13
Mutation Research, 45 (1977) 13--20 © Elsevier/North-Holland Biomedical Press
R E P A I R OF PYRIMIDINE DIMERS IN RADIATION-SENSITIVE MUTANTS rad3, rad4, rad6 AND rad9 OF SACCHAROMYCES CEREVISIAE
LOUISE PRAKASH Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, N. Y. 14642 (U.S.A.) (Received 12 April, 1977) (Accepted 16 May, 1977)
Summary The ability to remove ultraviolet (UV)-induced pyrimidine dimers was examined in four radiation-sensitive mutants of Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by either the T4 UV-endonuclease or an endonuclease activity found in crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad3 and rad4 mutants are shown to be defective in dimer excision whereas the rad6 and rad9 mutants are proficient in dimer excision.
Introduction Over twenty genetic loci are known to be involved in the repair of damage induced in DNA by ionizing radiation and ultraviolet (UV) light in the yeast Saccharomyces cerevisiae, a eucaryotic microorganism [1,5,8,12,15,20,26]. Dimer excision is known to occur in nuclear DNA of wild type yeast [19,27, 28], b u t is absent in mitochondrial DNA [19,29]. Both radl [19,27,28] and rad2 [22] mutants have been shown to be defective in the ability to excise UVinduced pyrimidine dimers. Double mutants involving various combinations of the radl, rad2, rad3 and rad4 loci show epistatic interactions, i.e. the sensitivity of the double mutant to UV is no greater than that of the more sensitive of the t w o single mutants [7]. This suggests that all four genes are in the same repair pathway. In addition, rad3 and rad4 mutants can be photoreactivated after dark holding [16], indicating that dimers are n o t removed during dark holding and that these two mutants are probably excision-defective. However, there is no direct evidence that the rad3 and rad4 mutants are in fact excision-defective.
14 The only other mutant whose defect has been partially identified is rad52, shown to be deficient in the capacity to repair X-ray-induced double strand breaks [6,21]. In this study, we have measured the fate of UV-induced dimers in four radiation-sensitive mutants of yeast, tad3, rad4, rad6 and rad9, by using a sensitive assay based on the ability of bacteriophage T4 UV endonuclease [9] and a UV endonuclease found in crude extracts prepared from Micrococcus luteus to produce a single-strand breaks in DNA containing pyrimidine dimers [30]. Evidence is presented that the rad3 and rad4 mutants are excision-defective while the rad6 and rad9 mutants are excision-proficient. Materials and methods
Strains The rad3, rad4, rad6-1 and rad9 mutants were isolated by Cox and Parry [5]. The ability to take up exogenous thymidine monophosphate (dTMP), was introduced into our strains, since a radioactive label specific for DNA was required for the experiments reported here. Strain MB1017-3A, a lysl-1 ade2-1 his5-2 RAD÷ tup [2], was crossed to LP3-18B, a cyc1-131 his1-1 tad6-1. The resulting diploid, LP-99, was sporulated and one of the meiotic segregants, LP99-18B, a his1-1 his5-2 rad6-1 tup, was the rad6 mutant used in this study. LP98-1D, a lysl-1 his5-2 tup, a strain previously described [19], was crossed to tad3, rad4 and rad9 mutants to generate the following haploid strains: LP397B, a ade2-1 rad3 tup; LP459-8B, a ilv3 rad4 tup; and LP457-6C, a hisS.2 lysl-1 rad9 tup. Media The media were as previously described [ 19]. Labeling o f cells, UV irradiation, preparation of spheroplasts and centrifugation in neutral CsCl gradients The details of these procedures were as described before [19]. Preparation o f UV endonuclease from Micrococcus luteusand bacteriophage T4 The nuclease specific for DNA containing dimers was prepared from M. luteus (Miles Chemical Co., Elkhart, Ind.). The enzyme was partially purified according to a modification [23] of the m e t h o d described by Carrier and Setlow [3]. Although extracts prepared in this way also contain activity for X-rayirradiated DNA [17], they do n o t have activity against DNA obtained from untreated cells. The bacteriophage T4 UV endonuclease was generously supplied by Dr. E.C. Friedberg. The enzyme preparation had been purified through the phosphocellulose step and was in 10% polyethylene glycol plus 33% glycerol. The activity of this preparation was such that 0.01 ml of stock solution would nick a b o u t 1.8 pmol of dimers in irradiated T7 DNA, as nucelotide/h (Friedberg, personal communication). This preparation was the same one as that used in our previous studies [19]. Endonuclease treatment of nuclear DNA of yeast. Treatment with T4 UV endonuclease was as previously described [19].
