Curr Genet (1992)22:277-282
9 Springer-Verlag 1992
The REV2 gene of Saccharomyces cerevisiae: cloning and D N A sequence E Ahne, M. Baur, and E Eckardt-Schupp GSF-Forsehungszentrum fiir Umwelt und Gesundheit, Institut ffir Strahlenbiologie,Ingolst/idter Landstrasse 1, W-8042 Neuherberg, Federal Republic of Germany Received April 21, 1992
Summary. The R E V 2 gene of Saccharomyces cerevisiae was cloned and sequenced; it contains an open reading frame of 1986 bp with a coding potential of 662 amino acids. Interruption of the chromosomal R E V 2 gene by integrating the URA3 gene coupled with partial deletion of the 3' terminal region produced viable haploid rev2A mutants. This indicates that the R E V 2 gene is non-essential for growth. The rev2A mutant is slightly more UVsensitive than strains carrying various rev2 alleles (rev2-1, rev2x, rad5-1, rad5-8). The putative Rev2 protein is probably a globular protein containing a highly conserved nucleotide-binding site and two zinc-finger domains. Key words: Yeast - DNA-repair - Mutation-deficient mutant - Nucleotide-binding consensus
We have analysed the R E V 2 gene which belongs to the R A D 6 epistasis group. The R E V 2 gene was isolated and characterized as a gene controlling the damage-induced reversion of certain ochre alleles (Lemontt 1971; Lawrence and Christensen 1978). Later, a specificity for frameshift alleles was also described (Lawrence et al. 1985). A number of studies have been performed using a temperature-sensitive rev2 mutant to characterize the REV2-controlled processes (Siede et al. 1983 a, b; Siede and Eckardt 1986a, b). The R E V 2 gene is of particular interest since we regard it as a candidate for a damage-inducible gene in yeast. In yeast there is no SOSqike repair system as found in E. coli (Walker et al. 1985). However, indirect evidence deduced from biological experiments has been published postulating inducible components of repair, mutation, and recombination processes in yeast (Siede and Eckardt 1984). Recently, the regulation of some cloned genes in response to DNA damage has been shown by molecular techniques (Friedberg et al. 1991). However, none of these genes seem to be required for an inducible mutagenic process. Thus, the R E V 2 gene is of interest with respect to the following two questions. First, does the R E V 2 gene encode a function required specifically in the process of mutation fixation? Answers to this question would help to elucidate the molecular and biochemical mechanism of damage-induced mutagenesis in yeast. Second, can it be shown by molecular techniques that the expression of the R E V 2 gene is regulated in response to DNA damage and possibly other stress factors? Previously we have reported the isolation of an yeast genomic clone which complements the rev2-I mutation (Siede and Eckardt-Schupp 1986 a), as well as a preliminary molecular characterization of this clone (Ahne et al. 1990). Here we present evidence that the gene that we have cloned is the R E V 2 gene, and provide details of its molecular structure.
In the yeast Saccharomyces cerevisiae the cellular response to DNA damage has been well studied. Three largely non-overlapping, so-called epistasis groups of genes controlling resistance towards radiation and chemicals have been classified, which probably correspond, respectively, to different DNA damage repair and tolerance mechanisms (Haynes and Kunz 1981; Friedberg 1988). The R A D 3 epistasis group controls nucleotide excision repair (Friedberg 1988) and genes in the R A D 5 2 epistasis group are mainly involved in DNA strand-break repair and recombination (Petes et al. 1991). The majority of the genes of the R A D 6 group are involved in DNA damage-induced mutagenesis; mutations in these genes cause hypo-mutability in response to DNA damage as compared to wild-type yeast. In most of the mutants, mutation deficiency is found for certain marker alleles only (Lemontt 1980; Lawrence 1982). This allele-specific control of damage-induced mutagenesis seems to be typical for yeast; however, its molecular basis is not clear.
Materials and methods
Correspondence to: E Ahne
Strains. The followinghaploid and diploid strains of the yeast S. cerevisiae were constructed by standard gcneticalmethods:
278 WS8100-1A a, REV2, ade2-1, arg4-17, trpl-289, ura 3-52 WS8100-3A ~, rev2-1, ade2-1, arg4-17, trpl-289, ura 3-52 WS8105 a/s, REV2/REV2, ade2-1/ade2-1, trpl-289/trpl-289, ura352/ura3-52 WS8105-1C ~, REV2, ade2-1, trpl-289, ura 3-52 WS8069/110 ~, REV2, ade2-1, arg4-17, trpl-289, ura3-52 ES106/14 a, rev2x, arg4-17, ura3-52 ESI04/2 a, radS-1, ade2-1, arg4-17, ura3-52 ES101/2 a, rad5-8, arg4-17, ura3-52 The bacterial strain HB101 (Boyer and Roulland-Dussoix 1969) was used as the recipient for amplification of the plasmids in E. coli.
