241
Mutation Research, 41 ( 1 9 7 6 ) 2 4 1 - - 2 4 8 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
THE R E L A T I O N BETWEEN REPAIR OF DNA AND RADIATION AND CHEMICAL MUTAGENESIS IN S A C C H A R O M Y C E S C E R E V I S I A E *
LOUISE PRAKASH
Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 (U.S.A.) ( R e c e i v e d M a r c h 3rd, 1 9 7 6 ) ( R e v i s i o n received J u n e 9th, 1 9 7 6 ) ( A c c e p t e d J u n e 23rd, 1 9 7 6 )
Summary The effect of various genes involved in DNA repair functions on radiation and chemical mutagenesis in E s c h e r i c h i a c o l i is discussed and compared to similar studies done in yeast. Results of the effect of various genes conferring radiation-sensitivity on mutation induction in yeast are presented and related to current ideas of mutagenesis.
Procaryotic and eucaryotic organisms possess mechanisms which enable them to repair damage induced in their DNA by a wide variety of chemical and physical agents. The manner in which DNA damage is repaired influences both the survival of the organism as well as the mutagenic effectiveness of the agent. The best studied repair processes in both eucaryotes and procaryotes are those which operate on ultraviolet light (UV)-induced damage of DNA. Strains of E s c h e r i c h i a c o l i which have normal radiation-sensitivity possess, in addition to photoreactivation, two pathways for the repair of pyrimidine dimers induced by UV [9,10,12]. The predominant one is excision-repair, in which pyrimidine dimers are enzymatically excised from the DNA. The gap generated by this process is sealed by repair replication in the region of the gap, using the opposite strand as a template [9]. The dimers which escape excision-repair are repaired during and after DNA replication by processes called post-replication repair. One of these processes is recombinational repair [33]; another is error-prone repair and generates mutations [45]. Both the r e c A ÷ a n d l e x + gene functions are necessary for both post-replicational repair and UV-induced mutations. In l e x - strains, no UV-induced mutations are produced [3,20,42] even though * By acceptance of this article, t h e p u b l i s h e r a n d / o r r e c i p i e n t a c k n o w l e d g e s the U.S. Government's right to retain a nonexclusive, royalty-free license in and to any c o p y r i g h t c o v e r i n g this paper.
242 some post-replication repair can be demonstrated [47], suggesting that the lex ~ gene function is involved in error-prone repair. R e c A - strains, which are also refractory to UV-induced mutation [14,19,43], do not undergo post-replication repair [ 41], suggesting that post-replication repair generates mutations. It is now believed that UV mutability results from the induction of errorprone repair of excision gaps or post-replication gaps and depends on at least the recA ÷ and lex + functions [27,44,46]. The protein synthesis inhibitor chloramphenicol has been shown to inhibit some a m o u n t of post-replication repair in excision-defective E. coli carrying a u v r A mutation but no inhibition is detected if a 20-min growth period occurs subsequent to the irradiation and prior to treatment with chloramphenicol [33]. According to current ideas, the presence of pyrimidine dimers in DNA acts as an inducer of the error-prone mode of post-replication repair, which results in enhanced mutation frequency observed in excision-defective strains at UV doses showing no demonstrable effect in excision-proficient strains. In uvrA strains which are deficient in excision, a large number of dimers remain in the DNA so that even at low UV doses, error-prone repair is induced. P o l A mutants having very low DNA polymerase I activity also exhibit enhanced mutation frequencies at doses below 5 J/m 2 [45,46.] The p o I A - single m u t a n t and u v r A - p o l A - double m u t a n t presumably induced error-prone repair at low doses because of their lowered rate of repair. Most of the work on m u t a t i o n induction in repair-deficient mutants of E. coli has dealt with radiation-induced mutations and the models relating repair to mutation have, of necessity, dealt primarily with data obtained from radiation mutagenesis. The few chemical mutagens examined have included mainly those whose effects parallel UV and X-ray. Damage induced by 4-nitroquinoline-l-oxide (NQO), a p o t e n t carcinogen, seems to be subject, in part at least, to the same excision-repair system operating on dimers. Uvr- strains are sensitive to the lethal effect of NQO and also show enhanced susceptibility to the mutagenic effects of NQO [13,14]. R e c A - strains, which presumably are involved in some step of error-prone repair, are immutable to UV, NQO, X-ray, and the monofunctional alkylating agent methyl methanesulfonate (MMS) [13, 14]. However, mutability induced by ethyl methanesulfonate (EMS), or nitrosoguanidine (NTG) is normal [13,14]. Therefore, the recA function, although it is required for radiation mutagenesis, is apparently not required for mutation induction by some chemicals. While the study of radiation mutagenesis and its genetic control as revealed by the elegant studies in E. coli has elucidated certain aspects of the role of repair in mutation, there may well be other u n k n o w n important processes. If we now examine similar studies in the eucaryotic organism, Saccharom y c e s cerevisiae, the situation becomes more complex. First of all, 32 distinct genetic loci have been identified so far which confer radiation-sensitivity. The mutations radl through rad22 were isolated as genes conferring sensitivity primarily to UV [6,28,37], while the mutations rad50 through r a d 5 7 confer sensitivity primarily to X-ray [7,34]. Cross-sensitivities to UV and X-ray exist for m u t a n t s within each of the t~vo groups. In addition, the mutations r e v l , rev2 (= rad5) and rev3 were isolated for reducing the UV-induced mutability of the highly revertible arg4-17 site [18]. Table I lists the various radiation sensitive
243 TABLE
I
PROPERTIES
OF RADIATION-SENSITIVE
MUTANTS
OF SACCHAROMYCES
CEREVISIAE
M M S - s e n s i t i v i t y o f r a d S O t h r o u g h t a d 5 7 m u t a n t s w a s d e t e r m i n e d b y w h e t h e r or n o t t h e strain g r e w o n 1 % y e a s t e x t r a c t , 2% p e p t o n e , 2% g l u c o s e s o l i d m e d i a c o n t a i n i n g 0 . 0 3 5 % M M S ( P r a k a s h , u n p u b l i s h e d results). Mutant
Sensitivity o f UV [6]
NQO [25]
~'-ray [ 6 ] or X-ray
MMS [49]
HNO 2 [49]
EMS [25]
tad1 rad2
++ +++
rad3 rad4 tad6 tad 7 rad8 tad9 radlO tad11 rad13 radl4 rod15 tad16 rad l 7 tad18 tad19 tad20 tad21
++++ +++ +++ ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ [28] + ++ +
+++ ++ ++ +++ +++
0 0 0 0 ++ 0 + 0 ++ 0 0 0 + 0 0 ++ [28] 0 0 0
0 0 0 [41] 0 +++ [25] 0 +]-++(0) [25] +/-+/-+[-+1-0 0 +/-++ [2] 0 + ++
++ [25] +]-++ [25,41] +]-++ [25] 0 +l-++(0) [25] +l-0 ++ 0 0(++) [25] 0 [25]
0 0 0 [25,41] 0 ++
revl rev2=rad5 rev3
+ [18] ++ +
0
+
tad50 tad51 tad52 tad53 tad54 tad55 rad56 rad5 7
+ [7] + 0 + 0 0 0 0
+++ [7] +++ +++ +++ +++ ++ + ++
++ ++ ++ [2] ++ ++ ++ ++ ++
0 +++
0 ++ 0 0 ++
0 0 0
0
+ 0 ++ ++ 0 [25] 0 [25] 0 [25]
+
0
0 + 0 0 0
Effect on sporulation [6]
normal normal normal normal eliminated normal normal normal normal normal normal normal normal normal normal normal reduced reduced reduced
0 0 0
++
normal [7] reduced reduced normal normal reduced normal normal
0, S e n s i t i v i t y l i k e R A D + ( o r R E V +) strain + --* + + + + , Increasing d e g r e e s o f sensitivity + / - - , s o m e w h a t m o r e s e n s i t i v e than R A D + ; m a y o r m a y n o t be significantly d i f f e r e n t f r o m R A D +
mutants of yeast and some of their properties. The specific lesion has been identified in only five mutants; radl, rad2, rad3 and rad4 are excision-defective [ 2 4 , 3 1 , 3 2 , 3 9 , 4 0 ] . Like their excision-defective counter-parts in E. coli, these 4 mutants showed enhanced UV mutability [1,17,21,22,29,48] and enhanced NQO mutability [25]. Recently, it has been shown that although R A D ÷ strains are able to repair X-ray induced double-strand breaks, rad52 mutants are deficient in this capacity [11,30]. In order to examine the effect of the various rad genes on mutations induced by radiation and chemical agents, a series of diploid strains homozygous for a particular rad gene was constructed in collaboration with Dr. Christopher Lawrence. The strains were also homozygous for cyc1-131, a chain initiation mutant in the structural gene determining iso-l-cytochrome c [38]. This mutant
244
