25
Mutation Research, 52 (1978) 25--35
© Elsevier/North-Holland Biomedical Press
MUTAGENIC INTERACTION BETWEEN NEAR-(365 nm) AND FAR-(254 nm)ULTRAVIOLET RADIATION IN REPAIR-PROFICIENT AND EXCISION-DEFICIENT STRAINS OF E s c b e r i c b i a c o l i
REX M. TYRRELL Instituto de Biofisica, Centro de Ci~ncias da Safide (Bloco G), Universidade Federal do Rio de Janeiro (Brazil)
(Received 6 December 1977) (Revision received 18 April 1978) (Accepted 15 May 1978) Summary The mutational interaction between radiation at 365 and 254 nm was studied in various strains of E. c o l i by a m u t a n t assay based on reversion to amino-acid independence in full nutrient conditions. In the two repair-proficient strains (K12 AB 1157 and B/r), pre-treatment with radiation at 365 nm strongly suppressed the induction of mutations by far-UV, a p h e n o m e n o n accompanied by a strong positive lethal interaction. The frequency of mutations induced by far-UV progressively declined with increasing dose of near-UV. FarUV-induced mutagenesis to T5 resistance was almost unaltered by pre-treatm e n t with near-UV. In AB 1886 u v r A there was no lethal interaction between the two wavelengths but the mutagenic interaction was synergistic. This synergism was maximal at a 365-nm dose of 8 × 10 s J m -2. It is proposed that in the wild-type strain, cells containing potentially mutagenic lesions are selectively eliminated from the population because of abortive excision of an error-prone repair-inducing signal. In excisionless strains, 365-nm radiation m a y be less damaging to the error-prone than to the error-free postreplication repair system. Alternatively, m u t a t i o n may be enhanced because of the occurrence of error-prone repair of 365-nm lesions by a system that is not induced in the absence of 254-nm radiation.
Introduction
Near-UV radiation modifies the lethal action of various physical and chemical agents on the bacterium E s c h e r i c h i a coll. (See reviews 18 and 22.) The Work su pported by the following Brazilian grants and funding agencies: CNPq, CNEN, CPEG/ UFRJ, FINEP/FNDCT-375/CT.
26 strong synergistic interaction observed between near-UV and ionizing radiation [16], heat [17] or chemicals (Correia and Tyrrell, in preparation) has usually been attributed to a disruption of DNA-repair systems by the long-wavelength radiation. No studies are available on the mutagenic interaction of monochromatic near-UV radiation with other radiations or DNA-damaging agents. The mutagenic interaction between other types of radiation has been studied to a limited extent (for a review of radiation interactions see ref. 20). Combinations of far-UV and ionizing radiation have been tested in several systems with varied results. An interaction was seen in the fungus Aspergillus terreus but not in Aspergillus nidulans (results cited in ref. 3). Several workers have observed a strong mutagenic interaction between ionizing radiation and UV in E. coli [2,3,7] but in a recent study no evidence was found for such an interaction in haploid yeast (D. Sweet and R.H. Haynes, personal communication). Current hypotheses of far-UV mutagenesis are based on the proposal that mutagenic events arise as a result of misrepair of the lesion by an error-prone repair mode (see ref. 26, for review) which may itself be UV-inducible [9] (and see ref. 27, for review). Since near-UV apparently strongly influences the repair of far-UV damage in bacteria [18,21,22], it was of interest to test whether there exists a mutagenic interaction between the two wavelength regions. This paper describes the results of such a study. Monochromatic radiation in the near-UV range is itself lethal to bacterial cells and, under certain conditions, may also be mutagenic. (For reviews see refs. 4 and 22.) In particular, Webb [22] has observed 365-nm radiation-induced reversion to tryptophan independence in auxotrophic strains of E. coli B/r and a uvrA derivative irradiated in stationary phase. Mutations were observed only on plates containing semi-enriched media [25] and did not appear on minimal plates supplemented only with low levels of tryptophan. In this study, K12 strains were generally used, and the system developed for detecting reversion to amino-acid independence was different. Under these conditions mutation induced by far-UV but n o t by near-UV was observed in the dose-ranges used. Materials and methods
(1) Bacterial strains. E. coli K12 AB 1157 (uvrA * recA ÷) and AB 1886 (uvrA recA ÷) require threonine, leucine, proline, histidine, arginine and thiamine, cannot use galactose, arabinose, xylose or mannitol as an energy source and are resistant to bacteriophage T6 and mitomycin C. The sub-strain of E. coli B/r used in these experiments required t h y m i n e and tryptophan. (2) Growth and harvesting o f bacteria. Exponential-phase cultures were prepared by inoculation of one part of an overnight culture of the appropriate bacteria into 40 parts of medium pre-warmed to 37°C and containing M9 salts, Casamino acids (2.5 mg/ml), glucose (2 g/l), thiamine (2 mg/ml) (for AB 1157 and AB 1186 only), t r y p t o p h a n (10 pg/ml) (B/r only) and t h y m i n e (10 pg/ml) (B/r only). Growth was for precisely 2.25 h. The culture was suspended in M9 to a final concentration of 109 (365 nm) or 108 (254 nm) cells per ml depending upon the wavelength of irradiation. Cells were assayed for viability by appropriate dilution, plating on the surface of nutrient agar and scoring for colony formation after 2 days incubation at 37 ° C.