Plasmids. As a shuttle vector for E. coli and yeast the centromere plasmid YCp50 (Kuo and Campbell 1983) was used for subcloning. The plasmid pWS301 was derived from the YCp50-based genomic library (Siede and Eckardt-Schupp 1986a) containing an 8.7 kb chromosomal DNA insert. The plasmid YEp24 (Carlson and Botstein 1982) was used for preparation of the 1.2 kb HindIII fragment containing the URA3 gene. pBR322 (Bolivar et al. 1977) was used as an intermediate plasmid for gene disruption. Media. YEPD, consisting of 2% dextrose, 2% Bacto peptone (Difco), 1% yeast extract (Difco) solidified with 2% agar (Difco) if required, was used as complex growth medium for yeast. For glycerol medium, 30 ml of glycerol was used instead of glucose. Yeast spheroblasts were plated on medium containing 1 M sorbitol, 4% dextrose, 0.33% Yeast Nitrogen Base w/o amino acids and ammonium sulphate (Difco) and 2% agar, supplemented with 9.8 g ammonium sulphate, 5 mg adenine, 20 mg arginine, 20 mg histidine, 40 mg lysine and 20 mg tryptophan per liter. For top agar, a concentration of 1.5% agar was used. Plasmid-free uracil-requiring strains were grown in medium supplemented with 20 mg of uracil per liter.
Agarose gel electrophoresis. Restriction patterns were analysed on 1% agarose gels stained with 0.5 I-tg/1of ethidium bromide (Maniatis et al. 1982). Elution of DNA from gels after preparative gel electrophoresis was carried out with the glasmilk bandprep-kit purchased from Pharmacia. Pulsed-field gel electrophoresis. The separation of yeast genomic DNA by pulsed-field gel electrophoresis was carried out as described by Geigl and Eckardt-Schupp (1990). Nucleic acid hybridisations. Southern and Northern hybridizations were performed according to Maniatis et al. (1982). Oligonucleotide synthesis. Oligonucleotide synthesis was carried out by a 'Gene assembler plus | using PACTM amitides. NAP10 columns were used to purify the oligonucleotides as recommended by the manufacturer. The chemicals and equipment described here are trademarks of Pharmacia. DNA sequencing. Both strands of the DNA were sequenced by the dideoxynucleotide chain-termination method (Sanger et al. 1977) using wedge gradient gels and deoxyadenosine 5'-[(c~-35S)thio]triphosphate (Biggin et al. 1983). Sequence-specific oligonucleotides were synthesized to prime the sequence reactions starting 3' and 5' of the cloning site. Computer analysis. The sequence was compiled using the PC-GENE sequence analysis program (IntelliGenetics, Inc./GENOFIT SA. University of Geneva, Switzerland).
Results E. eoli was propagated in LB medium, containing 1% Bacto trypton (Difco), 1% sodium chloride, 0.5% yeast extract, pH 7.5, supplemented with 100 mg ampicillin and/or 15 mg tetracyclin per liter where appropriate. Transformation. Transformation of yeast spheroblasts was carried out essentially as described by Hinnen et al. (1978). Transformation orE. eoli was performed according to the standard calcium chloride procedure (Lederberg and Cohen 1974). Measurement of UV sensitivity. For testing the UV sensitivity of the transformants, approximately 1 • 107 cells/ml were streaked on uracil-free medium and gradually irradiated with 0-60 J/m 2 UV light (254 nm at a fluence rate of 2 J/m 2 per s). Quantitatively, surviving fractions were determined by plating appropriate dilutions of a suspension of stationary phase cells on YEPD, followed by UV irradiation with doses up to 100 J/m 2 on at least three parallel plates per dose. Colonies were counted after incubation for 3 days at 30 ~ and the surviving fractions calculated. Isolation of chromosomal yeast DNA. Genomic DNA from yeast was isolated according to Cryer et al. (1975). Preparation ofplasmids. Plasmids from yeast were prepared from a 250 ml cell culture grown to logarithmic phase in YEPD; spheroblasts were prepared and lysed as described by Sigurdson et al. (1981). The clear lysate was extracted with phenol-chloroform three times and the supernatant precipitated with ethanol. The pellet was dissolved in 5 ml TE (10 mM Tris-HC1, 1 mM EDTA, pH 8) and treated with RNase (100 Ixg/ml) at 37~ for 2 h. The plasmids were purified on a Quiagen column (Diagen GmbH, Diisseldorf) as recommended by the manufacturer. The DNA was collected as described and dissolved in 200 Ixl TE. Samples of 10 Ixl DNA were used to transform the E. coli strain HB101 to ampicillin resistance. Large and small scale preparation of plasmids from E. coli were carried out according to the modified alkaline-lysis procedure (Birnboim and Doly 1979). The plasmids were purified on Quiagen maxi- and/or mini-columns.