reverts to give the normal protein by a G : C to A : T transition [26,38] and it was the site used to measure reversion. The effect of various tad genes on the mutation process could then be determined by comparing reversion frequencies in R A D ÷ and rad strains. The effect of UV on reversion of cyc1-131 was examined in these strains by Lawrence and Christensen [17] while Prakash [25] measured reversion frequencies using EMS, NQO and HNO2. The tad strains showing reduced UV-induced mutations do not necessarily show reduced mutations induced by chemicals. In general, even when comparing two mutagens expected to exhibit responses, such as UV and NQO, such a parallel response was not observed consistently. The results of all these experiments are summarized qualitatively in Table II. Although rad9, rad18, revl and rev2 did not lower the UV-induced reversion of cyc1-131, they do lower reversion of other cycl alleles and other genes [ 1 8 , 2 1 , 2 9 ] . Rad8 lowered reversion of c y c l - 9 whereas rad15-1 had little effect. It was concluded from these studies that at least seven genes, namely, revl, rev2, rev3, rad6, rad8, rad9, and tad18 are required for normal UV mutagenesis to occur [17]. In addition, double mutant survival data indicate that at least revl, rev3, tad6, rad9, and rad18 are in the same error-prone pathway [17]. The products of the rad6 and rev3 genes were suggested to be required for the production of UV-induced mutations throughout the genome whereas the effects of the other genes did not extend to all loci tested. A1-
TABLE
II
INDUCED CERE
cyc1--131
V1SIA
--+ C Y C 1 R E V E R S I O N
IN RADIATION-SENSITIVE
DIPLOID
SACCHAROMYCES
E
UV results were obtained
from Lawrence
Strains
UV
NQO
rad l tad2 tad3 tad4 tad6
+ +
and Christensen
[17].
EMS
HNO 2
+
0
0
+
0
+
+
0
+
+
0
.
.
tad8
.
0
.
(--)
tad9
0(--)
--
rad l O
+
+
tad13 tad 14 tad15 rad16 rad l 7 rad18
0
--
--
0
0
0
0
--
--
--
0
+
0
0
0
--
0
0(--)
--
0
+
tad50 tad52
0 --
0
rev l rev2 rev3
0(--)
--
0
0
0(--)
--
0
0
--
--
0
0
0
O, R e v e r s i o n +, R e v e r s i o n --, Reversion
f r e q u e n c y l i k e R A D +. g r e a t e r t h a n R A D ÷. reduced compared to RAD+ Blank indicates strain was not tested entry in parentheses loci.
refers to results for other cycl
alleles or other
245 though there appears to be one error-prone pathway for UV mutagenesis in yeast, three pathways are thought to be involved in repair of UV damage [5]. However, when looking at results obtained with chemical mutagens, two genes, rad6 and rad9, greatly reduced reversion of cyc1-131. This was true not only for NQO, EMS and HNO2, b u t also for diethyl sulfate (DES), MMS, dimethyl sulfate (DMS), NTG, nitrogen mustard (HN2), fl-propiolactone, tritiated uridine decay and ionizing radiation [23]. At low doses, HNO2 and nitrosoimidazolidone (NIL) revert cyc1-131 in rad6 and rad9 strains to the same extent as in R A D ÷ strains b u t at high doses, reversion frequencies obtained with both HNO2 and NIL are much lower in rad6 and rad9 than in R A D + strains {23]. This occurs over a dose range which causes essentially no lethality in either the R A D ÷, rad6 or rad9 strains. It therefore seems that the products of the rad6 and rad9 genes are essential for mutations induced by a wide variety of agents and not merely restricted to UV mutagenesis. The other rad genes and the three rev genes examined seem to affect induced mutation with one agent and not another. For example, rad18 does not alter UV-induced reversion of cyc1-131 but reduces c y c l - 9 [16] reversion, reduces NQO-induced reversion of c y c l 131, does not affect EMS-induced reversion of cyc1-131, and increases HNO2induced reversion of cyc1-131. Although similarities in the mutagenic processes were revealed from the increased rates of mutation induced by both UV light and NQO in E. coli mutants that are unable to excise pyrimidine dimers, the parallelism between UV and NQO breaks d o w n when one examines NQO reversion of cyc1-131. Some similarities do exist since the excision-defective mutants radl, rad2, rad3 and rad4 are sensitive to the lethal effects of both UV and NQO and also show increased mutability induced by UV and NQO. RadlO shows enhanced UV and NQO reversion. The rad6 and rad18 mutants also respond to UV and NQO in the same direction, showing sensitivity to killing and reduced mutability. Rad15, rad16 and radl 7, on the other hand, do not affect UV mutability but tad15 and rad17 exhibit reduced NQO mutability while rad16 exhibits enhanced NQO mutability. The remaining strains showing a differential response to UV and NQO with the cyc1-131 site are the revl and rev2 strains. None of the rev mutants are sensitive to the lethal effect of NQO b u t NQO-reversion in all three rev strains is reduced 100-fold in revl and rev3 and about 10-fold in rev2. In addition to rad6 and rad9, rad15 and rad52 lowered EMS-induced revertibility. EMS-induced reversion of cyc1--131 in a rad52 strain was a b o u t 500 times less than in the R A D ÷ strain. The effect of rad15 was more pronounced at higher EMS doses than lower doses. The rad15 gene also reduces HNO~ reversion a b o u t 300--400 fold. However, the response to UV mutability is unaffected [25]. If one considers mutability results as a whole, it would seem that only rad6, rad9 and perhaps rad15 are involved in error-prone repair which affects all mutagens. Several points emerge from the results presented in Table II. ( 1 ) M a n y rad genes affect mutations induced by NQO b u t relatively few rad genes affect either EMS- or HNO2-induced mutation. (2) No correlation exists between sensitivity to lethality and ability to mutate; e.g. revl, rev2 and rev3 mutants are not sensitive to NQO but show very low NQO mutability. This suggests that the
246 R E V + gene product, in a R E V + cell, does not carry out much repair of NQO-induced damage; therefore, in rev mutants, no increase in sensitivity is observed.
Presumably, the small a m o u n t of repair of NQO damage carried out by the R E V ~ gene product is highly error-prone; therefore, in rev mutants, whatever
repair occurs is relatively error-free and NQO-induced reversion is greatly decreased. (3) All the repair mutants known in E. coli respond to the mutagenic effect of EMS and NTG. However, NQO, which mimics some effects of UV, and MMS, considered to mimic some effects of X-rays, do not show mutagenic effects in some repair mutants of E. coli. The E. coli data would therefore suggest that both EMS and NTG mutagenesis is independent of the known repair functions. However, in yeast, the situation is quite different. At least two ( R A D 6 and R A D 9 ) , and possibly three ( R A D 1 5 ) genes, virtually abolish mutations induced by a wide variety of chemical agents, including EMS and NTG. In E. coli, only five loci concerned with repair have been studied in any depth for their effect on mutation. These loci are the uvrA, uvrB, recA, lex and p o I A loci. The number of loci examined in yeast for their effect on mutation is much more extensive. Therefore, whether the apparent difference observed, namely t h a t EMS and NTG mutagenesis is independent of repair in E. coli but dependent on repair in Saccharornyces cerevisiae is due to the fact that very few genes involved in repair have been examined in E. coli for their effect on mutation compared to yeast, or whether the distinction is due to a fundamental difference between repair in procaryotic and eucaryotic organisms remains to be seen. (4) One important question we must be asking ourselves concerns the physiological role of these DNA repair genes in the cell. It is hard to imagine that these genes have evolved " s o l e l y " to repair DNA damage induced by radiation or chemicals. In E. coli, the p o l A gene, which codes for DNA polymerase I and is involved in repair replication, functions in normal DNA replication as well [15]. R e c A mutants are deficient in post-replication repair and also reduce the growth rate and viability of unirradiated cells [4], as do recB and recC mutants [4]. The recA function is required for viability in a p o I A - background [8], as is the uvrB ÷ function [35]. It has been postulated that the rec- strains do not repair single strand breaks or gaps in DNA produced during normal cell growth. Although it seems likely that many of the tad genes in yeast must also function even in unirradiated cells, their role still remains to be elucidated. Acknowledgement I wish to acknowledge Ms. Susan Mancuso for her technical assistance. The author was supported in part by a U.S. Public Health Service career developm e n t award (GM-00004) from the National Institutes of Health. This investigation is based on work supported by PHS Grant GM19261 and 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-3490-927.