15 Treatment with M. luteus extract was carried out in a total volume of 105 pl. The reaction mixtures consisted of 50 gl (0.2 to 0.4 t~g} DNA, 50 ttl 0.02 M EDTA (pH 7.5) and 5 pl of M. luteus extract, containing 12 mg protein per ml determined by a modification of the Lowry m e t h o d [13]. The mixtures were incubated at 36°C for 10 min. Control DNAs were treated in the same way, w i t h o u t the addition of enzyme. The reaction was terminated by layering 0.1 ml on an alkaline sucrose gradient. Alkaline sucrose sedimentation and molecular weight determinations Linear gradients of 5 to 20% (w/w) sucrose, in 0.8 N NaC1--0.2 N NaOH, were made in cellulose nitrate tubes pretreated with heat-denatured salmon sperm DNA. Each gradient contained 4.9 ml of sucrose solution plus a top layer of 0.1 ml 1 N NaOH. Gradients with enzyme-treated and untreated DNAs were kept at room temperature for 20 min before centrifugation. Samples were centrifuged in an SW50.1 rotor in a Beckman L5-65 preparative ultracentrifuge at 20 ° C for 3 h at 40,000 rpm. 14C-labelled T7 bacteriophage DNA was used as a marker for all gradients, and fractions were collected and precipitated as described previously [19]. The method described by Rupp and Howard-Flanders [24] was used to determine the number average molecular weight; only the peak fractions were included in the calculations. Results Retention o f endonuclease-sensitive sites in the DNA o f rad3 and rad4 mutants Cells of the rad3 mutant irradiated with 6.7 J/m 2 (0.82% survival) were incubated in the dark in growth medium for 4 h. The nuclear DNA, which had been purified by CsC1 centrifugation, was treated with T4 UV endonuclease. The irradiation and conditions of treatment were the same as those used with a radl mutant which was shown by this method to be excision defective [19]. The nuclear DNAs obtained either from unirradiated cells, from irradiated cells, or from irradiated cells which had been given a 4 h period of dark repair in growth medium all showed the same sedimentation pattern in alkaline sucrose gradients (Fig. 1). The molecular weight of these nuclear DNAs corresponds very closely to the T7 DNA used as a marker and is equivalent to an M, value of about 1.1 X l 0 T. Assuming that there is only one DNA molecule per chromosome and that all 17 chromosomes of yeast are the same size, the single-stranded molecular weight of the DNA is expected to be a b o u t 3.5 X 10 ~, based on a DNA content of 1.2 X 101° to 1.3 X 101° [10] per haploid yeast cell. Thus, the nuclear DNA purified by CsC1 centrifugation is sheared since it is a b o u t 1/30 the expected size. Treatment of these nuclear DNA preparations with T4 UV endonuclease indicates that unirradiated DNA lacks endonuclease-sensitive sites, b u t that DNA from both irradiated cells given no period of dark repair as well as DNA obtained from cells given a 4 h dark repair period contain endonucleasesensitive sites (Fig. 2). The DNA from these cells has an Mr value of about 5.1 X 104; our previous results suggested that this fluence is in the range wherein there is fairly good agreement between the number of breaks observed and the number of breaks expected [19]. Since these sites are lost upon photoreac-
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Fig. 1. S e d i m e n t a t i o n in alkaline sucrose g r a d i e n t s o f n u c l e a r D N A f r o m u n i r r a d i a t e d ( ) a n d irradia t e d y e a s t cells of a t a d 3 m u t a n t given a 4 h p e r i o d of d a r k r e p a i r in g r o w t h m e d i u m ( . . . . . . ); T 7 m a r k e r (. . . . . . ). T h e s e d i m e n t a t i o n p a t t e r n s of t h e n u c l e a z D N A f r o m u n i r r a d i a t e d a n d i r r a d i a t e d cells w e r e s u p e r i m p o s a h l e a n d o n l y the p a t t e r n o b t a i n e d f o r D N A f r o m u n l r r a d i a t e d cells is given. T h e cells w e r e l a b e l e d w i t h [ 3 H ] d T M P a n d i r r a d i a t e d a t a f l u e n c e of 6.7 J / m 2 . Fig. 2. S e d i m e n t a t i o n in alkaline s u c r o s e g r a d i e n t s of t h e s a m e n u c l e a r D N A s a m p l e s s h o w n in Fig. 1 b u t t r e a t e d w i t h 5/~1 of T 4 U V e n d o n u c l e a s e p r i o r t o s e d i m e n t a t i o n ; ( ) D N A f r o m i r r a d i a t e d cells.
tivation, they are very likely to be dimers. Similar results were Obtained when the source of enzyme was a partially-purified UV endonuclease prepared from M. luteus. The rad4 m u t a n t was irradiated at 33 J/m 2 (0.44% survival). The irradiated cells were processed as described for the rad3 mutant. Purified DNA preparations were then treated with M. luteus extract. The results are given in Fig. 3. As expected, unirradiated DNA gives a sedimentation pattern with a peak corresponding to that of the T7 marker DNA, indicating that there are no UV endonuclease-sensitive sites present. Such sites are present, however, in DNA obtained from irradiated cells, which has an Mr value of about 1.5 X 106. The expected number of dimers for this Mr value is 7.38, assuming that 120 dimers are formed per yeast genome with an Mr value of 6 X 109 per Jm -2 [27] and that the M. luteus extract produces one break per dimer [23]. The observed number of breaks per 1.1 X 107 daltons, determined according to the m e t h o d of Ganesan [9], is 6.33. Inherent in the m e t h o d of calculation are several possible sources of error, including the molecular weight determinations and number of dimers induced. The lower observed figure may be a reflection of these calculations. Most of the endonuclease-sensitive sites are retained in the DNA obtained from irradiated cells given a 4 h period of dark repair. There appears to be a slight loss of endonuclease-sensitive sites, as indicated by the somewhat faster sedimentation of this DNA compared to the DNA from irradiated cells given no period of dark repair. The DNA from irradiated cells given a period of dark repair has an approximate Mr value of 2.0 X 106 compared to an Mr value
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Fig. 3. R e t e n t i o n o f endonuelease-sensitive sites in the nuclear D N A o f the r a d 4 m u t a n t irradiated at a fluence o f 33 J / m 2. A l k a l i n e sucrose gradient s e d i m e n t a t i o n patterns o f nuclear D N A : D N A f r o m u ~ a d i a t e d cells treated w i t h 5 #1 M. l u t e u s nuclease ( ); D N A f r o m irradiated cells treated w i t h 5 # l M. l u t e u s nuclease ( ); D N A f r o m irradiated cells given a 4 h d a r k i n c u b a t i o n p e r i o d in g r o w t h m e d i u m , treated w i t h 5 #1 M. l u t e u s n u c l e ~ ( ...... ); T 7 m a r k e r D N A ( . . . . . . ). T h e nuclear D N A s o b t a i n e d f r o m unirradiated cells, f r o m irradiated cells, or from i ~ a d i a t e d cells given a dark i n c u b a t i o n p e r i o d in g r o w t h m e d i u m all had an a p p r o x i m a t e M r value of 1.1 X 1 0 7 , like that of the T7 m a r k e r DNA. Fig. 4. L o s s o f endonucleaseosensitive sites in the nuclear D N A o f a t a d 6 m u t a n t irradiated at a fluence o f 67 J / m 2. A l k a l i n e sucrose gradient s e d i m e n t a t i o n patterns o f nucleax D N A : D N A f~om u n i ~ a d i a t e d cells t~eated w i t h 5 #1 T 4 U V e n d o n u c l e a s e ( ); D N A f r o m irradiated cells treated w i t h 5 #1 T 4 U V endonuclease ( ); D N A f r o m irradiated cells given a 4 h dark i n c u b a t i o n period in g r o w t h m e d i u m , treated w i t h 5 #1 T 4 U V e n d o n u c l e a s e ( . . . . . . ); T7 marker D N A ( . . . . . . ). The nuclear D N A s o b t a i n e d f r o m unirradiated cells, f r o m irradiated cells, or from irradiated cells given a dark i n c u b a t i o n p e r i o d in g r o w t h m e d i u m all had an a p p r o x i m a t e M r value of 1.1 X 1 0 7 , like that of the T7 m a r k e r D N A .
of about 1.5 X 106 for DNA from irradiated cells given no dark repair period. Endonuclease-sensitive sites are also retained in a rad4-3 mutant irradiated with 5 J/m 2 even after 6 h of incubation [23]. The rad4 mutant is apparently defective in removing dimers from DNA following irradiation.