Characterization o f the plasmid p W S 3 0 1 I n a p r e v i o u s p a p e r the i s o l a t i o n o f a p l a s m i d ( p W S 3 0 1 ) was d e s c r i b e d w h i c h r e s t o r e d the E V 2 ( R A D 5 ) w i l d - t y p e p h e n o t y p e w h e n t r a n s f o r m e d into a rev2-1 m u t a n t ( W S 8 1 0 0 - 3 A , Siede a n d E c k a r d t - S c h u p p 1986a). I n the m e a n t i m e we h a v e o b t a i n e d evidence t h a t the p l a s m i d also c o m p l e m e n t s o t h e r rev2 alleles. Strains c a r r y i n g the rad5-1, rad5-8 alleles (Berkeley S t r a i n C o l l e c t i o n ) o r a recently i s o l a t e d allele rev2x (the N498 strain was k i n d l y given to us b y C. L a w r e n c e , R o c h e s t e r ) were t r a n s f o r m e d to a R E V 2 w i l d - t y p e p h e n o t y p e b y p W S 3 0 1 ( d a t a n o t shown). T h e p l a s m i d p W S 3 0 1 c o u l d n o t be p r o p a g a t e d in the strain 490 rec A - o f E. coli w h e n p l a s m i d D N A was p u r i f i e d o n a c e s i u m - c h l o r i d e g r a d i e n t (Siede a n d E c k a r d t - S c h u p p 1986a). H o w e v e r p l a s m i d D N A p u r i fied on a Q u i a g e n c o l u m n was able to t r a n s f o r m the HB101 E. coli strain ( A h n e et al. 1990). D u e to u n k n o w n r e a s o n s the efficiency o f t r a n s f o r m a t i o n in E. coli was very low. N e v e r t h e l e s s , the 8.7 k b insert o f p l a s m i d p W S 3 0 1 c o u l d be c h a r a c t e r i z e d b y r e s t r i c t i o n analysis a n d s u b c l o n e d . A r e s t r i c t i o n m a p o f the insert o f p W S 3 0 1 is s h o w n in Fig. 1. To localize the R E V 2 gene w i t h i n the 8.7 k b insert, p a r t s o f the insert were s u b c l o n e d b y d i g e s t i n g p l a s m i d s w i t h the r e s t r i c t i o n e n z y m e s SaII a n d ClaI respectively. R e - l i g a t i o n after r e m o v a l o f the 3.8 k b SalI f r a g m e n t c r e a t e d p l a s m i d p F A 3 0 1 3 a n d r e - l i g a t i o n after r e m o v a l o f two ClaI f r a g m e n t s (1.35 k b a n d 3.5 k b ) c r e a t e d plasm i d s p F A 3 0 1 4 - 1 a n d p F A 3 0 1 4 - 2 . T h e s e c o n s t r u c t s were t r a n s f o r m e d into the strain W S 8 1 0 0 - 3 A c a r r y i n g the rev2-1 allele a n d into the strain ES101/2 c a r r y i n g the
1 ti:' 1
ORFX 1.4 k b
1 kb .,"
REV2 1.g kb
E ooR V
URA 3 pFA3014-2 pFA3013
10 -1 x
Fig. 1. Restriction map of the plasmid pWS30I. The thick black line represents the 8.7 kb insert of plasmid pWS301, the BamHI cloning sites of plasmid yCP50 are indicated by black arrows. The open reading frame of the REV2 gene is indicated by a hatched arrow, ORFX by a blank arrow. The strategy for the gene-disruption is demonstrated in the middle part of the figure; in the lower part, the three inserts of the subclones derived from plasmid pWS301 are shown. The rev2 complementing fragment is indicated by a thick black line .