247
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. L e h m a n n - B r a u n s , F o u r r a d i a t i o n sensitive m u t a n t s o f S a c c h a z o m y c e s . S u r v i v a l a f t e r U V - a n d X r a y - i r r a d i a t i o n as well as U V - i n d u c e d r e v e r s i o n r a t e s f r o m i s o l e u c i n e - v a l i n e d e p e n d e n c e t o 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., N . A . K h a n a n d R . H . H a y n e s , C o m m o n s t e p s in t h e r e p a i r o f a l k y l a t i o n a n d r a d i a t i o n d a m a g e in y e a s t , M o l e c . G e n . G e n e t . , 1 0 6 ( 1 9 7 0 ) 2 8 9 - - 2 9 5 . 3 B r i d g e s , B r y n A . , R a c h e l E. D e n n i s a n d R . J . M u n s o n , D i f f e r e n t i a l i n d u c t i o n a n d r e p a i r o f u l t r a v i o l e t d a m a g e l e a d i n g t o t r u e r e v e r s i o n s a n d e x t e r n a l s u p p r e s s o r m u t a t i o n s o f a n o c h r e c o d o n in E s c h e r i c h i a coli B / r W P 2 , G e n e t i c s , 5 7 ( 1 9 6 7 ) 8 9 7 - - 9 0 8 . 4 C a p a l d o , F . N . a n d S . D . B a r b o u r , T h e role o f t h e rec g e n e s in t h e v i a b i l i t y o f Escherichia coli K 1 2 , in P.C. H a n a w a l t a n d R . B . S e t l o w (eds.) 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 , 5 A , P l e n u m Press, N.Y., 1975, pp. 405--418. 5 C o x , B. 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 R e s . , 2 6 ( 1 9 7 4 ) 2 5 7 - - 2 6 4 . 6 C o x , B.S. a n d J . M . Par~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 . 7 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 o f 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 R e s . , 2 4 (1974) 281--292. 8 G r o s s , J., J . G r u n s t e i n a n d E.M. W i t k i n , I n v i a b i l i t y o f r e c A - d e r i v a t i v e s o f t h e D N A p o l y m e r a s e m u t a n t o f D e L u c i a a n d C a i r n s , J. Mol. Biol., 5 8 ( 1 9 7 1 ) 6 3 1 - - 6 3 4 . 9 H a n a w a l t , P.C., M o l e c u l a r m e c h a n i s m s i n v o l v e d in D N A r e p a i r , G e n e t i c s , 7 9 ( 1 9 7 5 ) 1 7 9 - - 1 9 7 . 1 0 H a y n e s , R d b e r t H . , T h e i n f l u e n c e o f r e p a i r p r o c e s s e s o n r a d i o b i o l o g i e a l survival c u r v e s , in T. A l p e r ( e d . ) Cell S u r v i v a l a f t e r L o w D o s e s o f R a d i a t i o n : T h e o r e t i c a l a n d C l i n i c a l I m p l i c a t i o n s , J o h n Wiley and Sons, Ltd; N.Y., 1975, pp. 197--208. 11 H o , K a r e n 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 a 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 R e s . , 3 0 ( 1 9 7 5 ) 3 2 7 - - 3 3 4 . 12 Howard-Flanders, Paul, DNA repair, Ann. Rev. Biochem., 37 (1968) 175--200. 13 Ishii, Y. a n d S. K o n d o , S p o n t a n e o u s a n d r a d i a t i o n - i n d u c e d d e l e t i o n m u t a t i o n s in Escherichia coli s t r a i n s w i t h d i f f e r e n t D N A r e p a i r c a p a c i t i e s , M u t a t i o n R e s . , 16 ( 1 9 7 2 ) 1 3 - - 2 5 . 1 4 K o n d o , S., H . I c h i k a w a , K . I w o a n d T. K a t o , B a s e - c h a n g e m u t a g e n e s i s a n d p h o p h a g e i n d u c t i o n in s t r a i n s o f E s c h e r i c h i a coli w i t h d i f f e r e n t D N A r e p a i r c a p a c i t i e s , G e n e t i c s , 6 6 ( 1 9 7 0 ) 1 8 7 - - 2 1 7 . 1 5 K o r n b e r g , A., D N A s y n t h e s i s , W . H . F r e e m a n a n d Co., 1 9 7 4 . 1 6 L a w r e n c e , C.W., J . W . S t e w a r t , F. S h e r m a n a n d R . C h r i s t e n s e n , S p e c i f i c i t y a n d f r e q u e n c y o f u l t r a v i o l e t - i n d u c e d r e v e r s i o n o f a n i s o - l - c y t o c h r o m e c o c h r e m u t a n t in r a d i a t i o n - s e n s i t i v e s t r a i n s o f y e a s t , J . Mol. Biol., 8 5 ( 1 9 7 4 ) 1 3 7 - - 1 6 2 . 1 7 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 8 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 light, G e n e t i c s , 6 8 ( 1 9 7 1 ) 21--33. 19 M i u r a , A . a n d J. T o m i z a w a , S t u d i e s o n 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 o f E. coli. III. P a r t i c i p a t i o n o f t h e rec s y s t e m in i n d u c t i o n o f m u t a t i o n b y u l t r a v i o l e t i r r a d i a t i o n , Mol. G e n . G e n e t . , 1 0 3 ( 1 9 6 8 ) 1 - - 1 0 . 2 0 M o u n t , D., K . B . L o w a n d S. E d m i s t o n , D o m i n a n t m u t a t i o n s ( l e x ) in E s c h e r i c h i a coli K - 1 2 w h i c h aff e c t r a d i a t i o n s e n s i t i v i t y a n d f r e q u e n c y o f u l t r a v i o l e t - l i g h t - i n d u c e d m u t a t i o n s , J. B a e t e r i o l . , 1 1 2 (1972) 886--893. 21 M o u s t a c c h i , E., C y t o p l a s m i c a n d n u c l e a r g e n e t i c e v e n t s i n d u c e d b y U V l i g h t in s t r a i n s o f S a c c h a r o . m y c e s cerevisiae w i t h d i f f e r e n t U V sensitivities, M u t a t i o n R e s . , 7 ( 1 9 6 9 ) 1 7 1 - - 1 8 5 . 2 2 M o u s t a c c h i , E., E v i d e n c e f o r n u c l e u s i n d e p e n d e n t s t e p s in c o n t r o l o f r e p a i r o f m i t o c h o n d r i a l d a m a g e . I. U V - i n d u c t i o n o f t h e c y t o p l a s m i c " p e t i t e " m u t a t i o n i n U V - s e n s i t i v e n u c l e a r m u t a n t s o f Saccharom y c e s cerevisiae, Mol. G e n . G e n e t . , 1 1 4 ( 1 9 7 2 ) 5 0 - - 5 8 . 2 3 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 , 7 8 (1974) 1101--1118. 2 4 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 5 P r a k a s h , L., E f f e c t o f g e n e s c o n t r o l l i n g r a d i a t i o n s e n s i t i v i t y o n 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 s in Sacc h a r o m y c e s cerevisiae, G e n e t i c s , 8 3 ( 1 9 7 6 ) 2 9 5 - - 3 0 1 . 26 P r a k a s h , L. a n d F . S h e r m a n , M u t a g e n i c s p e c i f i c i t y : r e v e r s i o n o f i s o - l - c y t o c h r o m e c m u t a n t s o f y e a s t , J. Mol. Biol., 7 9 ( 1 9 7 3 ) 6 5 - - 8 2 . 2 7 R a d m a n , M., S O S R e p a i r h y p o t h e s i s : p h e n o m e n o l o g y o f a n i n d u c i b l e D N A r e p a i r w h i c h is a c c o m p a n i e d b y m u t a g e n e s i s , in P.C. H a n a w a l t a n d R . B . S e t l o w (eds.) 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 , 5 A , P l e n u m Press, N . Y . , 1 9 7 5 , p p . 3 5 5 - - 3 6 7 . 2 8 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. 2 9 R e s n i c k , M., I n d u c t i o n o f m u t a t i o n s in S a c c h a r o m y c e s cerevisiae b y u l t r a v i o l e t l i g h t , M u t a t i o n R e s . , 7 (1969) 315--332.