Loss of endonuclease-sensitive sites in the DNA o f rad6 and rad9 mutants Both rad6 and rad9 are known to be involved in error-prone repair [4,11, 18]. Rad6 and rad9 mutants show reduced mutation frequencies with a wide variety of mutagens, including UV [11] and numerous chemical agents [18]. Since rad6 is very sensitive to the lethal effects of UV, although less so than radl or rad3 mutants, initial experiments involved irradiating the rad6 mutant with low UV fluences. However, when low UV fluences were used, i.e. less than 33 J/m ~, endonuclease-sensitive sites in the DNA could not be detected (results not shown). This is also found in R A D + strains. Apparently, excision occurs rapidly enough in a R A D + strain or a rad6 mutant so that unless high UV fluences are used, dimers are not detectable. Presumably dimers are detectable at low fluences in radl, rad3 or rad4 mutants because these strains are excision-
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Fig. 5. Loss of e n d o n u c l e a s e - s e n s i t i v e sites in t h e n u c l e a r D N A of a rad9 m u t a n t i r r a d i a t e d at a f l u e n c e of 33 J / m 2 . Alkaline s u c r o s e g r a d i e n t s e d i m e n t a t i o n p a t t e r n s o f n u c l e a r D N A : D N A f r o m u n i ~ a d i a t e d cells t r e a t e d w i t h 5 ~tl M. luteus e x t r a c t ( ); D N A f r o m i r r a d i a t e d cells t r e a t e d w i t h 5, ~1 M. luteus e x t r a c t ( ); D N A f r o m i r r a d i a t e d cells given a 4 h d a r k i n c u b a t i o n p e r i o d in g r o w t h m e d i u m , t r e a t e d w i t h 5 ~1 M. l u t e u s e x t r a c t ( . . . . . . ); T7 m a r k e r D N A ( . . . . . . ). T h e nuclear D N A s o b t a i n e d f r o m u n i r r a d i a t e d cells, f r o m i r r a d i a t e d cells, or f r o m i r r a d i a t e d cells given a d a r k i n c u b a t i o n p e r i o d in g r o w t h m e d i u m all h a d a n a p p r o x i m a t e M r v a l u e o f 1.1 × 10 ?, like t h a t of t h e T 7 m a ~ k e r D N A .
defective and the dimers remain in the DNA. Therefore, rad6 was irradiated with a fluence of 67 J/m 2 which resulted in less than 0.01% survival. The DNA was prepared and treated as described above. In spite of the extremely low survival, the results clearly show that the tad6 m u t a n t is excision-proficient (Fig. 4). Endonuclease-sensitive sites are absent in unirradiated DNA, they occur in irradiated DNA which, after enzyme treatment, has an Mr value of about 1.5 × 106, but are lost in DNA from irradiated cells which have been given a 4 h dark repair period. All of the endonuclease-sensitive sites as detected by this method, are lost during this time period. This loss of endonuclease-sensitive sites is occurring in a population of cells which is composed mainly of dead cells. The rad9 m u t a n t is also capable of carrying out excision repair (Fig. 5). The results are similar to that obtained for the rad6 mutant. In this experiment, a fluence of 33 J / m 2 resulting in 7.0% survival was used. Discussion
The results presented in this paper provide evidence that the rad3 and rad4 mutants are defective in the ability to remove UV-induced pyrimidine dimers from their DNA. Both mutants may be deficient in a UV-endonuclease rather than a subsequent step in dimer excision, since the DNA from irradiated cells does n o t contain nicks but does contain UV endonuclease-sensitive sites, or dimers. Alternatively, dimer-specific endonuclease activity could still be present in rad3 and/or rad4 mutants and not be active on yeast chromatin. Extracts of xeroderma pigmentosum can incise purified E. coli DNA containing dimers but show no endonuclease activity on their own chromatin [14]. Another possibility is that in a R A D + cell, the R A D 3 + and/or R A D 4 + products could interfere
19 with ligase activity such that once the UV endonuclease introduces a nick at the site of a dimer, ligase is prevented from sealing that nick so that dimer excision takes place. However, in the rad3 and/or rad4 mutants, if this function is missing, dimer excision would not occur and ligase would seal the nick produced by the UV endonuclease. This explanation has been proposed for the action of the uvrC gene, which is thought to control the activity of ligase [25]. The rad6 and rad9 mutants, on the other hand, are both proficient in dimer excision. Neither mutant appears to be defective in any step involved in excision-repair. A defect in repair synthesis would be detected by the slower sedimentation of DNA obtained from cells given a period of dark incubation in growth medium compared to the sedimentation of DNA from unirradiated cells. Both of these mutants are involved in the same error-prone pathway which results in UV-induced mutations [ 11 ]. In addition, both mutants seem to be involved in an error-prone repair pathway leading to chemically-induced mutations [18]. Since the double mutant radl rad6 is more sensitive to UV than either single mutant alone [11], suggesting that tad6 is n o t in the same pathway as radl, an excision-defective mutant, it is not surprising that rad6 can in fact excise UV-induced pyrimidine dimers. It appears to do so at a rate similar to the R A D ÷ strain. When UV fluences of less than 33 J/m 2 are used to irradiate either the R A D ÷ strain or the rad6 mutant, dimer excision apparently occurs rapidly enough, during the period required to prepare spheroplasts, that the DNA obtained from irradiated cells, and then treated with either the T4 UV endonuclease or the M. luteus extract shows no evidence of dimers. At this point, it is n o t known what the functions of rad6 and rad9 are, b u t a feasible possibility is that they are involved in some aspect of post-replication repair processes, which are believed to function in yeast [4]. Acknowledgments I am most grateful to Dr. E.C. Friedberg, who very generously provided the phage T4 UV endonuclease and to Dr. R.J. Reynolds, who made his results available to us. I gratefully acknowledge the expert technical assistance of Ms. Susan Mancuso. The author was supported in part by a U.S. Public Health Service career development award (GM-00004) from the National Institutes of Health. This investigation was supported in part by the U.S. Public Health Service (research grant GM19261 f r o m the National Institutes of Health) and in part by the U.S. Energy Research and Development Administration at the University of Rochester Biomedical and Environmental Research Project. This paper has been designated report No. UR-3490-965. References 1 A v e r b e c k , D., W. L a s k o w s k i , F. E c k a z d t a n d E. Lehmann-Brauns, F o u r radiation sensitive m u t a n t s of S a c c h a r o m y c e s . Survival after U V - and X-ray i r r a d i a t i o n as well as U V - i n d u c e d reversion rates f r o m isoleucine-valine d e p e n d e n c e to i n d e p e n d e n c e , Mol. G e n . G e n e t . , 1 0 7 ( 1 9 7 0 ) 1 1 7 - - 1 2 7 . 2 B r e n d e l , M. a n d R . H . Haynes, Kinetics and genetic c o n t r o l o f the i n c o r p o r a t i o n o f t h y m i d i n e m o n o p h o s p h a t e in y e a s t D N A , MoL G e n . O e n e t . , 1 1 7 ( 1 9 7 2 ) 3 9 - - 4 4 .