27
(3) Irradiations. Pure monochromatic radiations were obtained at 254 nm (0.1--1.0 W m -2) and 365 nm (1000 W m -s) as previously described [17], and the dose was measured by a Latarjet meter or radiometer. In mixed-radiation treatments, the second irradiation was carried out as soon as practicable (2--3 min) after the first. Scoring for mutations After appropriate irradiation treatment, 100-pl samples were taken immediately for viability assay on nutrient agar plates, and 500-pl samples were mixed with an equal volume of twice-concentrated nutrient broth and incubated for 2 days at 37°C. The incubated samples were centrifuged and resuspended in 1 ml of M9 salts medium. K12 strains were then assayed for revertants to histidine independence by plating between 5 × 106 and 5 × 107 cells on minimal afar plates containing M9 salts, glucose (4 g/l), threonine, proline and arginine (50 pg/ml), leucine (25 pg/ml) and thiamine (0.4 pg/ml). Concentrated salt solutions and thiamine were added after the autoclaving. Mutations to T5 resistance were scored by mixing and spreading 10 s T5 bacteriophage with a sample of the 2-day-grown culture on the surface of nutrient agar plates. B/r was assayed for t r y p t o p h a n revertants by plating the 2-day-grown cells on minimal plates containing M9 salts, glucose, Casamino acids and t h y m i n e (10 gg/ml). The two-day-grown cultures (both K12 and B/r) were also assayed for viability on nutrient afar plates to allow calculation of the mutation frequency. All plates for m u t a n t assay were grown for 3 days at 37°C before they were scored for revertants. The technique used in this study differed somewhat from the widely used m e t h o d of scoring for amino acid revertants on low-level broth plates [25-27] which allows for mutation and survival to be scored on the same medium. However, since the cells were cultivated for m u t a t i o n expression in nutrient broth, plating for immediate survival on nutrient agar appears to be a valid control. Furthermore, this is only important in Fig. 4 where mutants per survivor are plotted against surviving fraction. The m u t a n t yield itself was calculated as a fraction of the total viable cells in the 2-day-grown population, and this latter value was the same in minimal or enriched media (data from this laboratory). The argument that there will be selection for or against the revertants in the 2-day culture has been vigorously eliminated. With low-level (0.75 pg/ml) histidine-supplemented plates used for survival controls, the frequency of reversion to histidine independence induced by 1 J m -2 at 254 nm as measured on his plates (at hourly intervals for 6 h and then 12-h intervals} showed no change after 1 h in AB 1157 and after less than one cell division in AB 1886 (about 2 h) (Morais and Tyrrell, unpublished results, this laboratory). In this experimental protocol for the m u t a n t assay, the number of spontaneous mutants in the unirradiated controls was negligible. All procedures were carried out under fluorescent yellow light (Phillips, 30 W gold).
28
Results Some years ago it was reported that pre-irradiating freshly harvested exponential phase cells of the repair-proficient bacterium E. coli B/r with 365-nm radiation, strongly sensitized them to subsequent exposure to 254-nm radiation [21]. This experiment was repeated several times in our current experimental set-up and the result was always identical (Fig. 1) with that originally published. Far-UV induction of mutation to tryptophan independence, with and without pre~xposure to 365-nm radiation, was measured at the same time. The mutation curve for far-UV alone appears in Fig. 1. No data appears for the prenear-UV-treated samples since no detectable mutation was observed under these conditions. Mutation induction by the dose of near-UV radiation used {106 J m -2 at 365 nm) was at the extreme lower limit of detection of the assay (3--4 × 10 -8 mutants per survivor). Populations of the repair-proficient E. coli K12 strain AB 1157 pre-treated with 365-nm radiation showed a smaller increase in sensitivity to 254-nm radiation than the strain B/r (see Fig. 2). The interaction was apparently limited to the removal of the shoulder from the 254-nm-radiation survival curve. A strain difference also appeared for the mutagenic interaction. Fig. 2 demonstrates that, although induction of mutations by 254 nm radiation was again markedly suppressed by the near-UV treatment, the mutation level (reversion to histidine
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F i g . 2. T h e e f f e c t o f p r e - e x p o s u r e o f t h e r e p a i r - p r o f i c i e n t K 1 2 s t r a i n A B 1 1 5 7 t o 1 0 6 J m - 2 o f 3 6 5 - n m radiation on the lethal and mutagenic response of the cells to 254-nm radiation. Survival after 254-nm radiation only (o); survival after 365-nm + 254-nm radiation (o); mutation to histidine independence after 254-nm radiation only (~); mutation to histidine independence after 365-nm + 254-nm radiation (A); m u t a t i o n t o T 5 r e s i s t a n c e a f t e r 2 5 4 - n m r a d i a t i o n o n l y ( o ) ; m u t a t i o n t o T 5 r e s i s t a n c e a f t e r 3 6 5 - n m + 254-nm radiation
