only partial, i.e., did not restore wild-type phenotype for all sensitivities caused by the defective repair gene (Table 4). This partial complementation clearly shows that the H Y R genes are not identical with any of the nine repair genes; it also implies that the H Y R genes are not part of any of the three D N A repair pathways that are assumed for yeast (Friedberg 1988). The better survival of the H Y R transformants did not depend on the toleration of more mutagenic lesions in the D N A since such a mechanism should have led to increased mutagen-induced mutability. Instead, H Y R transformants containing p E H 1 6 ( S N Q 3 ) and p E H 2 2 ( S N Q 2 ) had a reduced sensitivity to 4-NQO and also a lowered mutability (Fig. 2). When over-expressed in a yeast cell, each of the three genes S N Q 1 (G6mpel-Klein etal. 1989), S N Q 2 , and S N Q 3 leads to the H Y R to 4-NQO phenotype (Fig. 1). Each S N Q gene was able to suppress the 4-NQO-sensitive phenotype caused by disruption of the other S N Q loci (Table 6) thus suggesting that their encoded proteins are not organized in a common metabolic pathway but rather affect 4-NQO and other chemical mutagens via independent protective activities. The resistance of wildtype cells to D N A damaging agents depends on the function of the D N A repair systems that are phenotypically well characterized by a host of mutagen-sensitive mutant strains and genetically by a large number of molecularly cloned repair genes (Friedberg 1988). On the other hand, many chemical compounds are subject to biotransformation, i.e., to enzymatical activation or de-activation within the cell. Several mechanisms that lead to "drug resistance" via multi-copy caused over-expression of genes are known in mammalian cells (Schimke 1984; Curt et al. 1984). A particularly well-known example is the export permease (Kartner et al. 1983), a P-glyco-protein encoded by the M D R gene (Shen et al. 1986), which by an unspecific export activity makes the cell resistant to a variety of structurally unrelated chemicals (Endicott and Ling 1989). A yeast gene ( P D R 1 ) responsible for

324 pleiotropic drug resistance encodes a regulatory protein which by an altered m e m b r a n e permeability m a y influence the resistance of the cells to chemicals (Balzi et al. 1987). Also, the enzymes of glutathione metabolism, as well as glutathione itself (Meister and Anderson 1983; Pickett and Lu 1989), m a y lead to improved resistance to chemicals, i.e., to the H Y R phenotype of mutagen-treated cells. Likewise, altered activities of cytochrome P-450 m a y influence cellular survival after mutagen treatment (Gonzalez et al. 1986). Both systems are important in the cellular detoxification of mutagens but also play a role in the toxification of some pro-mutagens (Meister and Anderson 1983, Kistler et al. 1986; Callen and Philpot 1977; Gonzalez et al. 1986). We assume that the described genes that confer the phenotype of mutagen H Y R if present on multi-copy vectors in S. cerevisiae, most likely lead to lowered intracellular levels of chemical mutagens either by increased export from the cell (e.g., SNQ1 G6mpel-Klein et al. 1989; K a n a z a w a et al. 1988), or by degradation to nonmutagenic compounds (e.g., SNQ2, SNQ3, and SNG1). This was concluded f r o m the mutagen-sensitive phenotype of the non-functional disrupted m u t a n t alleles snql::LEU2 (G6mpel-Klein and Brendel 1990), snq2::TnlO-LUK, snq3::TnlO-LUK, and sngl::LEU2 (this report). Over-expression of the yeast gene SNQ3 leads to a pleiotropic H Y R phenotype, its disruption likewise to a pleiotropic sensitivity phenotype (Fig. 1) and, as reported by Hertle et al. (1991), affects the toxicity of m a n y chemicals. Thus, screening for different resistance phenotypes has led to the independent cloning of the genes PAR1 (Schnell and Entian 1991), PDR4 (Hussain and Lenard 1991), and YAP1 (Moye-Rowley et al. 1989), all allelic with SNQ3. The similarity of the encoded protein with the transcriptional activator Ap-1 (Lee et al. 1987) readily suggests its regulatory influence on m a n y metabolic processes and thus the pleiotropic phenotype of its mutant alleles (Schnell et al. 1992). A more substantial discussion of the function of the H Y R genes SNQ2 and SNGI m a y be possible after establishing their respective base sequences.

Acknowledgements. We thank Ms M Niesen and M Marcovie for technical assistance. The reported data come from the Doctoral Thesis of E. H. This research was partially supported by the Deutsche Forschungsgemeinschaft.

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Isolation and characterization of additional genes influencing resistance to various mutagens in the yeast Saccharomyces cerevisiae.

Screening of a multi-copy vector-based yeast genomic library in haploid cells of wild-type Saccharomyces cerevisiae yielded transformants hyper-resist...
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