20 3 C a r r i e r , W . L . a n d R . B . S e t l o w , E n d o n u c l e a s e f r o m M i c r o c o c c u s luteus w h i c h h a s a c t i v i t y t o w a r d u l t r a v i o l e t - i r r a d i a t e d d e o x y r i b o n u c l e i c acid: p u r i f i c a t i o n a n d p r o p e r t i e s , J. B a c t e r i o l . , 1 0 2 ( 1 9 7 0 ) 178--186. 4 C o x , B.S. a n d J. G a m e , R e p a i r s y s t e m s in S a c c h a r o m y c e s , M u t a t i o n Res., 2 6 ( 1 9 7 4 ) 2 5 7 - - 2 6 4 . 5 C o x , B.S. a n d J . M . P a r r y , T h e i s o l a t i o n , g e n e t i c s a n d survival c h a r a c t e r i s t i c s o f u l t r a v i o l e t - l i g h t sensitive m u t a n t s in y e a s t , M u t a t i o n R e s . , 6 ( 1 9 6 8 ) 3 7 - - 5 5 . 6 H o , K . S . Y . , I n d u c t i o n o f D N A d o u b l e - s t r a n d b r e a k s b y X - r a y s in r a d i o s e n s i t i v e s t r a i n o f t h e y e a s t S a c c h a r o m y c e s cerevisiae, M u t a t i o n Res., 3 0 ( 1 9 7 5 ) 3 2 7 - - 3 3 4 . 7 G a m e , J . C . a n d B.S. C o x , E p i s t a t i c i n t e r a c t i o n s b e t w e e n f o u r t a d l o c i in y e a s t , M u t a t i o n Res., 1 6 (1972) 353--362. 8 G a m e , J . C . a n d R . K . M o r t i m e r , A g e n e t i c s t u d y of X - r a y sensitive m u t a n t s in y e a s t , M u t a t i o n Res., 24 (1974) 281--292. 9 G a n e s a n , A . K . , A m e t h o d f o r d e t e c t i n g p y r i d i m i n e d i m e r s in t h e D N A o f b a c t e r i a i r r a d i a t e d w i t h l o w d o s e s o f u l t r a v i o l e t l i g h t , P r o c . N a t l . A c a d . Sci. U . S . A . , 7 0 ( 1 9 7 3 ) 2 7 5 3 - - 2 7 5 6 . 10 Hartwell, L.H., Biochemical genetics of yeast, Ann. Rev. Genet., 4 (1970) 373--396. 11 L a w r e n c e , C.W. a n d R . C h r i s t e n s e n , U V m u t a g e n e s i s in r a d i a t i o n sensitive s t r a i n s o f y e a s t , G e n e t i c s , 82 (1976) 207--232. 1 2 L e m o n t t , J., M u t a n t s o f y e a s t d e f e c t i v e in m u t a t i o n i n d u c e d b y u l t r a v i o l e t l i g h t , G e n e t i c s , 6 8 ( 1 9 7 1 ) 21--33. 13 Lowry, O.H., N.J. Rosebrough, A.L. Fair and R.J. Randall, Protein measurement with the Folin p h e n o l r e a g e n t , J . Biol. C h e m . , 1 9 3 ( 1 9 5 1 ) 2 6 5 - - 2 7 5 . 1 4 M o r t e l m a n s , K., E . C . F r i e d b e r g , H. F l o r , G. T h o m a s a n d J . E . Cleaver, D e f e c t i v e t h y m i n e d i m e r e x c i s i o n b y cell free e x t r a c t s o f X e r o d e r m a p i g m e n t o s u m . P r o c . N a t l . A c a d . Sci., U . S . A . 7 3 ( 1 9 7 6 ) 2 7 5 7 - 2761. 1 5 N a k a i , S. a n d S. M a t s u m o t o , T w o t y p e s o f r a d i a t i o n - s e n s i t i v e m u t a n t s in y e a s t , M u t a t i o n Res., 4 (1967) 129--136. 1 6 P a r r y , J . M . a n d E.M. P a r r y , T h e e f f e c t s o f U V - l i g h t p o s t - t r e a t m e n t s o n t h e survival c h a r a c t e r i s t i c s o f 21 UV-sensitive m u t a n t s o f S a c c h a r o m y c e s cerevisiae, M u t a t i o n Res. 8 ( 1 9 6 9 ) 5 4 5 - - 5 5 6 . 17 P a t e r s o n , M.C. a n d R . B . S e t l o w , E n d o n u c l e o l y t i c a c t i v i t y f r o m M i c r o c o c c u s luteus t h a t a c t s o n X - r a y i n d u c e d d a m a g e in p l a s m i d D N A o f E s c h e r i c h i a coli m i n i c e l i s , P r o c . N a t l . A c a d . Sci., U . S . A . 6 9 (1972) 2927--2931. 1 8 P r a k a s h , L . , L a c k o f c h e m i c a l l y i n d u c e d m u t a t i o n in r e p a i r - d e f i c i e n t m u t a n t s o f y e a s t , G e n e t i c s , 78 (1974) 1101--1118. 19 P r a k a s h , L., R e p a i r o f p y r i m i d i n e d i m e r s in n u c l e a r a n d m i t o c h o n d r i a l D N A o f y e a s t i r r a d i a t e d w i t h l o w d o s e s o f u l t r a v i o l e t light, J. Mol. Biol., 9 8 ( 1 9 7 5 ) 7 8 1 - - 7 9 5 . 2 0 R e s n i c k , M., G e n e t i c c o n t r o l o f r a d i a t i o n s e n s i t i v i t y in S a c c h a r o m y c e s cerevisiae, G e n e t i c s , 6 2 ( 1 9 6 9 ) 519--531. 21 R e s n i c k , M . A . a n d P. M a r t i n , T h e r e p a i r o f d o u b l e - s t x a n d b r e a k s in t h e n u c l e a r D N A o f S a e c h a r o m y ces cerevisiae a n d its g e n e t i c c o n t r o l , Mol. G e n . G e n e t . , 1 4 3 ( 1 9 7 6 ) 1 1 9 - - 1 2 9 . 