\ \ \
\ u~lO 9
\ \ \
U V D o s e ( J m -2)
Fig. 2a-c. The surviving fractions plotted against UV dose are shown, a for the haploid REV2 wild-type (0) and mutant strains carrying the rev2-1 (e) and tad5-8 (V) alleles. The mutants were
complemented with pFA3013 (Q), V) and pFA3014-1/2 (Q, V); b for the haploid integrant rev2A (O) in comparison to the haploid
rad5-8 allele. As shown in Fig. 2 a, full complementation to a R E V 2 wild-type phenotype was achieved in both
mutants by transformation with subclone pFA3013, whereas no reduction of UV sensitivity could be achieved with plasmids pFA3014-1 and pFA3014-2. The same results were obtained by transforming strain ES106/14 carrying the rev2x allele which was complemented by plasmid pFA3013 but not by pFA3014-1 and pFA3014-2 (data not shown). This last result contradicts our former report (Ahne et al. 1990) where we presented preliminary evidence that the R E V 2 gene is located on the SalI fragment subcloned in pFA3014-1. This fragment has been shown by sequencing to carry a 1425 bp open reading frame ('ORF X' in Fig. 1). However, chromosomal interruption o f this reading frame did not yield a rev2 mutant
UV Dose(Ira -2)
U V Oose(Jm -2)
REV2 wild-type (0) and mutants carrying the alleles rev2-1 (e), rev2x (@), rad5-1 (A) and tad5-8 (V); e for the haploid REV2 wild-type (0) and the rev2A (O) and rev2-1 (e) mutants. After UV irradiation, the strains were plated on glucose (solid lines) or glycerol (dashed lines) medium
phenotype (manuscript in preparation). These unexpected, but clear, results forced us to repeat the subcloning of pWS301 using the strategy which we describe here and gave us the results presented in Fig. 2 a. Chromosomal mapping
The R E V 2 gene has been mapped genetically on chromosome XII (Mortimer and Schild 1985). Various probes from the 8.7 kb insert of plasmid p W S 3 0 ! were labelled and hybridized to Southern blots of chromosomal yeast D N A which had been separated by pulsed-field gel electrophoresis. The insert fragments hybridized specifically to the band representing chromosome XII (data not shown).
280 Gene disruption
Final proof was required that U V resistance of the rev2 mutants transformed with pFA3013 reflects the expression of the REV2 gene, and that the gene is localized on the ClaI fragments of pWS301. Therefore, we created rev2A mutants according to Rothstein (1983) by interruption of the ClaI-EeoRV fragment of pFA3013 using a fragment which carries the URA3 gene of yeast. The rev2A mutants were constructed as follows (see also Fig. 1). The 1.1 kb ClaI-EcoRV fragment from plasmid pFA3013 was isolated and inserted into plasmid pBR322 at the Cla! and EcoRV sites. This plasmid was cut with HindIII and a 1.2 kb fragment containing the URA3 gene (isolated from plasmid YEp24) was inserted into this site. The plasmid produced (pFA3015) was digested with ClaI and EcoRV and the resulting linear fragment carrying the URA3 gene was transformed into a diploid REV2 wildtype strain (WS8105) and two haploid REV2 strains (WS8105-1C and WS8069/110). URA + integrants from all three strains were isolated on selective medium lacking uracil. Unexpectedly, the URA + transformants of the diploid strain failed to sporulate due to unknown, but not further investigated, reasons. Fortunately, the URA + integrants of both haploid strains were viable and could be analyzed. As tested in a fast streak test, four out of a total of five integrants were UV-sensitive. Quantitative survival assays were performed with one of the integrants. The U V sensitivity of this mutant, presumably exhibiting a completely destroyed REV2 function, is as high as that found for the rev2-1 and rev2x mutants (Fig. 2b). Furthermore, the rev2A mutant shows the same enhanced U V resistance as the rev2-1 mutant when plated on glycerol medium after U V irradiation (Fig. 2 c). As yet it is not understood why growth on glycerol or lactate (Lawrence and Christensen 1978), as compared to growth on glucose, of UV-irradiated rev2 cells reduces the U V sensitivity of rev2 mutants but does not influence the U V response of repair-competent wild-type yeast. Chromosomal D N A extracted from UV-sensitive URA + integrants was cut with ClaI and separated on agarose gels. As compared to REV2 and rev2 reference strains, the band hybridizing to a probe obtained from the 1.35 kb Clal fragment was shifted considerably in its molecular weight, as is expected due to the insert of 1.2 kb (data not shown). This was taken as further evidence that the fragment carrying the URA3 gene did interrupt the chromosomal REV2 gene located on the 1.35 kb and 3.5 kb ClaI fragments.