248 3 0 R e s n i c k , M., T h e r e p a i r o f d o u b l e - s t r a n d b r e a k s in c h r o m o s o m a l D N A o f y e a s t , in P.C. H a n a w a l t a n d R . B . S e t l o w (eds.) 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 of D N A , 5B, P l e n u m Press, N . Y . , 1 9 7 5 , p p . 5 4 9 - 556. 3 1 R e s n i c k , M. a n d J a n e K . S e t l o w , R e p a i r of 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 . 3 2 R e y n o l d s , R i c h a r d , E x c i s i o n - r e p a i r in S a e c h a r o m y c e s cerevisiae, P h . D . thesis, U n i v e r s i t y o f T e n e s s e e , 1976. 3 3 R u p p , W . D . , C . E . Wilde, III, D . L . R e n o a n d P. H o w a r d - F l a n d e r s , E x c h a n g e s b e t w e e n D N A s t r a n d s in u l t r a v i o l e t - i r r a d i a t e d Escherichia coli, J. Mol. Biol., 6 1 ( 1 9 7 1 ) 2 5 - - 4 4 . 3 4 S e d g w i c k , S t e v e n G., I n d u c i b l e e r r o r - p r o n e r e p a i r in Eseherichia eoli, P r o c . N a t l . A c a d . Sci., 7 2 ( 1 9 7 5 ) 2753--2757. 3 5 S h i z u y a , H i r o a k i a n d D a n i e l D y k b u i z e n , C o n d i t i o n a l l e t h a l i t y o f d e l e t i o n s w h i c h i n c l u d e uvrB in s t r a i n s o f Escheriehia coli l a c k i n g d e o x y r i b o n u c l e i c a c i d p o l y m e r a s e I, J. B a c t e r i o l . , 1 1 2 ( 1 9 7 2 ) 6 7 6 - 681. 3 6 S m i t h , K e n d r i c C. a n d D i e t e r H . C . M e u n , R e p a i r o f r a d i a t i o n - i n d u c e d d a m a g e in Escherichia coli. I. E f f e c t o f rec m u t a t i o n s o n p o s t - r e p l i c a t i o n r e p a i r o f d a m a g e d u e t o u l t r a v i o l e t r a d i a t i o n , J. Mol. Biol., 51 ( 1 9 7 0 ) 4 5 9 - - 4 7 2 . 3 7 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 light, J. B a c t e r i o l , 9 4 ( 1 9 6 7 ) 5 7 1 - - 5 7 5 . 3 8 S t e w a r t , J . W . , F. S h e r m a n , N . A . S h i p m a n a n d M. J a c k s o n , I d e n t i f i c a t i o n a n d m u t a t i o n a l r e l o c a t i o n o f t h e A U G c o d o n i n i t i a t i n g t r a n s l a t i o n o f i s o - l - c y t o c h r o m e c in y e a s t , J. Biol. C h e m . , 2 4 6 ( 1 9 7 1 ) 7429--7445. 3 9 U n r a u , P., 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 irr 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 . 4 0 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. 41 Waters, R. and J.M. Parry, The response to chemical mutagens of the individual haploid and homoallelic d i p l o i d U V - s e n s i t i v e m u t a n t s o f t h e tad3 l o c u s o f S a c c h a r o m y c e s cerevisiae, Moi. G e n . G e n e t . , 124 (1973) 135--143. 4 2 W i t k i n , E., M u t a t i o n - p r o o f a n d m u t a t i o n - p r o n e m o d e s o f survival i n d e r i v a t i v e s o f Escherichia coli B d i f f e r i n g in s e n s i t i v i t y t o u l t r a v i o l e t light, B r o o k h a v e n S y r u p . in Biol., 2 0 ( 1 9 6 7 ) 1 7 - - 5 5 . 4 3 W i t k i n , E., T h e m u t a b i l i t y t o w a r d u l t r a v i o l e t l i g h t o f r e c o m b i n a t i o n - d e f i c i e n t s t r a i n s o f Escherichia coli, M u t a t i o n R e s . , 8 ( 1 9 6 9 ) 9 - - 1 4 . 4 4 W i t k i n , E., T h e r m a l e n h a n c e m e n t o f u l t r a v i o l e t m u t a b i l i t y i n a tif-1 u v r A d e r i v a t i v e o f Escherichia coli B / r : e v i d e n c e t h a t u l t r a v i o l e t m u t a g e n e s i s d e p e n d s o n a n i n d u c i b l e f u n c t i o n , P r o c . N a t l . A c a d . Sci., 71 ( 1 9 7 4 ) 1 9 3 0 - - 1 9 3 4 . 4 5 W i t k i n , E., E l e v a t e d m u t a b i l i t y o f p o l A and u v r A p o l A d e r i v a t i v e s o f E s c h e r i c h i a coli B / r a t s u b l e t h a l d o s e s o f u l t r a v i o l e t light: e v i d e n c e f o r a n i n d u c i b l e e r r o r - p r o n e r e p a i r s y s t e m ( " S O S r e p a i r " ) a n d its a n o m a l o u s e x p r e s s i o n in t h e s e s t r a i n s , G e n e t i c s , 7 9 ( 1 9 7 5 ) 1 9 9 - - 2 1 3 . 4 6 w i t k i n , E. a n d D o n n a L. G e o r g e , U l t r a v i o l e t m u t a g e n e s i s in p o l A and u v r A p o l A d e r i v a t i v e s o f Escherichia coli B / r : e v i d e n c e f o r a n i n d u c i b l e e r r o r * p r o n e r e p a i r s y s t e m , G e n e t i c s , 7 3 ( 1 9 7 3 ) 9 1 - - 1 0 8 . 4 7 Y o u n g s , D . A . a n d K . C . S m i t h , E v i d e n c e f o r t h e c o n t r o l b y e x r A and p o l A g e n e s o f t w o b r a n c h e s o f t h e uvr g e n e - d e p e n d e n t e x c i s i o n r e p a i r p a t h w a y i n E s c h e r i e h i a coli K - 1 2 , J. B a e t e r i o l . , 1 1 6 ( 1 9 7 3 ) 175--182. 4 8 Z a k h a r o v , t . A . , T . N . K o z i n a a n d I.V. F e d o r o v a , E f f e t s d e s m u t a t i o n s vers las sensibilit~ r a y o n n e m e n t u l t r a v i o l e t c h e z la l e v u r e , M u t a t i o n R e s . , 9 ( 1 9 7 0 ) 3 1 - - 3 9 . 4 9 Z i m m e r m a n n , F . K . , S e n s i t i v i t y t o m e t h y l m e t h a n e - s u l f o n a t e a n d n i t r o u s a c i d o f u l t r a v i o l e t light-sensitive m u t a n t s i n S a c c h a r o r n y c e s cerevisiae, M o l e c . G e n . G e n e t . , 1 0 2 ( 1 9 6 8 ) 2 4 7 - - 2 5 6 .