2 2 R e s n i c k , M. and J a n e K . S e t l o w , R e p a i r o f p y r i m i d i n e d i m e r d a m a g e i n d u c e d in y e a s t b y u l t r a v i o l e t light, J. B a c t e r i o l . , 1 0 9 ( 1 9 7 2 ) 9 7 9 - - 9 8 6 . 2 3 R e y n o l d s , R . , E x c i s i o n r e p a i r in S a c e h a r o r n y e e s eerevisiae. P h . D . thesis. U n i v e r s i t y o f T e n n e s s e e , Knoxville, Tenn., 1976. 2 4 R u p p , W.D. a n d P. H o w a r d - F l a n d e r s , D i s c o n t i n u i t i e s in t h e D N A s y n t h e s i z e d i n a n e x c i s i o n - d e f e c t i v e s t r a i n o f E s c h e r i c h i a coli f o l l o w i n g u l t r a v i o l e t i r r a d i a t i o n , J. Mol. Biol., 3 1 ( 1 9 6 8 ) 2 9 1 - - 3 0 2 . 2 5 S e e b e r g , E. a n d W.D. R u p p , E f f e c t o f m u t a t i o n s in lig and p o l A o n U V - i n d u c e d s t r a n d c u t t i n g in a avrC s t r a i n o f Escherichia coli, in M o l e c u l a r M e c h a n i s m s f o r R e p a i r o f D N A , V o l . 5B, P.C. H a n a w a l t a n d R . B . S e f l o w (eds.) P l e n u m Press, N . Y . , 1 9 7 5 , p p . 4 3 9 - - 4 4 1 . 26 S n o w , R . , M u t a n t s o f y e a s t sensitive t o u l t r a v i o l e t l i g h t , J. B a c t e r i o l . , 9 4 ( 1 9 6 7 ) 5 7 1 - - 5 7 5 . 27 U n r a u , R., R . W h e a t c r o f t a n d B.S. C o x , T h e e x c i s i o n o f p y r i m i d i n e d i m e r s f r o m D N A o f u l t r a v i o l e t i r r a d i a t e d y e a s t , Mol. G e n . G e n e t . 1 1 3 ( 1 9 7 1 ) 3 5 9 - - 3 6 2 . 2 8 W a t e r s , R . a n d E. M o u s t a c c h i , T h e d i s a p p e a r a n c e o f u l t r a v i o l e t - i n d u c e d p y r i m i d i n e d i m e r s f r o m t h e n u c l e a r D N A o f e x p o n e n t i a l a n d s t a t i o n a r y p h a s e cells o f S a c e h a r o m y c e s cerevisiae f o l l o w i n g v a r i o u s post-irradiation treatments, Biochim. Biophys. Acta, 353 (1974) 407--419. 29 W a t e r s , R . a n d E. M o u s t a c c h i , T h e f a t e o f u l t r a v i o l e t - i n d u c e d p y r i m i d i n e d i m e r s in t h e m i t o c h o n d r i a l D N A o f S a c c h a r o r n y c e s cerevisiae, B i o c h i m . B i o p h y s . A c t a , 3 6 6 ( 1 9 7 4 ) 2 4 1 - - 2 5 0 . 3 0 Wilkins, R . J . , E n d o n u c l e a s e - s e n s i t i v e sites in t h e D N A o f i r r a d i a t e d b a c t e r i a : a r a p i d a n d sensitive assay, Biochim. Biophys. Acta, 312 (1973) 33--37.
Mutation Research, 45 (1977) 13--20 © Elsevier/North-Holland Biomedical Press
R E P A I R OF PYRIMIDINE DIMERS IN RADIATION-SENSITIVE MUTANTS rad3, rad4, rad6 AND rad9 OF SACCHAROMYCES CEREVISIAE
LOUISE PRAKASH Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, N. Y. 14642 (U.S.A.) (Received 12 April, 1977) (Accepted 16 May, 1977)
Summary The ability to remove ultraviolet (UV)-induced pyrimidine dimers was examined in four radiation-sensitive mutants of Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by either the T4 UV-endonuclease or an endonuclease activity found in crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad3 and rad4 mutants are shown to be defective in dimer excision whereas the rad6 and rad9 mutants are proficient in dimer excision.
Introduction Over twenty genetic loci are known to be involved in the repair of damage induced in DNA by ionizing radiation and ultraviolet (UV) light in the yeast Saccharomyces cerevisiae, a eucaryotic microorganism [1,5,8,12,15,20,26]. Dimer excision is known to occur in nuclear DNA of wild type yeast [19,27, 28], b u t is absent in mitochondrial DNA [19,29]. Both radl [19,27,28] and rad2 [22] mutants have been shown to be defective in the ability to excise UVinduced pyrimidine dimers. Double mutants involving various combinations of the radl, rad2, rad3 and rad4 loci show epistatic interactions, i.e. the sensitivity of the double mutant to UV is no greater than that of the more sensitive of the t w o single mutants [7]. This suggests that all four genes are in the same repair pathway. In addition, rad3 and rad4 mutants can be photoreactivated after dark holding [16], indicating that dimers are n o t removed during dark holding and that these two mutants are probably excision-defective. However, there is no direct evidence that the rad3 and rad4 mutants are in fact excision-defective.