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29 independence) arising from the mixed-radiation treatments remained at the level of detection. When the two induction curves were replotted on logarithmic axes as in Fig. 3, they appeared nearly parallel and with slopes close to 2. Since the level of inactivation by a given far-UV dose was much higher for populations pre-treated with near-UV, it is meaningful to plot the m u t a t i o n frequency against surviving fraction. From Fig. 4A it is evident that, at survival levels higher than 15%, the frequency of far-UV mutagenesis (reversion to histidine independence) as a function of survivors (i.e. per lethal event} was 17 times higher in the cells of AB 1157 not exposed to near-UV. This suggests either that cells containing potentially mutagenic lesions may be selectively inactivated as a result of the interaction between the two types of radiation or that the mutagenic potential of the lesions themselves has been (directly or indirectly) modified. With B/r (see above) the effect was even more: marked since there were no detectable mutations in the survivors of the combined treatments. When plotted as a function of far-UV dose, 254-nm radiation-induced mutation to T5 resistance in AB 1157 seemed to be only slightly modified by 365-nm treatment. However, it is clear from Fig. 4B that, for the same survival level, doubly treated cells were much less likely to be mutated. No mutation to T5 resistance was observed in the repair-proficient strain after a dose of 106 J m -2 at 365 nm, and no m u t a t i o n to histidine independence was induced in either the repair-proficient or excision-deficient (AB ] 8 8 6 uvrA) strain by doses as high as 2 × 106 J m -2 at 365 nm (d~ta not shown). ~ .... . . Graded doses of 365-nm radiation caused a progressive decline in the fre-
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quency of mutations (reversion to histidine independence) induced by a constant dose of far-UV (30 J m -2) in the repair-proficient strain (see Fig. 5). The curve shows a marked shoulder and then declines more sharply, a pattern similar to that for loss of colony-forming ability after 365-nm radiation. A curve
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AB 1 1 5 7 as a f u n c t i o n of t h e (o) p o s t - e x p o s u r e t o 2 5 4 - n m 30 J m - 2 (¢). T h e d o t t e d / / n e b u t c o r r e c t e d f o r t h e survival
31 corrected for survival after far-UV alone is depicted by the dotted line in Fig. 5. The 365-nm radiation seemed to have a maximum lethal sensitizing effect in the middle (0.8--1.4 J m -2) of the dose range used. To determine the influence of excision-repair on the mutational and lethal interaction, similar experiments were carried out with an excisionless strain. Fig. 6 illustrates that not only is there no mutational antagonism in a doubly irradiated uvrA strain but that there exists a marked positive interaction [20] between the long- and short-wavelength UV radiations. No lethal synergism was observed at the 365-nm radiation dose used. By analogy with the repair-proficient strain, the curve of far-UV mutation induction plotted as a function of the square of the dose used shows similar kinetics with or without 365-nm radiation pre-treatment (Fig. 3). When the frequency of mutation induced by far UV in the strain AB 1886 uvrA is plotted as a function of surviving fraction (see Fig. 4A), the curve coincides closely with that for the repair-proficient strain. This is an indication that the probability of occurrence of this type of mutation per lethal event is not modified by excision-repair and would also be expected if, at the same survivor level, the same number of error-prone repair events had occurred. However, the mutation probability more than doubled when the uvrA strain was pre-treated with 106 J m -2 at 365 nm. The change in far-UV-induced mutation frequency as a function of 365-nm dose in AB 1886 uvrA did not follow a simple pattern. Fig. 7 illustrates that, with a constant far-UV dose (1 J m-2), the mutation frequency rose sharply until a pre-exposure dose of 8 X 10 s J m-: at 365 nm ~vas reached and then
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Fig. 6. T h e e f f e c t o f p r e - e x p o s u r e o f t h e e x c i s i o n l e s s K 1 2 strain A B 1 8 8 6 t o 1 0 6 J m - 2 o f 3 6 5 - n m radiat i o n o n t h e l e t h a l a n d m u t a g e n i e r e s p o n s e s o f t h e cells t o 2 5 4 - n m r a d i a t i o n . Survival a f t e r 2 5 4 - n m radiat i o n o n l y ( o ) ; survival ,after 3 6 5 - n m + 2 5 4 - n m r a d i a t i o n ( e ) ; m u t a t i o n t o h i s t i d i n e i n d e p e n d e n c e a f t e r 2 5 4 - n m r a d i a t i o n o n l y ( ~ ) ; m u t a t i o n a f t e r 3 6 5 - n m + 2 5 4 - n m r a d i a t i o n (A). Fig. 7 . F a r - U V - i n d u c e d m u t a t i o n t o h i s t i d i n e i n d e p e n d e n c e a n d survival in A B 1 8 8 6 u u r A as a t h e p r e - e x p o s u r e d o s e o f r a d i a t i o n at 3 6 5 n m . Survival w i t h ( e ) a n d w i t h o u t ( o ) p o s t - e x p o s u r e r a d i a t i o n at 1 J m - 2 . M u t a t i o n a f t e r p o s t - e x p o s u r e t o 2 5 4 - n m r a d i a t i o n at 1 J m - 2 ( 0 ) . T h e p l o t s t h e s a m e d a t a as r e p r e s e n t e d b y t h e c l o s e d circles ( 2 5 4 n m + 3 6 5 n m ) b u t c o r r e c t e d f o r after 254-nm radiation alone.