-540 -480 -420 -380 -320 -280 -240 -180 -120 -60
A~GAGATC~GTGAAGAGATTTCTGCTACT~AGTATATGACTCACGTGGTAG~AGAA AGCGTCCATGGCTAGTTTGGT~G~TTTTTGATATCC~TATGATAGAGAAATTGGCAG AGTTTCGG~GACATTGCTCAAATACTATACCCTCTTTT~GTTCACACG~%AT~GTTT TG~GTTACATTGATTTTTTGTGAT~TAAACGGTTGAGTATAGGTGATAGCTTTATTCT AC~TTGGATTGCTTTTT~CATCTCTCATTTTTGAGG~CGT~TGATGGAG~TCCTT GATGAAAAG~GACGTACAGAGGGAGGAAATAAAAGAGAGAAAGAC~TGGAAATTTTGG ~G~CATTGACTGAAACTGATG~GAGCTAG~GCCGCTCGAAAAGACTGGCTCTACTA ~GTTATTTGAT~-AATTGAGGCTAAAGCCTATTTTGGACGAGCAG~GGCATTAGAAAAG CATAAAATAGAGCTT~TAGTGACCCCGAAATCATTGATTTAGAT~CGACGAGATTTGC TCT~TC~GCGACTG~GTCCAT~C~TCTCCGAGATACTCAGCACG~G~GAAACA
ATG~CTTG~TC~TTG~CATTTTAT~GGCCGCAC~TCATCAG~TCTTTAAAA M N L N Q L K T F Y K A A Q S S E S L K
AGTTTGCCTGAAACAG~CCTTCTCGCGATGTCTTC~GCTAG~CT~GAAATTATC~ S L P E T E P S R D V F K L E L R N Y Q
~GC~GGTCTTACTTGGATGCT~GGAGGGAGC~GAGTTTGCCAAAGCAGCCTCTGAT K Q G L T W M L R R E Q E F A K A A S D
GGTGAGGCTTCAGA~CGGGTGCT~TATGAT~L%ACCCATTATGG~GCGGTTCA~TGG G E A S E T G A N M I N P L W K R F K W
CCA~TGATATGTCGTGGGCAGCTCAAAATTTGCAGCAGGACCATGTAAACGTTG~GAT P N D M S W A A Q N L Q Q D H V N V E D
GGCATATTCTTTTATGCG~CTTACATTCTGGTG~TTTTCGCTAGCAAAACCTATATTA G I F F Y A N L H S G E F S L A K P I L
A~ACTATGATAAAGGGTGGCATATTATCAGATGAAATGGGGTTGGGTAA~AACAGTGGCA K T M I K G G I L S D E M G L G K T V A
GCGTATTCTTTAGTTTTATCTTGTCCTCACGATAGTGATGTTGTTGAC~GA~CTGTTT A Y S L V L S C P H D S D V V D K K L F
GATATTGAG~CACAGCAGTCTCAGAT~TCTTCC~GCACTTGGC~GAT~T~GAAA D I E N T A V S D N L P S T W Q D N K K
CCATATGCTTC~A/~AAC~CGCT~TCGTGGTCCC~TGTCTTTGCT~CGCAGTGGAGT P Y A S K T T L I V V P M S L L T Q W S
~CGAGTTTAC~d~AGCT~T~TTCCCCCGATATGTATCATGAGGTGTATTATGGTGGG N E F T K A N N S P D M Y H E V Y Y G G
~TGTTTCCAGTTTG~CCCTATT~CC~GACA~CCCTCC~CTGTAGTCCTT N V S S L K T L L T K T K N P P T V V
ACTACATATGGTATTGTTC~AAATG~TGGACT~AACATTCC~GGG~GGATGACAGAT T T Y G I V Q N E W T K H S K G R M T D
GAGGACGTC~TATATCTTCAGGCTTATTTTCTGTC~TTTTTATCGCAT~T~TCGAT E D V N I S S G L F S V N F Y R I I I D
GAGGGTCAT~CATTAGAAACAG~CGACAGTTACATCTAAAGCAGTCATGGCTTTAC~ E G H N I R N R T T V T S K A V M A L Q
GGCA~TGTA~TGGGTTTT~CAGG~CACC~TTATT~CAGGCTTGACGATTTATAC G K C K W V L T G T P I I N R L D D L Y
AGTCTGGTT~GTTTTTAGAGTTAGATCCCTGGCGGCAAATT~TTACTGG~GACCTTT S L V K F L E L D P W R Q I N Y W K T F
1021 G T A T C ~ C G C C T T T T G A G A G T A A A A A T T A C A A A C ~ G C A T T T G A T G T G G T G ~ T G C ~ T T V S T P F E S K N Y K Q A F D V V N A I 1081 T T G G ~ C C C G T A T T A T T ~ G ~ G G A C A A A A C A ~ I T G A A A G A T A A A G A T G G T ~ G C C A T T A L E P V L L R R T K Q M K D K D G K P L 1141 G T A G A G T T G C C A C C A A A G G A G G T C G T T A T T A / ~ k A G A C T C C C C T T C A G T A A A T C T C ~ G A T V E L P P K E V V I K R L P F S K S Q D 1201 C T T C T A T A C ~ G T T T C T G T T G G A T ~ G G C T G A G G T T T C T G T T A A A T C G G G T A T T G C A C G C L L Y K F L L D K A E V S V K S G I A R 1261 G G T G A T T T A T T G A A 2 ~ A A G T A C T C C A C T A T C C T T G T C C A T A T T T T ~ G A T T G A G G C ~ G T C G D L L K K Y S T I L V H I L R L R Q V 1321 T G T T G C C A T C C C A G T C T T A T T G G ~ G T C ~ G A T G A G ~ C G A T G A G G A T T T A T C T A A A A A T C C H P S L I G S Q D E N D E D L S K N 1381 ~ T A A A T T G G T T A C G G ~ C A A A C G G T G G A G C T T G A C T C T T T ~ T G C G T G T T G T T T C C G A G N K L V T E Q T V E L D S L M R V V S E 1441 A G A T T C G A T ~ C T C A T T T T C T ~ G G A G G ~ T T A G A T G C ~ T G A T A C A A A G C T T A A A A G T T R F D N S F S K E E L D A M I Q S L K V 1501 A A A T A T C C A G A C ~ T A A A T C G T T T C A G T C C T T A G A G T G C T C C A T C T G C A C ~ C G G ~ C C T K Y P D N K S F Q S L E C S I C T T E ~ 1561 A T G G A T T T G G A C ~ G G C T T T A T T T A C A G ~ T G C G G C C A C A G T T T T T G T G A G A A A T G T T T A M D L D K A L F T E C G H S F C E K C ~ 1621 T T T G ~ T A T A T T G A G T T T C A G ~ C A G T ~ G ~ T T T G G G T T T A A A G T G C C C C ~ T T G C C G T F E Y I ~ F Q M S E N L G L K C P N C R