14 The only other mutant whose defect has been partially identified is rad52, shown to be deficient in the capacity to repair X-ray-induced double strand breaks [6,21]. In this study, we have measured the fate of UV-induced dimers in four radiation-sensitive mutants of yeast, tad3, rad4, rad6 and rad9, by using a sensitive assay based on the ability of bacteriophage T4 UV endonuclease [9] and a UV endonuclease found in crude extracts prepared from Micrococcus luteus to produce a single-strand breaks in DNA containing pyrimidine dimers [30]. Evidence is presented that the rad3 and rad4 mutants are excision-defective while the rad6 and rad9 mutants are excision-proficient. Materials and methods
Strains The rad3, rad4, rad6-1 and rad9 mutants were isolated by Cox and Parry [5]. The ability to take up exogenous thymidine monophosphate (dTMP), was introduced into our strains, since a radioactive label specific for DNA was required for the experiments reported here. Strain MB1017-3A, a lysl-1 ade2-1 his5-2 RAD÷ tup [2], was crossed to LP3-18B, a cyc1-131 his1-1 tad6-1. The resulting diploid, LP-99, was sporulated and one of the meiotic segregants, LP99-18B, a his1-1 his5-2 rad6-1 tup, was the rad6 mutant used in this study. LP98-1D, a lysl-1 his5-2 tup, a strain previously described [19], was crossed to tad3, rad4 and rad9 mutants to generate the following haploid strains: LP397B, a ade2-1 rad3 tup; LP459-8B, a ilv3 rad4 tup; and LP457-6C, a hisS.2 lysl-1 rad9 tup. Media The media were as previously described [ 19]. Labeling o f cells, UV irradiation, preparation of spheroplasts and centrifugation in neutral CsCl gradients The details of these procedures were as described before [19]. Preparation o f UV endonuclease from Micrococcus luteusand bacteriophage T4 The nuclease specific for DNA containing dimers was prepared from M. luteus (Miles Chemical Co., Elkhart, Ind.). The enzyme was partially purified according to a modification [23] of the m e t h o d described by Carrier and Setlow [3]. Although extracts prepared in this way also contain activity for X-rayirradiated DNA [17], they do n o t have activity against DNA obtained from untreated cells. The bacteriophage T4 UV endonuclease was generously supplied by Dr. E.C. Friedberg. The enzyme preparation had been purified through the phosphocellulose step and was in 10% polyethylene glycol plus 33% glycerol. The activity of this preparation was such that 0.01 ml of stock solution would nick a b o u t 1.8 pmol of dimers in irradiated T7 DNA, as nucelotide/h (Friedberg, personal communication). This preparation was the same one as that used in our previous studies [19]. Endonuclease treatment of nuclear DNA of yeast. Treatment with T4 UV endonuclease was as previously described [19].
15 Treatment with M. luteus extract was carried out in a total volume of 105 pl. The reaction mixtures consisted of 50 gl (0.2 to 0.4 t~g} DNA, 50 ttl 0.02 M EDTA (pH 7.5) and 5 pl of M. luteus extract, containing 12 mg protein per ml determined by a modification of the Lowry m e t h o d [13]. The mixtures were incubated at 36°C for 10 min. Control DNAs were treated in the same way, w i t h o u t the addition of enzyme. The reaction was terminated by layering 0.1 ml on an alkaline sucrose gradient. Alkaline sucrose sedimentation and molecular weight determinations Linear gradients of 5 to 20% (w/w) sucrose, in 0.8 N NaC1--0.2 N NaOH, were made in cellulose nitrate tubes pretreated with heat-denatured salmon sperm DNA. Each gradient contained 4.9 ml of sucrose solution plus a top layer of 0.1 ml 1 N NaOH. Gradients with enzyme-treated and untreated DNAs were kept at room temperature for 20 min before centrifugation. Samples were centrifuged in an SW50.1 rotor in a Beckman L5-65 preparative ultracentrifuge at 20 ° C for 3 h at 40,000 rpm. 14C-labelled T7 bacteriophage DNA was used as a marker for all gradients, and fractions were collected and precipitated as described previously [19]. The method described by Rupp and Howard-Flanders [24] was used to determine the number average molecular weight; only the peak fractions were included in the calculations. Results Retention o f endonuclease-sensitive sites in the DNA o f rad3 and rad4 mutants Cells of the rad3 mutant irradiated with 6.7 J/m 2 (0.82% survival) were incubated in the dark in growth medium for 4 h. The nuclear DNA, which had been purified by CsC1 centrifugation, was treated with T4 UV endonuclease. The irradiation and conditions of treatment were the same as those used with a radl mutant which was shown by this method to be excision defective [19]. The nuclear DNAs obtained either from unirradiated cells, from irradiated cells, or from irradiated cells which had been given a 4 h period of dark repair in growth medium all showed the same sedimentation pattern in alkaline sucrose gradients (Fig. 1). The molecular weight of these nuclear DNAs corresponds very closely to the T7 DNA used as a marker and is equivalent to an M, value of about 1.1 X l 0 T. Assuming that there is only one DNA molecule per chromosome and that all 17 chromosomes of yeast are the same size, the single-stranded molecular weight of the DNA is expected to be a b o u t 3.5 X 10 ~, based on a DNA content of 1.2 X 101° to 1.3 X 101° [10] per haploid yeast cell. Thus, the nuclear DNA purified by CsC1 centrifugation is sheared since it is a b o u t 1/30 the expected size. Treatment of these nuclear DNA preparations with T4 UV endonuclease indicates that unirradiated DNA lacks endonuclease-sensitive sites, b u t that DNA from both irradiated cells given no period of dark repair as well as DNA obtained from cells given a 4 h dark repair period contain endonucleasesensitive sites (Fig. 2). The DNA from these cells has an Mr value of about 5.1 X 104; our previous results suggested that this fluence is in the range wherein there is fairly good agreement between the number of breaks observed and the number of breaks expected [19]. Since these sites are lost upon photoreac-
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Fig. 1. S e d i m e n t a t i o n in alkaline sucrose g r a d i e n t s o f n u c l e a r D N A f r o m u n i r r a d i a t e d ( ) a n d irradia t e d y e a s t cells of a t a d 3 m u t a n t given a 4 h p e r i o d of d a r k r e p a i r in g r o w t h m e d i u m ( . . . . . . ); T 7 m a r k e r (. . . . . . ). T h e s e d i m e n t a t i o n p a t t e r n s of t h e n u c l e a z D N A f r o m u n i r r a d i a t e d a n d i r r a d i a t e d cells w e r e s u p e r i m p o s a h l e a n d o n l y the p a t t e r n o b t a i n e d f o r D N A f r o m u n l r r a d i a t e d cells is given. T h e cells w e r e l a b e l e d w i t h [ 3 H ] d T M P a n d i r r a d i a t e d a t a f l u e n c e of 6.7 J / m 2 . Fig. 2. S e d i m e n t a t i o n in alkaline s u c r o s e g r a d i e n t s of t h e s a m e n u c l e a r D N A s a m p l e s s h o w n in Fig. 1 b u t t r e a t e d w i t h 5/~1 of T 4 U V e n d o n u c l e a s e p r i o r t o s e d i m e n t a t i o n ; ( ) D N A f r o m i r r a d i a t e d cells.