function of to 254-nm d o t t e d line t h e survival
32 declined, at least over the dose range used. No lethal synergism was observed in the doubly irradiated excisionless strain. Discussion An explanation of the preceding results must take into account n~)t only the mutational antagonism observed in the repair-proficient strain AB 1157 and the synergism in AB 1886 uvrA in doubly irradiated cells but also the lethal interaction in AB 1157 and lack of it in AB 1886. The widely accepted hypothesis that most UV mutations arise as a result of errors in post-replication repair [26] and that this error-prone repair system is probably inducible [9, for review see 27] should also be taken into account. Near-UV has been shown temporarily to suppress induced enzyme synthesis [15,23] and this almost certainly relates to the inhibition of protein synthesis seen at these wavelengths [10,15]. Radiation at 365 nm also leads to a strong, dose-dependent but reversible inhibition of DNA synthesis (author's unpublished results). The following hypothesis will take into account the probability that the induction of error-prone repair will be temporarily inhibited by 365-nm radiation and that DNA replication will also be transiently slowed or halted. The model has been developed primarily to explain the mutation data on reversion to p r o t o t r o p h y but is n o t incompatible with the data on m u t a t i o n to T5 resistance. Irradiation of the repair-proficient strain AB 1157 (and AB 1886 uvrA) with far-UV radiation is presumed to induce an error-prone repair pathway that acts upon unexcised lesions with a probability of error that is reflected by the m u t a t i o n frequency in Figs. 2, 3, 4 and 6. Excision-repair is probably involved in both the mutational antagonism and lethal synergism, since neither effect is seen in the excisionless strain (Fig. 6). The antagonistic effect of 365-nm radiation on 254-nm mutation frequency could be explained as an increase in the ratio of error-free (excision) to error-prone repair as a result of the near-UV-induced growth and macromolecular synthesis delay [6,13]. However, there is no apparent reason why this should also lead to a lethal synergism. It is known that dimer excision is slowed by 365-nm radiation [21], and the strong lethal interaction probably reflects damage to (or resulting from) excision-repair. One way this could occur is if a less balanced excision-repair process leads to enlarged gaps and a consequent higher probability of abortive excision because of a gap overlapping a dimer in the opposite strand. Sedgwick [ 12] has suggested that closely spaced lesions (in opposite strands) that reach the replication point interfere with constitutive post-replication repair and that the resulting formation of overlapping daughter-strand gaps is the inducing signal for synthesis of error-prone repair systems. The longer time available for excision after 365-nm radiation, possibly combined with an imbalance in the process itself (see above), may well lead to a higher probability of formation of a lethal double-strand break from closely spaced lesions [ 1,5,8 ] or to a dimer overlapping a gap (which would eventually lead to a double-strand break at the replication point). As a consequence, cells that contain potential inducing signals for error-prone repair are inactivated with a much higher probability in populations treated with a combination of 365- and 254-nm radia-
33 tions. This would not only lead to the lethal synergism observed (Figs. 1 and 2) but would also be expected to reduce the probability of m u t a t i o n in the survivors because a much smaller fraction of the surviving cells will be capable of inducing mutagenic DNA repair. A far greater lethal synergism was observed in the strain B/r than in the repair-proficient K12 strain (Fig. 1). According to the above hypothesis, this would mean that an even greater number of closely spaced lesions would be converted to lethal events. Since this will selectively inactivate a still higher fraction of the potentially m u t a n t cells, an even lower mutation frequency should be observed. This was indeed so, since no mutation was detectable in E. coli B/r after the combined radiation treatments. Because the technique used for detecting mutations involves post-irradiation growth of the treated cells before scoring for mutation, it may be argued that 365-nm radiation specifically reduces the growth of m u t a n t wild-type cells and thereby results in the reduced frequency of mutations induced by 254-nm radiation. Although this possibility cannot be eliminated by current data, such an interpretation is not consistent with the results obtained with similar nearUV doses with the excisionless strain. In an excision-defective strain, the closely spaced lesions will presumably arrive at the replication point intact. The lack of lethal synergism in the uvrA strain (Fig. 6) suggests that at any given far-UV dose, there was an equal chance of making a lethal mistake with or without 365-nm-radiation pre-treatment. The chance of an error leading to a mutation, however, was apparently increased (Figs. 3, 4 and 6) by 365-nm radiation so that, even though the induction of error-prone repair may be delayed (see above), it is evident that in the excisionless strain, the lesions remained stable enough to allow its eventual operation. At least two possibilities may be suggested to explain the apparent mutational synergism between the two types of radiation. The first is based on the proposal [11] that at least two types of post-replication repair occur in E. coli, one being constitutive and error-free and the other being an inducible error-prone process. There is evidence (see Introduction) that certain constitutive "rec"-related repair systems are inhibited by near-UV. If the cellular changes induced by 365-nm radiation favour operation of the error-prone post-replication repair system, but result in the