Nucleotide sequence of the REV2 gene
1681 ~ C C 2 ~ T A G A T G C T T G T C G G T T G T T G G C A T T G G C A C A A A C G ~ T A G C ~ C T C G A ~ T N Q I D A C R L L A L A Q T N S N S K N
To determine the nucleotide sequence of the insert of pFA3013, we applied the dideoxynucleotide chain-termi-
1741 T T G G ~ T T C A A A C C A T A T T C A C C A G C C T C C A A A T C ~ G C A A A A T C A C T G C T T T A T T A A A G L E F K P Y S P A S K S S K I T A L L K 1801 G A G C T T C ~ T T G C T A C A G G A T A G T T C G G C A G G C G ~ C ~ G T T G T C A T T T T T T C C C ~ T T T E L Q L L Q D S S A G E Q V V I F S Q F
Fig. 3. Nucleotide sequence of the REV2 gene and the derived predicted amino-acid sequence. The glycine, lysine and sefine residues of the potential nucleotide-binding site are underlined, the potential zinc-binding, DNA-binding, finger domains are double underlined. The end of the open reading frame is indicated by an arrow. Consensus sequences for the putative binding sites for HSTF ( - 3 6 4 - - 3 5 7 ) and CCBF (SWI) ( - 4 3 4 - - 4 2 8 ) are underlined italic
1861 T C C A C A T A C T T G G A T A T C C T G G A G ~ J % A G A G C T ~ C T C A T A C T T T C T C A ~ G A T G T T G C A S T Y L D I L E K E L T H T F S K D V A 1921 A / ~ A A T T T A T A A A T T C G A T G G A C G T C T C T C A T T A ~ G 2 L A A G ~ C T A G T G T A T T A G C A G A T K I Y K F D G R L S L K E R T S V L A D 1981 T T T C G T T A A A G A C T A T A G C A G G C A ~ k A A A T C C T A T T A C T C T C T C T G ~ G G C T G G T G G C G T F R % 2041 G G G T T T G ~ T C T ~ C G T G T G C T T C C C A C G C T T A T A T G A T G G A C C C A T T G G T G G T C A C C C A 2101 G T A T G G ~ G A T C A G G C ~ T C G A T A G A C T G C A T A G ~ T T G G C C A G A C ~ J ~ A C A G C G T C ~ G G 2161 T T A T G A G A T T T A T C A T A C A G A T A G C A T A G ~ G ~ A A ~ J % A T G C T A C G C A T T C ~ G A A A A G ~ 2221 GAG~CTATCGGTGAGGCCATGGACACAGACG~GACGAGAG~GA~kAAGGAG~TTGA
nation method of Sanger et al. (1977). The insert was sequenced both from the 5' and the 3' end starting with specific oligonucleotide primers on the vector and processing via 'primer hopping'; both strands were sequenced. An open reading frame of 1986 bp was found; in addition, 500 bp of the 5' promoter and the 3' terminating regions were determined (see Fig. 3). Northern blot analysis of total RNA extracted from different R E V 2 wild-type and rev2 mutant strains revealed a transcript of approximately 2.1 kb in length hybridizing to an internal R E V 2 probe (1.2 kb ClaI fragment; data not shown). A potential translational start (ATG) codon, preceded by an A at position -3 and followed by an A at position + 4, is present. Thus the heptanucleotide ANNAUGA, which is found at the translational start site for eukaryotic mRNAs (Kozak 1983), is present. No typical TATA boxes 5' to the translational start site were found. The gene is terminated by an ochre codon and an additional ochre codon is present 6 bp downstream from the first. Neither the proposed consensus sequence for polyadenylation (AATAAA) (Fitzgerald and Shenk 1981) nor the TAG...TATG/TATGT...(AT rich)...TTT motive, proposed to be a signal for transcription termination in yeast (Zaret and Sherman 1982), could be detected. However, similar AT-rich motives exist downstream from the 3' end of the R E V 2 open reading frame. Discussion