tivation, they are very likely to be dimers. Similar results were Obtained when the source of enzyme was a partially-purified UV endonuclease prepared from M. luteus. The rad4 m u t a n t was irradiated at 33 J/m 2 (0.44% survival). The irradiated cells were processed as described for the rad3 mutant. Purified DNA preparations were then treated with M. luteus extract. The results are given in Fig. 3. As expected, unirradiated DNA gives a sedimentation pattern with a peak corresponding to that of the T7 marker DNA, indicating that there are no UV endonuclease-sensitive sites present. Such sites are present, however, in DNA obtained from irradiated cells, which has an Mr value of about 1.5 X 106. The expected number of dimers for this Mr value is 7.38, assuming that 120 dimers are formed per yeast genome with an Mr value of 6 X 109 per Jm -2 [27] and that the M. luteus extract produces one break per dimer [23]. The observed number of breaks per 1.1 X 107 daltons, determined according to the m e t h o d of Ganesan [9], is 6.33. Inherent in the m e t h o d of calculation are several possible sources of error, including the molecular weight determinations and number of dimers induced. The lower observed figure may be a reflection of these calculations. Most of the endonuclease-sensitive sites are retained in the DNA obtained from irradiated cells given a 4 h period of dark repair. There appears to be a slight loss of endonuclease-sensitive sites, as indicated by the somewhat faster sedimentation of this DNA compared to the DNA from irradiated cells given no period of dark repair. The DNA from irradiated cells given a period of dark repair has an approximate Mr value of 2.0 X 106 compared to an Mr value
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Fig. 3. R e t e n t i o n o f endonuelease-sensitive sites in the nuclear D N A o f the r a d 4 m u t a n t irradiated at a fluence o f 33 J / m 2. A l k a l i n e sucrose gradient s e d i m e n t a t i o n patterns o f nuclear D N A : D N A f r o m u ~ a d i a t e d cells treated w i t h 5 #1 M. l u t e u s nuclease ( ); D N A f r o m irradiated cells treated w i t h 5 # l M. l u t e u s nuclease ( ); D N A f r o m irradiated cells given a 4 h d a r k i n c u b a t i o n p e r i o d in g r o w t h m e d i u m , treated w i t h 5 #1 M. l u t e u s n u c l e ~ ( ...... ); T 7 m a r k e r D N A ( . . . . . . ). T h e nuclear D N A s o b t a i n e d f r o m unirradiated cells, f r o m irradiated cells, or from i ~ a d i a t e d cells given a dark i n c u b a t i o n p e r i o d in g r o w t h m e d i u m all had an a p p r o x i m a t e M r value of 1.1 X 1 0 7 , like that of the T7 m a r k e r DNA. Fig. 4. L o s s o f endonucleaseosensitive sites in the nuclear D N A o f a t a d 6 m u t a n t irradiated at a fluence o f 67 J / m 2. A l k a l i n e sucrose gradient s e d i m e n t a t i o n patterns o f nucleax D N A : D N A f~om u n i ~ a d i a t e d cells t~eated w i t h 5 #1 T 4 U V e n d o n u c l e a s e ( ); D N A f r o m irradiated cells treated w i t h 5 #1 T 4 U V endonuclease ( ); D N A f r o m irradiated cells given a 4 h dark i n c u b a t i o n period in g r o w t h m e d i u m , treated w i t h 5 #1 T 4 U V e n d o n u c l e a s e ( . . . . . . ); T7 marker D N A ( . . . . . . ). The nuclear D N A s o b t a i n e d f r o m unirradiated cells, f r o m irradiated cells, or from irradiated cells given a dark i n c u b a t i o n p e r i o d in g r o w t h m e d i u m all had an a p p r o x i m a t e M r value of 1.1 X 1 0 7 , like that of the T7 m a r k e r D N A .
of about 1.5 X 106 for DNA from irradiated cells given no dark repair period. Endonuclease-sensitive sites are also retained in a rad4-3 mutant irradiated with 5 J/m 2 even after 6 h of incubation [23]. The rad4 mutant is apparently defective in removing dimers from DNA following irradiation.