same total magnitude of repair, a higher m u t a t i o n rate will result. No lethal synergism would be expected. These considerations will not apply to the excision-proficient strain since the error-prone repair-inducing lesion itself will have been selected out of the population. At higher doses of 365-nm radiation, the inhibition of error-prone repair induction m a y become effectively irreversible and lead to the observed decline in 254-nm radiationinduced mutation (Fig. 7). One factor that is not simply explained by this first suggestion is the lack of 365-nm mutagenesis in the uvrA strain under these experimental conditions. The uvrA strain was inactivated by a dose of 365-nm radiation very close to 106 times greater than the dose necessary to give the same surviving fraction after 254-nm radiation (over the dose range tested; see Figs. 6 and 7). This factor of 106 approximately coincides with the ratio of total lesions known to be induced at the two wavelengths: dimers at 254 nm and dimers and single-
34 strand breaks at 365 nm [18]. So, if the 365-nm lesions are pre-mutagenic, considerable mutagenesis would be expected after 106 J m -2 at 365 nm (equivalent to 1 J m -2 at 254 nm; see Fig. 6), particularly if this radiation does indeed increase the proportion of error-prone compared with error-free repair. It has been suggested by Witkin [27] that near-UV lesions are cryptic, that is, they are susceptible to error-prone repair (but see below) although they are not capable of inducing it. This would then explain why 365-nm radiation alone is poorly mutagenic. Furthermore, when a mixture of 254- and 365-nm radiations are used (in doses equivalent in terms of lesions), the 254-nm-induced error-prone repair would act on the " c r y p t i c " 365-nm lesions and be expected to lead to an increase in the mutation rate, as observed in Fig. 6. Mutation would increase above the 254-nm background as a function of 365-nm dose and be expected to decline at higher doses if the induction of error-prone repair is eventually irreversibly inhibited (Fig. 7). Alternatively, the complete lack of mutagenesis may simply mean that the lesions are not susceptible to errorprone repair. This latter possibility is favoured by experiments (this laboratory) that have shown that 365-nm-induced lesions in phage are not susceptible to Weigle reactivation [ 24] in host cells (repair-proficient or uvrA ) preirradiated at 254 nm. Further evidence is being sought to distinguish between these possible explanations of the mutational interaction in the excisionless strains. The anti-mutagenic action of near-UV radiation in repair-proficient cells and the synergistic interaction in excisionless strains may have significance b e y o n d these fundamental considerations. It has been pointed out [19] that, although dependent on many factors, the daily terrestrial sunlight dose of wavelengths in the range 334--405 nm will often exceed the near-UV doses used in this study. A possible mutagenic interaction between natural sunlight and the growing list of natural or artificially produced mutagens should be considered. Acknowledgements This work was supported by the following Brazilian granting agents: CNPq (National Research Council), CNEN (National Nuclear Energy Council), C P E G / U F R J (University Council for Post-Graduate studies) and FINEP/ FNDCT-375/CT (Study and Project Grants/National Fund for Scientific and Technological Development). The author thanks Dr. L.R. Caldas for his interest and encouragement and Dr. R.D. Ley for helpful discussion and comments on the manuscript. References 1 Bonura, T., and K.C. Smith, Enzymatic production o f deoxyribonucleic acid double-strand breaks after ultraviolet irradiation of Escherichia coli, J. Bacteriol., 121 (1975) 511--517. 2 Bridges, B.A., R.J. M u n s o n , C.F. Arlett and D.R. Davies, Interaction between ultraviolet light and X-radiation d a m a g e in the induction of mutants in Escherichia coli: The effect of s o m e modifying treatments, J. Gen. Microbiol., 46 (1967) 339--346. 3 Davies, D.R., C.F. Arlett, R~I. M u n s o n and B.A. Bridges, Interaction between ultraviolet light and X-radiation d a m a g e in the induction of mutants in Escherichia coli: The response in strains with normal and reduced ability to repair ultraviolet damage, J. Gen. Microbiol., 46 (1967) 329--338. 4 Eisenstark, A., Mutagenic and lethal effects of visible and near-ultraviolet light on bacterial cells, in:
35 E.W. Caspari (Ed.), A d v a n c e s in Genetics, Vol. 12, A c a d e m i c Press, New Y o r k , 1 9 7 1 , p p . 1 6 7 - - 1 9 8 . 5 H a r m , W., Effects of d o s e - f r a c t i o n a t i o n o n ultraviolet survival of E s c h e r i c h i a coli, P h o t o c h e m . P h o t o biol., 7 ( 1 9 6 8 ) 7 3 - - 8 6 . 6 Jagger, J., W. Curtis-Wise a n d R.S. S t a f f o r d , Delay in g r o w t h a n d division i n d u c e d b y near-ultraviolet r a d i a t i o n in Escherichia coli B a n d its role in p h o t o p r o t e c t i o n a n d l i q u i d - h o l d i n g r e c o v e r y , P h o t o chem. Photobiol., 3 (1964) 11--24. 7 K a d a , T., C.O. D o u d n e y a n d F.L. Hass, M u t a t i o n f r e q u e n c y response of i r r a d i a t e d b a c t e r i a to a s e c o n d r a d i a t i o n e x p o s u r e , M u t a t i o n Res., 3 ( 1 9 6 6 ) 1 1 8 - - 1 2 8 . 8 Moss, S.H., a n d D.J.G. Davies, I n t e r r e l a t i o n s h i p of repair m e c h a n i s m 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. Bacteriol., 1 2 0 ( 1 9 7 4 ) 1 5 - - 2 3 . 9 R a d m a n , M., SOS repair h y p o t h e s i s : p h e n o m e n o l o g y of a n inducible D N A repair 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. Setlow (Eds.), Molecular Mechanisms for repair of D N A , p a r t A, P l e n u m , New Y o r k , 1 9 7 5 , p p . 3 4 7 - - 3 5 5 . 