We present evidence that the plasmid pWS301, which complements four different rev2 (radS) alleles (rev2-1, rev2x, radS-i and radS-8) to wild-type UV resistance, harbours the R E V 2 gene of the yeast S. cerevisiae. Interruption and partial deletion of the chromosomal R E V 2 gene is not lethal for the cells, indicating that the R E V 2 gene is not essential for growth. Abolition of the R E V 2 gene function causes the complex rev2 phenotype as described earlier. An i~termediate UV sensitivity is found which differs depending on whether UV-irradiated rev2 cells are plated on fermentable or non-fermentable carbon sources. Sequencing of the gene reveals an open reading frame of 1986 bp. A computer search indicated no significant homologies of the R E V 2 0 R F with any other sequences available in the data bases, either at the nucleotide, or at the amino-acid levels. Obviously, the R E V 2 gene is a new, as yet undescribed gene, but partial homologies have been found. As our main interest in the molecular analysis of the R E V 2 gene came from postulates that the gene is DNA damage-inducible (Siede and Eckardt-Schupp 1986b), we searched for elements described as binding sites for specific regulatory proteins in this region. We found some consensus sequences for putative binding sites (Fig. 3), for example for HSTE a heat shock-factor, and for CCBF (SWI), a general activator of some genes in late G 1 of the cell cycle (Verdier 1990). We have already isolated the R E V 2 promoter and fused it to the lacZ reporter gene, and deletions of various length are under construction. These constructs will enable us to experimentally determine which agents or treatments increase R E V 2 gene expression, and which of the putative binding sites have biological function in the regulation of the
R E V 2 gene in response to DNA damage, heat shock, and carbon source supply. The putative Rev2 protein (Rev2p) has an estimated molecular weight of 75 kDa and contains a total of 662 amino-acid residues: 31.4% polar, 41.2% non-polar, 14.4% basic and 12.3% acidic residues. Baslc and acidic residues are distributed uniformly and no distinct acidic stretches are present. A prediction of the putative secondary structure of Rev2p according to the method of Garnier et al. (1978) reveals 54.2% helical conformation, 27.3% extended conformation, 10% turns as well as 8.4% coils, suggesting that Rev2p is a globular protein. Comparing the amino-acid sequence of Rev2p with those of other repair genes from yeast and E. coli, some sequence homologies are obvious. Rev2p contains a conserved sequence, between amino-acid residues 124 and 144, that is also present in different proteins binding and hydrolyzing ATP (Walker et al. 1982), such as the E. coli DNA repair and recombination proteins UvrA (Hussain et al. 1986), UvrD (Finch and Emmerson 1984), RecA (Walker et al. 1982) and RecB (Finch et al. 1986). In yeast the conserved nucleotide-binding consensus sequence was found in Rad3p (Reynolds et al. 1985), Radl8p (Chanet et al. 1988; Jones et al. 1988), Rad54p (Emery et al. 1991), and in Pifp (Foury and Lahaye 1987). The Rad3 protein was purified to physical homogeneity (Sung et al. 1987b). The purified protein catalyzes the hydrolysis of ATP or dATP in the presence of Mg + + (or Mn + +) and single-stranded DNA, and possesses a DNA helicase activity which can unwind long regions of double-stranded DNA (Sung et al. 1987a, b). The RAD54 gene is involved in the control of both DNA repair and mitotic recombination. The rad54 mutant is highly X ray-sensitive (Game and Mortimer 1974), it is unable to repair DNA double-strand breaks (Budd and Mortimer 1982) and shows decreased spontaneous and induced mitotic recombination (Saeki et al. 1981). The P I F gene is required for both repair of mitochondrial DNA and the recognition of a recombinogenic signal (Foury and Lahaye 1987). As shown in Table 1, 11 of 21 residues of the nucleotide-binding consensus are identical between Rev2p and Rad54p. Another interesting feature of the putative Rev2 protein is apparent from the sequence of amino-acid residues from 513 to 531 and 533 to 569, where two potential DNA-binding sequences, known as zinc-fingers, are present (Miller et al. 1985). These sequences have been described for several transcription factors and Table 1. H o m o l o g y between the putative nucleotide-binding sequence of the Rev2 protein and other known or putative nucleotidebinding proteins from yeast and E. eoli. Conserved residues between Rev2p and the other proteins are underlined in bold Protein
Rev2p Rad54p Rad3p Rad18p Pifp UvrAp UvrDp
124-144 328-348 35- 55 353-373 251-273 633-653 22- 42
I K GGI L S D EMG LGK A Y GCI MAD EMG LGK GGNS ILEMPS GTGK GG I SI(LMI MKS NGK GHNIFYTGSAGTGK GL FTCI TGVS G S GK R S NLLVLA GAG S GK
TVAAYS L TLQCI AL TVS LL S L SSS YR KL SI LLR EM STL I NDT TRVLV HR
282 Table 2. Potential metal-binding domains in the Rev2 and Rad18 proteins Position
X 2 -C-
X14 - C -
533-569 26-48 190-210 Consensus
X2 - C X z -CX2 -CX2_ 4 C/H
X16 - C - X z - C Xll - C - X 4 - C X12 - H - X 3 - C X2_15 C/H X2_ 4 C/H
Rev2p Rev2p Radl8p Radl8p
References: Rad54p (Emery et al. 1991), Rad3p (Reynolds et al. 1985), Rad18p (Chanet et al. 1988; Jones et al. 1988), Pifp (Foury and Lahaye 1987), UvrAp (Hussain et al. 1986), UvrDp (Finch and Emmerson 1984). Consensus-sequence (Berg 1986; Klug and Rhodes 1987) nucleic acid-binding proteins (Klug and R h o d e s 1987) and they have also been f o u n d in D N A repair proteins like the U v r A protein o f E. coli, and R a d l 8p o f S. cerevisiae ( C h a n e t et al. 1988; Jones et al. 1988). The two different zinc-finger d o m a i n s o f the putative Rev2 protein are shown in Table 2 in c o m p a r i s o n to those f o u n d in R a d l 8 p . A l t h o u g h we have no experimental evidence, we speculate that the putative zinc-finger domains in Rev2p and R a d l 8 p m a y be o f i m p o r t a n c e for the biological role o f the two genes. The rev2 and tad18 m u t a n t s share a c o m m o n p h e n o t y p e which, so far, has n o t been described for any other radiation-sensitive m u tant o f yeast. B o t h m u t a n t diploids are sensitive t o w a r d s ionizing irradiation b u t they repair D N A double-strand breaks (DBS) as efficiently as repair-competent diploid strains. However, b o t h m u t a n t s are unable to repair another type o f ionizing radiation damage, the so-called Sl nuclease-sensitive sites (SSS) which are induced approximately twice as frequently as D S B (Geigl a n d EckardtS c h u p p 1991). The molecular structure o f SSS is n o t known; they m a y represent sites o f disturbed D N A helical structure possibly caused by clustered base d a m a g e (Geigl and E c k a r d t - S c h u p p 1990). Thus one might speculate as to whether the SSS repair-specific functions o f the R E V 2 and R A D 1 8 gene are related to the zinc-finger functions o f the Rev2 and R a d l 8 proteins. Acknowledgements. We thank Mrs. U. Nevries, Mrs. U. Hoffmann and Mr. E. Kurtkaya for excellent technical assistance, and Drs. W. Giinzburg and B. Salmons for critically reading the manuscript.
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