Mutation Research, 41 ( 1 9 7 6 ) 2 4 1 - - 2 4 8 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
THE R E L A T I O N BETWEEN REPAIR OF DNA AND RADIATION AND CHEMICAL MUTAGENESIS IN S A C C H A R O M Y C E S C E R E V I S I A E *
LOUISE PRAKASH
Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 (U.S.A.) ( R e c e i v e d M a r c h 3rd, 1 9 7 6 ) ( R e v i s i o n received J u n e 9th, 1 9 7 6 ) ( A c c e p t e d J u n e 23rd, 1 9 7 6 )
Summary The effect of various genes involved in DNA repair functions on radiation and chemical mutagenesis in E s c h e r i c h i a c o l i is discussed and compared to similar studies done in yeast. Results of the effect of various genes conferring radiation-sensitivity on mutation induction in yeast are presented and related to current ideas of mutagenesis.
Procaryotic and eucaryotic organisms possess mechanisms which enable them to repair damage induced in their DNA by a wide variety of chemical and physical agents. The manner in which DNA damage is repaired influences both the survival of the organism as well as the mutagenic effectiveness of the agent. The best studied repair processes in both eucaryotes and procaryotes are those which operate on ultraviolet light (UV)-induced damage of DNA. Strains of E s c h e r i c h i a c o l i which have normal radiation-sensitivity possess, in addition to photoreactivation, two pathways for the repair of pyrimidine dimers induced by UV [9,10,12]. The predominant one is excision-repair, in which pyrimidine dimers are enzymatically excised from the DNA. The gap generated by this process is sealed by repair replication in the region of the gap, using the opposite strand as a template [9]. The dimers which escape excision-repair are repaired during and after DNA replication by processes called post-replication repair. One of these processes is recombinational repair [33]; another is error-prone repair and generates mutations [45]. Both the r e c A ÷ a n d l e x + gene functions are necessary for both post-replicational repair and UV-induced mutations. In l e x - strains, no UV-induced mutations are produced [3,20,42] even though * By acceptance of this article, t h e p u b l i s h e r a n d / o r r e c i p i e n t a c k n o w l e d g e s the U.S. Government's right to retain a nonexclusive, royalty-free license in and to any c o p y r i g h t c o v e r i n g this paper.
242 some post-replication repair can be demonstrated [47], suggesting that the lex ~ gene function is involved in error-prone repair. R e c A - strains, which are also refractory to UV-induced mutation [14,19,43], do not undergo post-replication repair [ 41], suggesting that post-replication repair generates mutations. It is now believed that UV mutability results from the induction of errorprone repair of excision gaps or post-replication gaps and depends on at least the recA ÷ and lex + functions [27,44,46]. The protein synthesis inhibitor chloramphenicol has been shown to inhibit some a m o u n t of post-replication repair in excision-defective E. coli carrying a u v r A mutation but no inhibition is detected if a 20-min growth period occurs subsequent to the irradiation and prior to treatment with chloramphenicol [33]. According to current ideas, the presence of pyrimidine dimers in DNA acts as an inducer of the error-prone mode of post-replication repair, which results in enhanced mutation frequency observed in excision-defective strains at UV doses showing no demonstrable effect in excision-proficient strains. In uvrA strains which are deficient in excision, a large number of dimers remain in the DNA so that even at low UV doses, error-prone repair is induced. P o l A mutants having very low DNA polymerase I activity also exhibit enhanced mutation frequencies at doses below 5 J/m 2 [45,46.] The p o I A - single m u t a n t and u v r A - p o l A - double m u t a n t presumably induced error-prone repair at low doses because of their lowered rate of repair. Most of the work on m u t a t i o n induction in repair-deficient mutants of E. coli has dealt with radiation-induced mutations and the models relating repair to mutation have, of necessity, dealt primarily with data obtained from radiation mutagenesis. The few chemical mutagens examined have included mainly those whose effects parallel UV and X-ray. Damage induced by 4-nitroquinoline-l-oxide (NQO), a p o t e n t carcinogen, seems to be subject, in part at least, to the same excision-repair system operating on dimers. Uvr- strains are sensitive to the lethal effect of NQO and also show enhanced susceptibility to the mutagenic effects of NQO [13,14]. R e c A - strains, which presumably are involved in some step of error-prone repair, are immutable to UV, NQO, X-ray, and the monofunctional alkylating agent methyl methanesulfonate (MMS) [13, 14]. However, mutability induced by ethyl methanesulfonate (EMS), or nitrosoguanidine (NTG) is normal [13,14]. Therefore, the recA function, although it is required for radiation mutagenesis, is apparently not required for mutation induction by some chemicals. While the study of radiation mutagenesis and its genetic control as revealed by the elegant studies in E. coli has elucidated certain aspects of the role of repair in mutation, there may well be other u n k n o w n important processes. If we now examine similar studies in the eucaryotic organism, Saccharom y c e s cerevisiae, the situation becomes more complex. First of all, 32 distinct genetic loci have been identified so far which confer radiation-sensitivity. The mutations radl through rad22 were isolated as genes conferring sensitivity primarily to UV [6,28,37], while the mutations rad50 through r a d 5 7 confer sensitivity primarily to X-ray [7,34]. Cross-sensitivities to UV and X-ray exist for m u t a n t s within each of the t~vo groups. In addition, the mutations r e v l , rev2 (= rad5) and rev3 were isolated for reducing the UV-induced mutability of the highly revertible arg4-17 site [18]. Table I lists the various radiation sensitive
243 TABLE
I
PROPERTIES
OF RADIATION-SENSITIVE
MUTANTS
OF SACCHAROMYCES
CEREVISIAE
M M S - s e n s i t i v i t y o f r a d S O t h r o u g h t a d 5 7 m u t a n t s w a s d e t e r m i n e d b y w h e t h e r or n o t t h e strain g r e w o n 1 % y e a s t e x t r a c t , 2% p e p t o n e , 2% g l u c o s e s o l i d m e d i a c o n t a i n i n g 0 . 0 3 5 % M M S ( P r a k a s h , u n p u b l i s h e d results). Mutant
Sensitivity o f UV [6]
NQO [25]
~'-ray [ 6 ] or X-ray
MMS [49]
HNO 2 [49]
EMS [25]
tad1 rad2
++ +++
rad3 rad4 tad6 tad 7 rad8 tad9 radlO tad11 rad13 radl4 rod15 tad16 rad l 7 tad18 tad19 tad20 tad21
++++ +++ +++ ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ [28] + ++ +
+++ ++ ++ +++ +++
0 0 0 0 ++ 0 + 0 ++ 0 0 0 + 0 0 ++ [28] 0 0 0
0 0 0 [41] 0 +++ [25] 0 +]-++(0) [25] +/-+/-+[-+1-0 0 +/-++ [2] 0 + ++
++ [25] +]-++ [25,41] +]-++ [25] 0 +l-++(0) [25] +l-0 ++ 0 0(++) [25] 0 [25]
0 0 0 [25,41] 0 ++
revl rev2=rad5 rev3
+ [18] ++ +
0
+
tad50 tad51 tad52 tad53 tad54 tad55 rad56 rad5 7
+ [7] + 0 + 0 0 0 0
+++ [7] +++ +++ +++ +++ ++ + ++
++ ++ ++ [2] ++ ++ ++ ++ ++
0 +++
0 ++ 0 0 ++
0 0 0
0
+ 0 ++ ++ 0 [25] 0 [25] 0 [25]
+
0
0 + 0 0 0
Effect on sporulation [6]
normal normal normal normal eliminated normal normal normal normal normal normal normal normal normal normal normal reduced reduced reduced
0 0 0
++
normal [7] reduced reduced normal normal reduced normal normal
0, S e n s i t i v i t y l i k e R A D + ( o r R E V +) strain + --* + + + + , Increasing d e g r e e s o f sensitivity + / - - , s o m e w h a t m o r e s e n s i t i v e than R A D + ; m a y o r m a y n o t be significantly d i f f e r e n t f r o m R A D +
mutants of yeast and some of their properties. The specific lesion has been identified in only five mutants; radl, rad2, rad3 and rad4 are excision-defective [ 2 4 , 3 1 , 3 2 , 3 9 , 4 0 ] . Like their excision-defective counter-parts in E. coli, these 4 mutants showed enhanced UV mutability [1,17,21,22,29,48] and enhanced NQO mutability [25]. Recently, it has been shown that although R A D ÷ strains are able to repair X-ray induced double-strand breaks, rad52 mutants are deficient in this capacity [11,30]. In order to examine the effect of the various rad genes on mutations induced by radiation and chemical agents, a series of diploid strains homozygous for a particular rad gene was constructed in collaboration with Dr. Christopher Lawrence. The strains were also homozygous for cyc1-131, a chain initiation mutant in the structural gene determining iso-l-cytochrome c [38]. This mutant
244