10 R a m a b h a d r a n , T.V., Effects o f n e a r - u l t r a v i o l e t a n d violet r a d i a t i o n s (313---405 n m ) o n D N A , R N A , a n d p r o t e i n s y n t h e s i s in E. coli B/r: I m p l i c a t i o n s f o r g r o w t h d e l a y , P h o t o c h e m . P h o t o b i o l . , 22 ( 1 9 7 5 ) 117--123. 11 Sedgwick, S.G., I n d u c i b l e e r r o r - p r o n e repair in Escherichia coli, Proc. Natl. A c a d . Sci. (U.S.A.), 72 (1975) 2753--2757. 12 S e d g w i c k , S.G., Misrepair of o v e r l a p p i n g d a u g h t e r s t r a n d gaps as a possible m e c h a n i s m for UV i n d u c e d m u t a g e n e s i s in U V R strains o f Escherichia coli: A general m o d e l for i n d u c e d m u t a g e n e s i s b y m i s r e p a i r (SOS repair) of closely s p a c e d D N A lesions, M u t a t i o n Res., 41 ( 1 9 7 6 ) 1 8 5 - - 2 0 0 . 13 S w e n s o n , P.A., Physiological r e s p o n s e s of E s c h e r i c h i a coli to far-ultraviolet r a d i a t i o n , in: K.C. S m i t h (Ed.), P h o t o c h e m . P h o t o b i o l . Reviews, Vol. 1, P l e n u m , New Y o r k , 1 9 7 6 , p p . 2 6 8 - - 3 8 7 . 14 S w e n s o n , P.A., a n d R.B. Setlow, I n h i b i t i o n of the i n d u c e d f o r m a t i o n of t r y p t o p h a n a s e in E s c h e r i c h i a coU b y near-ultraviolet r a d i a t i o n , J. Bacteriol., 102 ( 1 9 7 0 ) 8 1 5 - - 8 1 9 . 15 S w e n s o n , P.A., J.E. Ives a n d R.L. S c h e n l e y , P h o t o p r o t e c t i o n of E. coli B/r: R e s p i r a t i o n , g r o w t h , m a c r o m o l e c u l a r s y n t h e s i s a n d repair o f D N A , P h o t o c h e m . P h o t o b i o l . , 21 ( 1 9 7 5 ) 2 3 5 - - 2 4 1 . 16 Tyrrel], R.M., The i n t e r a c t i o n o f near-UV ( 3 6 5 n m ) a n d X-irradiations on w i l d - t y p e a n d repair-defic i e n t strains o f Escherichia coli K 1 2 : Physical a n d biological m e a s u r e m e n t s , Int. J. R a d i a t . Biol., 25 (1974) 373--390. 17 Tyrrell, R.M., Synergistic lethal a c t i o n of ultraviolet r a d i a t i o n s a n d mild h e a t in E s c h e r i c h i a coli, P h o t o c h e m . P h o t o b i o l . , 24 ( 1 9 7 6 ) 345---351. 18 Tyrrell, R.M., L e t h a l cellular changes i n d u c e d b y near-ultraviolet r a d i a t i o n , in: Proceedings of V I t h UV S y m p o s i u m , Ultraviolet R a d i a t i o n in Biology a n d Medicine, K i i h l u n g s b o r n , 1 9 7 7 . 19 Tyrrell, R.M., Solar d o s i m e t r y w i t h r e p a i r d e f i c i e n t b a c t e r i a l spores: A c t i o n s p e c t r a , p h o t o p r o d u c t m e a s u r e m e n t s a n d a c o m p a r i s o n w i t h o t h e r biological systems, P h o t o c h e m . P h o t o b i o l . , (in press). 20 Tyrrell, R.M., R a d i a t i o n synergism a n d a n t a g o n i s m , in: K.C. S m i t h (Ed.), P h o t o c h e m . P h o t o b i o l . Reviews, Vol. 3, P l e n u m , New Y o r k , 1 9 7 8 , pp. 3 5 - - 1 1 3 . 21 TyrreU, R.M., a n d R.B. Webb, R e d u c e d d i m e r excision in b a c t e r i a following n e a r u l t r a v i o l e t ( 3 6 5 n m ) r a d i a t i o n , M u t a t i o n Res., 19 ( 1 9 7 3 ) 3 6 1 - - 3 6 4 . 22 Webb, R.B., Lethal a n d m u t a g e n i c effects o f near-ultraviolet r a d i a t i o n , in: K.C. S m i t h (Ed.), P h o t o c h e m . P h o t o b i o l . Reviews, Vol. 2, P l e n u m , New Y o r k , 1 9 7 7 , p p . 1 6 9 - - 2 6 3 . 23 Webb, S.J., a n d J.S. Bhorjee, The effect of 3 0 0 0 t o 4 0 0 0 A light o n t h e synthesis of ~-galactosidases a n d b a c t e r i o p h a g e s b y Escherichia coli B, Can. J. Microbiol., 13 ( 1 9 6 7 ) 6 9 - - 7 9 . 24 Weigie, J., I n d u c t i o n of m u t a t i o n in a b a c t e r i a l virus, Proc. Natl. Acad. Sci. (U.S.A.), 3 9 ( 1 9 5 3 ) 6 2 8 - 636. 25 Witkin, E.M., The effect o f acriflavine o n p h o t o r e v e r s a l of lethal a n d m u t a g e n i e d a m a g e p r o d u c e d in b e c t e r i a b y u l t r a v i o l e t light, Proc. Natl. A c a d . Sci. (U.S.A.), 50 ( 1 9 6 3 ) 4 2 5 - - 4 3 0 . 26 Witkin, E.M., U l t r a v i o l e t - i n d u c e d m u t a t i o n a n d D N A repair, A n n u . Rev. Microbiol., 23 ( 1 9 6 9 ) 4 8 7 - 514. 27 Witkin, E.M., Ultraviolet m u t a g e n e s i s a n d i n d u c i b l e D N A repair in Escherichia coli, Bacteriol. Rev., 4 0 (1976) 869--907.
Mutation Research, 52 (1978) 25--35
© Elsevier/North-Holland Biomedical Press
MUTAGENIC INTERACTION BETWEEN NEAR-(365 nm) AND FAR-(254 nm)ULTRAVIOLET RADIATION IN REPAIR-PROFICIENT AND EXCISION-DEFICIENT STRAINS OF E s c b e r i c b i a c o l i
REX M. TYRRELL Instituto de Biofisica, Centro de Ci~ncias da Safide (Bloco G), Universidade Federal do Rio de Janeiro (Brazil)
(Received 6 December 1977) (Revision received 18 April 1978) (Accepted 15 May 1978) Summary The mutational interaction between radiation at 365 and 254 nm was studied in various strains of E. c o l i by a m u t a n t assay based on reversion to amino-acid independence in full nutrient conditions. In the two repair-proficient strains (K12 AB 1157 and B/r), pre-treatment with radiation at 365 nm strongly suppressed the induction of mutations by far-UV, a p h e n o m e n o n accompanied by a strong positive lethal interaction. The frequency of mutations induced by far-UV progressively declined with increasing dose of near-UV. FarUV-induced mutagenesis to T5 resistance was almost unaltered by pre-treatm e n t with near-UV. In AB 1886 u v r A there was no lethal interaction between the two wavelengths but the mutagenic interaction was synergistic. This synergism was maximal at a 365-nm dose of 8 × 10 s J m -2. It is proposed that in the wild-type strain, cells containing potentially mutagenic lesions are selectively eliminated from the population because of abortive excision of an error-prone repair-inducing signal. In excisionless strains, 365-nm radiation m a y be less damaging to the error-prone than to the error-free postreplication repair system. Alternatively, m u t a t i o n may be enhanced because of the occurrence of error-prone repair of 365-nm lesions by a system that is not induced in the absence of 254-nm radiation.