Loss of endonuclease-sensitive sites in the DNA o f rad6 and rad9 mutants Both rad6 and rad9 are known to be involved in error-prone repair [4,11, 18]. Rad6 and rad9 mutants show reduced mutation frequencies with a wide variety of mutagens, including UV [11] and numerous chemical agents [18]. Since rad6 is very sensitive to the lethal effects of UV, although less so than radl or rad3 mutants, initial experiments involved irradiating the rad6 mutant with low UV fluences. However, when low UV fluences were used, i.e. less than 33 J/m ~, endonuclease-sensitive sites in the DNA could not be detected (results not shown). This is also found in R A D + strains. Apparently, excision occurs rapidly enough in a R A D + strain or a rad6 mutant so that unless high UV fluences are used, dimers are not detectable. Presumably dimers are detectable at low fluences in radl, rad3 or rad4 mutants because these strains are excision-
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Fig. 5. Loss of e n d o n u c l e a s e - s e n s i t i v e sites in t h e n u c l e a r D N A of a rad9 m u t a n t i r r a d i a t e d at a f l u e n c e of 33 J / m 2 . Alkaline s u c r o s e g r a d i e n t s e d i m e n t a t i o n p a t t e r n s o f n u c l e a r D N A : D N A f r o m u n i ~ a d i a t e d cells t r e a t e d w i t h 5 ~tl M. luteus e x t r a c t ( ); D N A f r o m i r r a d i a t e d cells t r e a t e d w i t h 5, ~1 M. luteus e x t r a c t ( ); D N A f r o m i r r a d i a t e d cells given a 4 h d a r k i n c u b a t i o n p e r i o d in g r o w t h m e d i u m , t r e a t e d w i t h 5 ~1 M. l u t e u s e x t r a c t ( . . . . . . ); T7 m a r k e r D N A ( . . . . . . ). T h e nuclear D N A s o b t a i n e d f r o m u n i r r a d i a t e d cells, f r o m i r r a d i a t e d cells, or f r o m i r r a d i a t e d cells given a d a r k i n c u b a t i o n p e r i o d in g r o w t h m e d i u m all h a d a n a p p r o x i m a t e M r v a l u e o f 1.1 × 10 ?, like t h a t of t h e T 7 m a ~ k e r D N A .
defective and the dimers remain in the DNA. Therefore, rad6 was irradiated with a fluence of 67 J/m 2 which resulted in less than 0.01% survival. The DNA was prepared and treated as described above. In spite of the extremely low survival, the results clearly show that the tad6 m u t a n t is excision-proficient (Fig. 4). Endonuclease-sensitive sites are absent in unirradiated DNA, they occur in irradiated DNA which, after enzyme treatment, has an Mr value of about 1.5 × 106, but are lost in DNA from irradiated cells which have been given a 4 h dark repair period. All of the endonuclease-sensitive sites as detected by this method, are lost during this time period. This loss of endonuclease-sensitive sites is occurring in a population of cells which is composed mainly of dead cells. The rad9 m u t a n t is also capable of carrying out excision repair (Fig. 5). The results are similar to that obtained for the rad6 mutant. In this experiment, a fluence of 33 J / m 2 resulting in 7.0% survival was used. Discussion
The results presented in this paper provide evidence that the rad3 and rad4 mutants are defective in the ability to remove UV-induced pyrimidine dimers from their DNA. Both mutants may be deficient in a UV-endonuclease rather than a subsequent step in dimer excision, since the DNA from irradiated cells does n o t contain nicks but does contain UV endonuclease-sensitive sites, or dimers. Alternatively, dimer-specific endonuclease activity could still be present in rad3 and/or rad4 mutants and not be active on yeast chromatin. Extracts of xeroderma pigmentosum can incise purified E. coli DNA containing dimers but show no endonuclease activity on their own chromatin [14]. Another possibility is that in a R A D + cell, the R A D 3 + and/or R A D 4 + products could interfere
19 with ligase activity such that once the UV endonuclease introduces a nick at the site of a dimer, ligase is prevented from sealing that nick so that dimer excision takes place. However, in the rad3 and/or rad4 mutants, if this function is missing, dimer excision would not occur and ligase would seal the nick produced by the UV endonuclease. This explanation has been proposed for the action of the uvrC gene, which is thought to control the activity of ligase [25]. The rad6 and rad9 mutants, on the other hand, are both proficient in dimer excision. Neither mutant appears to be defective in any step involved in excision-repair. A defect in repair synthesis would be detected by the slower sedimentation of DNA obtained from cells given a period of dark incubation in growth medium compared to the sedimentation of DNA from unirradiated cells. Both of these mutants are involved in the same error-prone pathway which results in UV-induced mutations [ 11 ]. In addition, both mutants seem to be involved in an error-prone repair pathway leading to chemically-induced mutations [18]. Since the double mutant radl rad6 is more sensitive to UV than either single mutant alone [11], suggesting that tad6 is n o t in the same pathway as radl, an excision-defective mutant, it is not surprising that rad6 can in fact excise UV-induced pyrimidine dimers. It appears to do so at a rate similar to the R A D ÷ strain. When UV fluences of less than 33 J/m 2 are used to irradiate either the R A D ÷ strain or the rad6 mutant, dimer excision apparently occurs rapidly enough, during the period required to prepare spheroplasts, that the DNA obtained from irradiated cells, and then treated with either the T4 UV endonuclease or the M. luteus extract shows no evidence of dimers. At this point, it is n o t known what the functions of rad6 and rad9 are, b u t a feasible possibility is that they are involved in some aspect of post-replication repair processes, which are believed to function in yeast [4]. Acknowledgments I am most grateful to Dr. E.C. Friedberg, who very generously provided the phage T4 UV endonuclease and to Dr. R.J. Reynolds, who made his results available to us. I gratefully acknowledge the expert technical assistance of Ms. Susan Mancuso. The author was supported in part by a U.S. Public Health Service career development award (GM-00004) from the National Institutes of Health. This investigation was supported in part by the U.S. Public Health Service (research grant GM19261 f r o m the National Institutes of Health) and in part by the U.S. Energy Research and Development Administration at the University of Rochester Biomedical and Environmental Research Project. This paper has been designated report No. UR-3490-965. References 1 A v e r b e c k , D., W. L a s k o w s k i , F. E c k a z d t a n d E. Lehmann-Brauns, F o u r radiation sensitive m u t a n t s of S a c c h a r o m y c e s . Survival after U V - and X-ray i r r a d i a t i o n as well as U V - i n d u c e d reversion rates f r o m isoleucine-valine d e p e n d e n c e to i n d e p e n d e n c e , Mol. G e n . G e n e t . , 1 0 7 ( 1 9 7 0 ) 1 1 7 - - 1 2 7 . 2 B r e n d e l , M. a n d R . H . Haynes, Kinetics and genetic c o n t r o l o f the i n c o r p o r a t i o n o f t h y m i d i n e m o n o p h o s p h a t e in y e a s t D N A , MoL G e n . O e n e t . , 1 1 7 ( 1 9 7 2 ) 3 9 - - 4 4 .
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