reverts to give the normal protein by a G : C to A : T transition [26,38] and it was the site used to measure reversion. The effect of various tad genes on the mutation process could then be determined by comparing reversion frequencies in R A D ÷ and rad strains. The effect of UV on reversion of cyc1-131 was examined in these strains by Lawrence and Christensen [17] while Prakash [25] measured reversion frequencies using EMS, NQO and HNO2. The tad strains showing reduced UV-induced mutations do not necessarily show reduced mutations induced by chemicals. In general, even when comparing two mutagens expected to exhibit responses, such as UV and NQO, such a parallel response was not observed consistently. The results of all these experiments are summarized qualitatively in Table II. Although rad9, rad18, revl and rev2 did not lower the UV-induced reversion of cyc1-131, they do lower reversion of other cycl alleles and other genes [ 1 8 , 2 1 , 2 9 ] . Rad8 lowered reversion of c y c l - 9 whereas rad15-1 had little effect. It was concluded from these studies that at least seven genes, namely, revl, rev2, rev3, rad6, rad8, rad9, and tad18 are required for normal UV mutagenesis to occur [17]. In addition, double mutant survival data indicate that at least revl, rev3, tad6, rad9, and rad18 are in the same error-prone pathway [17]. The products of the rad6 and rev3 genes were suggested to be required for the production of UV-induced mutations throughout the genome whereas the effects of the other genes did not extend to all loci tested. A1-
TABLE
II
INDUCED CERE
cyc1--131
V1SIA
--+ C Y C 1 R E V E R S I O N
IN RADIATION-SENSITIVE
DIPLOID
SACCHAROMYCES
E
UV results were obtained
from Lawrence
Strains
UV
NQO
rad l tad2 tad3 tad4 tad6
+ +
and Christensen
[17].
EMS
HNO 2
+
0
0
+
0
+
+
0
+
+
0
.
.
tad8
.
0
.
(--)
tad9
0(--)
--
rad l O
+
+
tad13 tad 14 tad15 rad16 rad l 7 rad18
0
--
--
0
0
0
0
--
--
--
0
+
0
0
0
--
0
0(--)
--
0
+
tad50 tad52
0 --
0
rev l rev2 rev3
0(--)
--
0
0
0(--)
--
0
0
--
--
0
0
0
O, R e v e r s i o n +, R e v e r s i o n --, Reversion
f r e q u e n c y l i k e R A D +. g r e a t e r t h a n R A D ÷. reduced compared to RAD+ Blank indicates strain was not tested entry in parentheses loci.
refers to results for other cycl
alleles or other
245 though there appears to be one error-prone pathway for UV mutagenesis in yeast, three pathways are thought to be involved in repair of UV damage [5]. However, when looking at results obtained with chemical mutagens, two genes, rad6 and rad9, greatly reduced reversion of cyc1-131. This was true not only for NQO, EMS and HNO2, b u t also for diethyl sulfate (DES), MMS, dimethyl sulfate (DMS), NTG, nitrogen mustard (HN2), fl-propiolactone, tritiated uridine decay and ionizing radiation [23]. At low doses, HNO2 and nitrosoimidazolidone (NIL) revert cyc1-131 in rad6 and rad9 strains to the same extent as in R A D ÷ strains b u t at high doses, reversion frequencies obtained with both HNO2 and NIL are much lower in rad6 and rad9 than in R A D + strains {23]. This occurs over a dose range which causes essentially no lethality in either the R A D ÷, rad6 or rad9 strains. It therefore seems that the products of the rad6 and rad9 genes are essential for mutations induced by a wide variety of agents and not merely restricted to UV mutagenesis. The other rad genes and the three rev genes examined seem to affect induced mutation with one agent and not another. For example, rad18 does not alter UV-induced reversion of cyc1-131 but reduces c y c l - 9 [16] reversion, reduces NQO-induced reversion of c y c l 131, does not affect EMS-induced reversion of cyc1-131, and increases HNO2induced reversion of cyc1-131. Although similarities in the mutagenic processes were revealed from the increased rates of mutation induced by both UV light and NQO in E. coli mutants that are unable to excise pyrimidine dimers, the parallelism between UV and NQO breaks d o w n when one examines NQO reversion of cyc1-131. Some similarities do exist since the excision-defective mutants radl, rad2, rad3 and rad4 are sensitive to the lethal effects of both UV and NQO and also show increased mutability induced by UV and NQO. RadlO shows enhanced UV and NQO reversion. The rad6 and rad18 mutants also respond to UV and NQO in the same direction, showing sensitivity to killing and reduced mutability. Rad15, rad16 and radl 7, on the other hand, do not affect UV mutability but tad15 and rad17 exhibit reduced NQO mutability while rad16 exhibits enhanced NQO mutability. The remaining strains showing a differential response to UV and NQO with the cyc1-131 site are the revl and rev2 strains. None of the rev mutants are sensitive to the lethal effect of NQO b u t NQO-reversion in all three rev strains is reduced 100-fold in revl and rev3 and about 10-fold in rev2. In addition to rad6 and rad9, rad15 and rad52 lowered EMS-induced revertibility. EMS-induced reversion of cyc1--131 in a rad52 strain was a b o u t 500 times less than in the R A D ÷ strain. The effect of rad15 was more pronounced at higher EMS doses than lower doses. The rad15 gene also reduces HNO~ reversion a b o u t 300--400 fold. However, the response to UV mutability is unaffected [25]. If one considers mutability results as a whole, it would seem that only rad6, rad9 and perhaps rad15 are involved in error-prone repair which affects all mutagens. Several points emerge from the results presented in Table II. ( 1 ) M a n y rad genes affect mutations induced by NQO b u t relatively few rad genes affect either EMS- or HNO2-induced mutation. (2) No correlation exists between sensitivity to lethality and ability to mutate; e.g. revl, rev2 and rev3 mutants are not sensitive to NQO but show very low NQO mutability. This suggests that the
246 R E V + gene product, in a R E V + cell, does not carry out much repair of NQO-induced damage; therefore, in rev mutants, no increase in sensitivity is observed.
Presumably, the small a m o u n t of repair of NQO damage carried out by the R E V ~ gene product is highly error-prone; therefore, in rev mutants, whatever
repair occurs is relatively error-free and NQO-induced reversion is greatly decreased. (3) All the repair mutants known in E. coli respond to the mutagenic effect of EMS and NTG. However, NQO, which mimics some effects of UV, and MMS, considered to mimic some effects of X-rays, do not show mutagenic effects in some repair mutants of E. coli. The E. coli data would therefore suggest that both EMS and NTG mutagenesis is independent of the known repair functions. However, in yeast, the situation is quite different. At least two ( R A D 6 and R A D 9 ) , and possibly three ( R A D 1 5 ) genes, virtually abolish mutations induced by a wide variety of chemical agents, including EMS and NTG. In E. coli, only five loci concerned with repair have been studied in any depth for their effect on mutation. These loci are the uvrA, uvrB, recA, lex and p o I A loci. The number of loci examined in yeast for their effect on mutation is much more extensive. Therefore, whether the apparent difference observed, namely t h a t EMS and NTG mutagenesis is independent of repair in E. coli but dependent on repair in Saccharornyces cerevisiae is due to the fact that very few genes involved in repair have been examined in E. coli for their effect on mutation compared to yeast, or whether the distinction is due to a fundamental difference between repair in procaryotic and eucaryotic organisms remains to be seen. (4) One important question we must be asking ourselves concerns the physiological role of these DNA repair genes in the cell. It is hard to imagine that these genes have evolved " s o l e l y " to repair DNA damage induced by radiation or chemicals. In E. coli, the p o l A gene, which codes for DNA polymerase I and is involved in repair replication, functions in normal DNA replication as well [15]. R e c A mutants are deficient in post-replication repair and also reduce the growth rate and viability of unirradiated cells [4], as do recB and recC mutants [4]. The recA function is required for viability in a p o I A - background [8], as is the uvrB ÷ function [35]. It has been postulated that the rec- strains do not repair single strand breaks or gaps in DNA produced during normal cell growth. Although it seems likely that many of the tad genes in yeast must also function even in unirradiated cells, their role still remains to be elucidated. Acknowledgement I wish to acknowledge Ms. Susan Mancuso for her technical assistance. The author was supported in part by a U.S. Public Health Service career developm e n t award (GM-00004) from the National Institutes of Health. This investigation is based on work supported by PHS Grant GM19261 and 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-3490-927.