Introduction
Near-UV radiation modifies the lethal action of various physical and chemical agents on the bacterium E s c h e r i c h i a coll. (See reviews 18 and 22.) The Work su pported by the following Brazilian grants and funding agencies: CNPq, CNEN, CPEG/ UFRJ, FINEP/FNDCT-375/CT.
26 strong synergistic interaction observed between near-UV and ionizing radiation [16], heat [17] or chemicals (Correia and Tyrrell, in preparation) has usually been attributed to a disruption of DNA-repair systems by the long-wavelength radiation. No studies are available on the mutagenic interaction of monochromatic near-UV radiation with other radiations or DNA-damaging agents. The mutagenic interaction between other types of radiation has been studied to a limited extent (for a review of radiation interactions see ref. 20). Combinations of far-UV and ionizing radiation have been tested in several systems with varied results. An interaction was seen in the fungus Aspergillus terreus but not in Aspergillus nidulans (results cited in ref. 3). Several workers have observed a strong mutagenic interaction between ionizing radiation and UV in E. coli [2,3,7] but in a recent study no evidence was found for such an interaction in haploid yeast (D. Sweet and R.H. Haynes, personal communication). Current hypotheses of far-UV mutagenesis are based on the proposal that mutagenic events arise as a result of misrepair of the lesion by an error-prone repair mode (see ref. 26, for review) which may itself be UV-inducible [9] (and see ref. 27, for review). Since near-UV apparently strongly influences the repair of far-UV damage in bacteria [18,21,22], it was of interest to test whether there exists a mutagenic interaction between the two wavelength regions. This paper describes the results of such a study. Monochromatic radiation in the near-UV range is itself lethal to bacterial cells and, under certain conditions, may also be mutagenic. (For reviews see refs. 4 and 22.) In particular, Webb [22] has observed 365-nm radiation-induced reversion to tryptophan independence in auxotrophic strains of E. coli B/r and a uvrA derivative irradiated in stationary phase. Mutations were observed only on plates containing semi-enriched media [25] and did not appear on minimal plates supplemented only with low levels of tryptophan. In this study, K12 strains were generally used, and the system developed for detecting reversion to amino-acid independence was different. Under these conditions mutation induced by far-UV but n o t by near-UV was observed in the dose-ranges used. Materials and methods
(1) Bacterial strains. E. coli K12 AB 1157 (uvrA * recA ÷) and AB 1886 (uvrA recA ÷) require threonine, leucine, proline, histidine, arginine and thiamine, cannot use galactose, arabinose, xylose or mannitol as an energy source and are resistant to bacteriophage T6 and mitomycin C. The sub-strain of E. coli B/r used in these experiments required t h y m i n e and tryptophan. (2) Growth and harvesting o f bacteria. Exponential-phase cultures were prepared by inoculation of one part of an overnight culture of the appropriate bacteria into 40 parts of medium pre-warmed to 37°C and containing M9 salts, Casamino acids (2.5 mg/ml), glucose (2 g/l), thiamine (2 mg/ml) (for AB 1157 and AB 1186 only), t r y p t o p h a n (10 pg/ml) (B/r only) and t h y m i n e (10 pg/ml) (B/r only). Growth was for precisely 2.25 h. The culture was suspended in M9 to a final concentration of 109 (365 nm) or 108 (254 nm) cells per ml depending upon the wavelength of irradiation. Cells were assayed for viability by appropriate dilution, plating on the surface of nutrient agar and scoring for colony formation after 2 days incubation at 37 ° C.
27
(3) Irradiations. Pure monochromatic radiations were obtained at 254 nm (0.1--1.0 W m -2) and 365 nm (1000 W m -s) as previously described [17], and the dose was measured by a Latarjet meter or radiometer. In mixed-radiation treatments, the second irradiation was carried out as soon as practicable (2--3 min) after the first. Scoring for mutations After appropriate irradiation treatment, 100-pl samples were taken immediately for viability assay on nutrient agar plates, and 500-pl samples were mixed with an equal volume of twice-concentrated nutrient broth and incubated for 2 days at 37°C. The incubated samples were centrifuged and resuspended in 1 ml of M9 salts medium. K12 strains were then assayed for revertants to histidine independence by plating between 5 × 106 and 5 × 107 cells on minimal afar plates containing M9 salts, glucose (4 g/l), threonine, proline and arginine (50 pg/ml), leucine (25 pg/ml) and thiamine (0.4 pg/ml). Concentrated salt solutions and thiamine were added after the autoclaving. Mutations to T5 resistance were scored by mixing and spreading 10 s T5 bacteriophage with a sample of the 2-day-grown culture on the surface of nutrient agar plates. B/r was assayed for t r y p t o p h a n revertants by plating the 2-day-grown cells on minimal plates containing M9 salts, glucose, Casamino acids and t h y m i n e (10 gg/ml). The two-day-grown cultures (both K12 and B/r) were also assayed for viability on nutrient afar plates to allow calculation of the mutation frequency. All plates for m u t a n t assay were grown for 3 days at 37°C before they were scored for revertants. The technique used in this study differed somewhat from the widely used m e t h o d of scoring for amino acid revertants on low-level broth plates [25-27] which allows for mutation and survival to be scored on the same medium. However, since the cells were cultivated for m u t a t i o n expression in nutrient broth, plating for immediate survival on nutrient agar appears to be a valid control. Furthermore, this is only important in Fig. 4 where mutants per survivor are plotted against surviving fraction. The m u t a n t yield itself was calculated as a fraction of the total viable cells in the 2-day-grown population, and this latter value was the same in minimal or enriched media (data from this laboratory). The argument that there will be selection for or against the revertants in the 2-day culture has been vigorously eliminated. With low-level (0.75 pg/ml) histidine-supplemented plates used for survival controls, the frequency of reversion to histidine independence induced by 1 J m -2 at 254 nm as measured on his plates (at hourly intervals for 6 h and then 12-h intervals} showed no change after 1 h in AB 1157 and after less than one cell division in AB 1886 (about 2 h) (Morais and Tyrrell, unpublished results, this laboratory). In this experimental protocol for the m u t a n t assay, the number of spontaneous mutants in the unirradiated controls was negligible. All procedures were carried out under fluorescent yellow light (Phillips, 30 W gold).