247
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. L e h m a n n - B r a u n s , F o u r r a d i a t i o n sensitive m u t a n t s o f S a c c h a z o m y c e s . S u r v i v a l a f t e r U V - a n d X r a y - i r r a d i a t i o n as well as U V - i n d u c e d r e v e r s i o n r a t e s f r o m i s o l e u c i n e - v a l i n e d e p e n d e n c e t o 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., N . A . K h a n a n d R . H . H a y n e s , C o m m o n s t e p s in t h e r e p a i r o f a l k y l a t i o n a n d r a d i a t i o n d a m a g e in y e a s t , M o l e c . G e n . G e n e t . , 1 0 6 ( 1 9 7 0 ) 2 8 9 - - 2 9 5 . 3 B r i d g e s , B r y n A . , R a c h e l E. D e n n i s a n d R . J . M u n s o n , D i f f e r e n t i a l i n d u c t i o n a n d r e p a i r o f u l t r a v i o l e t d a m a g e l e a d i n g t o t r u e r e v e r s i o n s a n d e x t e r n a l s u p p r e s s o r m u t a t i o n s o f a n o c h r e c o d o n in E s c h e r i c h i a coli B / r W P 2 , G e n e t i c s , 5 7 ( 1 9 6 7 ) 8 9 7 - - 9 0 8 . 4 C a p a l d o , F . N . a n d S . D . B a r b o u r , T h e role o f t h e rec g e n e s in t h e v i a b i l i t y o f Escherichia coli K 1 2 , in P.C. H a n a w a l t a n d R . B . S e t l o w (eds.) 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 , 5 A , P l e n u m Press, N.Y., 1975, pp. 405--418. 5 C o x , B. 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 R e s . , 2 6 ( 1 9 7 4 ) 2 5 7 - - 2 6 4 . 6 C o x , B.S. a n d J . M . Par~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 . 7 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 o f 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 R e s . , 2 4 (1974) 281--292. 8 G r o s s , J., J . G r u n s t e i n a n d E.M. W i t k i n , I n v i a b i l i t y o f r e c A - d e r i v a t i v e s o f t h e D N A p o l y m e r a s e m u t a n t o f D e L u c i a a n d C a i r n s , J. Mol. Biol., 5 8 ( 1 9 7 1 ) 6 3 1 - - 6 3 4 . 9 H a n a w a l t , P.C., M o l e c u l a r m e c h a n i s m s i n v o l v e d in D N A r e p a i r , G e n e t i c s , 7 9 ( 1 9 7 5 ) 1 7 9 - - 1 9 7 . 1 0 H a y n e s , R d b e r t H . , T h e i n f l u e n c e o f r e p a i r p r o c e s s e s o n r a d i o b i o l o g i e a l survival c u r v e s , in T. A l p e r ( e d . ) Cell S u r v i v a l a f t e r L o w D o s e s o f R a d i a t i o n : T h e o r e t i c a l a n d C l i n i c a l I m p l i c a t i o n s , J o h n Wiley and Sons, Ltd; N.Y., 1975, pp. 197--208. 11 H o , K a r e n 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 a 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 R e s . , 3 0 ( 1 9 7 5 ) 3 2 7 - - 3 3 4 . 12 Howard-Flanders, Paul, DNA repair, Ann. Rev. Biochem., 37 (1968) 175--200. 13 Ishii, Y. a n d S. K o n d o , S p o n t a n e o u s a n d r a d i a t i o n - i n d u c e d d e l e t i o n m u t a t i o n s in Escherichia coli s t r a i n s w i t h d i f f e r e n t D N A r e p a i r c a p a c i t i e s , M u t a t i o n R e s . , 16 ( 1 9 7 2 ) 1 3 - - 2 5 . 1 4 K o n d o , S., H . I c h i k a w a , K . I w o a n d T. K a t o , B a s e - c h a n g e m u t a g e n e s i s a n d p h o p h a g e i n d u c t i o n in s t r a i n s o f E s c h e r i c h i a coli w i t h d i f f e r e n t D N A r e p a i r c a p a c i t i e s , G e n e t i c s , 6 6 ( 1 9 7 0 ) 1 8 7 - - 2 1 7 . 1 5 K o r n b e r g , A., D N A s y n t h e s i s , W . H . F r e e m a n a n d Co., 1 9 7 4 . 1 6 L a w r e n c e , C.W., J . W . S t e w a r t , F. S h e r m a n a n d R . C h r i s t e n s e n , S p e c i f i c i t y a n d f r e q u e n c y o f u l t r a v i o l e t - i n d u c e d r e v e r s i o n o f a n i s o - l - c y t o c h r o m e c o c h r e m u t a n t in r a d i a t i o n - s e n s i t i v e s t r a i n s o f y e a s t , J . Mol. Biol., 8 5 ( 1 9 7 4 ) 1 3 7 - - 1 6 2 . 1 7 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 8 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 light, G e n e t i c s , 6 8 ( 1 9 7 1 ) 21--33. 19 M i u r a , A . a n d J. T o m i z a w a , S t u d i e s o n 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 o f E. coli. III. P a r t i c i p a t i o n o f t h e rec s y s t e m in i n d u c t i o n o f m u t a t i o n b y u l t r a v i o l e t i r r a d i a t i o n , Mol. G e n . G e n e t . , 1 0 3 ( 1 9 6 8 ) 1 - - 1 0 . 2 0 M o u n t , D., K . B . L o w a n d S. E d m i s t o n , D o m i n a n t m u t a t i o n s ( l e x ) in E s c h e r i c h i a coli K - 1 2 w h i c h aff e c t r a d i a t i o n s e n s i t i v i t y a n d f r e q u e n c y o f u l t r a v i o l e t - l i g h t - i n d u c e d m u t a t i o n s , J. B a e t e r i o l . , 1 1 2 (1972) 886--893. 21 M o u s t a c c h i , E., C y t o p l a s m i c a n d n u c l e a r g e n e t i c e v e n t s i n d u c e d b y U V l i g h t in s t r a i n s o f S a c c h a r o . m y c e s cerevisiae w i t h d i f f e r e n t U V sensitivities, M u t a t i o n R e s . , 7 ( 1 9 6 9 ) 1 7 1 - - 1 8 5 . 2 2 M o u s t a c c h i , E., E v i d e n c e f o r n u c l e u s i n d e p e n d e n t s t e p s in c o n t r o l o f r e p a i r o f m i t o c h o n d r i a l d a m a g e . I. U V - i n d u c t i o n o f t h e c y t o p l a s m i c " p e t i t e " m u t a t i o n i n U V - s e n s i t i v e n u c l e a r m u t a n t s o f Saccharom y c e s cerevisiae, Mol. G e n . G e n e t . , 1 1 4 ( 1 9 7 2 ) 5 0 - - 5 8 . 2 3 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 , 7 8 (1974) 1101--1118. 2 4 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 5 P r a k a s h , L., E f f e c t o f g e n e s c o n t r o l l i n g r a d i a t i o n s e n s i t i v i t y o n 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 s in Sacc h a r o m y c e s cerevisiae, G e n e t i c s , 8 3 ( 1 9 7 6 ) 2 9 5 - - 3 0 1 . 26 P r a k a s h , L. a n d F . S h e r m a n , M u t a g e n i c s p e c i f i c i t y : r e v e r s i o n o f i s o - l - c y t o c h r o m e c m u t a n t s o f y e a s t , J. Mol. Biol., 7 9 ( 1 9 7 3 ) 6 5 - - 8 2 . 2 7 R a d m a n , M., S O S R e p a i r h y p o t h e s i s : p h e n o m e n o l o g y o f a n i n d u c i b l e D N A r e p a i r w h i c h is a c c o m p a n i e d b y m u t a g e n e s i s , in P.C. H a n a w a l t a n d R . B . S e t l o w (eds.) 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 , 5 A , P l e n u m Press, N . Y . , 1 9 7 5 , p p . 3 5 5 - - 3 6 7 . 2 8 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. 2 9 R e s n i c k , M., I n d u c t i o n o f m u t a t i o n s in S a c c h a r o m y c e s cerevisiae b y u l t r a v i o l e t l i g h t , M u t a t i o n R e s . , 7 (1969) 315--332.
248 3 0 R e s n i c k , M., T h e r e p a i r o f d o u b l e - s t r a n d b r e a k s in c h r o m o s o m a l D N A o f y e a s t , in P.C. H a n a w a l t a n d R . B . S e t l o w (eds.) 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 of D N A , 5B, P l e n u m Press, N . Y . , 1 9 7 5 , p p . 5 4 9 - 556. 3 1 R e s n i c k , M. a n d J a n e K . S e t l o w , R e p a i r of 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 . 3 2 R e y n o l d s , R i c h a r d , E x c i s i o n - r e p a i r in S a e c h a r o m y c e s cerevisiae, P h . D . thesis, U n i v e r s i t y o f T e n e s s e e , 1976. 3 3 R u p p , W . D . , C . E . Wilde, III, D . L . R e n o a n d P. H o w a r d - F l a n d e r s , E x c h a n g e s b e t w e e n D N A s t r a n d s in u l t r a v i o l e t - i r r a d i a t e d Escherichia coli, J. Mol. Biol., 6 1 ( 1 9 7 1 ) 2 5 - - 4 4 . 3 4 S e d g w i c k , S t e v e n G., I n d u c i b l e e r r o r - p r o n e r e p a i r in Eseherichia eoli, P r o c . N a t l . A c a d . Sci., 7 2 ( 1 9 7 5 ) 2753--2757. 3 5 S h i z u y a , H i r o a k i a n d D a n i e l D y k b u i z e n , C o n d i t i o n a l l e t h a l i t y o f d e l e t i o n s w h i c h i n c l u d e uvrB in s t r a i n s o f Escheriehia coli l a c k i n g d e o x y r i b o n u c l e i c a c i d p o l y m e r a s e I, J. B a c t e r i o l . , 1 1 2 ( 1 9 7 2 ) 6 7 6 - 681. 3 6 S m i t h , K e n d r i c C. a n d D i e t e r H . C . M e u n , R e p a i r o f r a d i a t i o n - i n d u c e d d a m a g e in Escherichia coli. I. E f f e c t o f rec m u t a t i o n s o n p o s t - r e p l i c a t i o n r e p a i r o f d a m a g e d u e t o u l t r a v i o l e t r a d i a t i o n , J. Mol. Biol., 51 ( 1 9 7 0 ) 4 5 9 - - 4 7 2 . 3 7 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 light, J. B a c t e r i o l , 9 4 ( 1 9 6 7 ) 5 7 1 - - 5 7 5 . 3 8 S t e w a r t , J . W . , F. S h e r m a n , N . A . S h i p m a n a n d M. J a c k s o n , I d e n t i f i c a t i o n a n d m u t a t i o n a l r e l o c a t i o n o f t h e A U G c o d o n i n i t i a t i n g t r a n s l a t i o n o f i s o - l - c y t o c h r o m e c in y e a s t , J. Biol. C h e m . , 2 4 6 ( 1 9 7 1 ) 7429--7445. 3 9 U n r a u , P., 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 irr 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 . 4 0 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. 41 Waters, R. and J.M. Parry, The response to chemical mutagens of the individual haploid and homoallelic d i p l o i d U V - s e n s i t i v e m u t a n t s o f t h e tad3 l o c u s o f S a c c h a r o m y c e s cerevisiae, Moi. G e n . G e n e t . , 124 (1973) 135--143. 4 2 W i t k i n , E., M u t a t i o n - p r o o f a n d m u t a t i o n - p r o n e m o d e s o f survival i n d e r i v a t i v e s o f Escherichia coli B d i f f e r i n g in s e n s i t i v i t y t o u l t r a v i o l e t light, B r o o k h a v e n S y r u p . in Biol., 2 0 ( 1 9 6 7 ) 1 7 - - 5 5 . 4 3 W i t k i n , E., T h e m u t a b i l i t y t o w a r d u l t r a v i o l e t l i g h t o f r e c o m b i n a t i o n - d e f i c i e n t s t r a i n s o f Escherichia coli, M u t a t i o n R e s . , 8 ( 1 9 6 9 ) 9 - - 1 4 . 4 4 W i t k i n , E., T h e r m a l e n h a n c e m e n t o f u l t r a v i o l e t m u t a b i l i t y i n a tif-1 u v r A d e r i v a t i v e o f Escherichia coli B / r : e v i d e n c e t h a t u l t r a v i o l e t m u t a g e n e s i s d e p e n d s o n a n i n d u c i b l e f u n c t i o n , P r o c . N a t l . A c a d . Sci., 71 ( 1 9 7 4 ) 1 9 3 0 - - 1 9 3 4 . 4 5 W i t k i n , E., E l e v a t e d m u t a b i l i t y o f p o l A and u v r A p o l A d e r i v a t i v e s o f E s c h e r i c h i a coli B / r a t s u b l e t h a l d o s e s o f u l t r a v i o l e t light: e v i d e n c e f o r a n i n d u c i b l e e r r o r - p r o n e r e p a i r s y s t e m ( " S O S r e p a i r " ) a n d its a n o m a l o u s e x p r e s s i o n in t h e s e s t r a i n s , G e n e t i c s , 7 9 ( 1 9 7 5 ) 1 9 9 - - 2 1 3 . 4 6 w i t k i n , E. a n d D o n n a L. G e o r g e , U l t r a v i o l e t m u t a g e n e s i s in p o l A and u v r A p o l A d e r i v a t i v e s o f Escherichia coli B / r : e v i d e n c e f o r a n i n d u c i b l e e r r o r * p r o n e r e p a i r s y s t e m , G e n e t i c s , 7 3 ( 1 9 7 3 ) 9 1 - - 1 0 8 . 4 7 Y o u n g s , D . A . a n d K . C . S m i t h , E v i d e n c e f o r t h e c o n t r o l b y e x r A and p o l A g e n e s o f t w o b r a n c h e s o f t h e uvr g e n e - d e p e n d e n t e x c i s i o n r e p a i r p a t h w a y i n E s c h e r i e h i a coli K - 1 2 , J. B a e t e r i o l . , 1 1 6 ( 1 9 7 3 ) 175--182. 4 8 Z a k h a r o v , t . A . , T . N . K o z i n a a n d I.V. F e d o r o v a , E f f e t s d e s m u t a t i o n s vers las sensibilit~ r a y o n n e m e n t u l t r a v i o l e t c h e z la l e v u r e , M u t a t i o n R e s . , 9 ( 1 9 7 0 ) 3 1 - - 3 9 . 4 9 Z i m m e r m a n n , F . K . , S e n s i t i v i t y t o m e t h y l m e t h a n e - s u l f o n a t e a n d n i t r o u s a c i d o f u l t r a v i o l e t light-sensitive m u t a n t s i n S a c c h a r o r n y c e s cerevisiae, M o l e c . G e n . G e n e t . , 1 0 2 ( 1 9 6 8 ) 2 4 7 - - 2 5 6 .