28
Results Some years ago it was reported that pre-irradiating freshly harvested exponential phase cells of the repair-proficient bacterium E. coli B/r with 365-nm radiation, strongly sensitized them to subsequent exposure to 254-nm radiation [21]. This experiment was repeated several times in our current experimental set-up and the result was always identical (Fig. 1) with that originally published. Far-UV induction of mutation to tryptophan independence, with and without pre~xposure to 365-nm radiation, was measured at the same time. The mutation curve for far-UV alone appears in Fig. 1. No data appears for the prenear-UV-treated samples since no detectable mutation was observed under these conditions. Mutation induction by the dose of near-UV radiation used {106 J m -2 at 365 nm) was at the extreme lower limit of detection of the assay (3--4 × 10 -8 mutants per survivor). Populations of the repair-proficient E. coli K12 strain AB 1157 pre-treated with 365-nm radiation showed a smaller increase in sensitivity to 254-nm radiation than the strain B/r (see Fig. 2). The interaction was apparently limited to the removal of the shoulder from the 254-nm-radiation survival curve. A strain difference also appeared for the mutagenic interaction. Fig. 2 demonstrates that, although induction of mutations by 254 nm radiation was again markedly suppressed by the near-UV treatment, the mutation level (reversion to histidine
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29 independence) arising from the mixed-radiation treatments remained at the level of detection. When the two induction curves were replotted on logarithmic axes as in Fig. 3, they appeared nearly parallel and with slopes close to 2. Since the level of inactivation by a given far-UV dose was much higher for populations pre-treated with near-UV, it is meaningful to plot the m u t a t i o n frequency against surviving fraction. From Fig. 4A it is evident that, at survival levels higher than 15%, the frequency of far-UV mutagenesis (reversion to histidine independence) as a function of survivors (i.e. per lethal event} was 17 times higher in the cells of AB 1157 not exposed to near-UV. This suggests either that cells containing potentially mutagenic lesions may be selectively inactivated as a result of the interaction between the two types of radiation or that the mutagenic potential of the lesions themselves has been (directly or indirectly) modified. With B/r (see above) the effect was even more: marked since there were no detectable mutations in the survivors of the combined treatments. When plotted as a function of far-UV dose, 254-nm radiation-induced mutation to T5 resistance in AB 1157 seemed to be only slightly modified by 365-nm treatment. However, it is clear from Fig. 4B that, for the same survival level, doubly treated cells were much less likely to be mutated. No mutation to T5 resistance was observed in the repair-proficient strain after a dose of 106 J m -2 at 365 nm, and no m u t a t i o n to histidine independence was induced in either the repair-proficient or excision-deficient (AB ] 8 8 6 uvrA) strain by doses as high as 2 × 106 J m -2 at 365 nm (d~ta not shown). ~ .... . . Graded doses of 365-nm radiation caused a progressive decline in the fre-
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Fig. 4. T h e i n f l u e n c e of 3 6 5 - n m r a d i a t i o n o n 2 5 4 - n m i n d u c e d m u t a t i o n . Results p l o t t e d as a f u n c t i o n of t h e f r a c t i o n surviving t h e t r e a t m e n t . A, M u t a t i o n to histidine i n d e p e n d e n c e in K 1 2 strains a f t e r 2 5 4 - n m r a d i a t i o n w i t h (AB 1 1 5 7 , A; AB 1 8 8 6 , e ) a n d w i t h o u t (AB 1 1 5 7 , a ; AB 1 8 8 6 , o) p r e - e x p o s u r e t o 3 6 5 - n m r a d i a t i o n at 106 J m - 2 ; B, M u t a t i o n t o T 5 r e s i s t a n c e in K 1 2 AB 1 1 5 7 a f t e r 2 5 4 - n m r a d i a t i o n w i t h (m) a n d w i t h o u t (~) p r e - e x p o s u r e to 106 J m -2 at 3 6 5 n m .
quency of mutations (reversion to histidine independence) induced by a constant dose of far-UV (30 J m -2) in the repair-proficient strain (see Fig. 5). The curve shows a marked shoulder and then declines more sharply, a pattern similar to that for loss of colony-forming ability after 365-nm radiation. A curve
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Fig. 5. F a x - U V - i n d u c e d m u t a t i o n t o histidine i n d e p e n d e n c e a n d survival in p r e - e x p o s u r e d o s e o f r a d i a t i o n a t 3 6 5 n m . Survival w i t h ( e ) a n d w i t h o u t r a d i a t i o n a t 30 J m - 2 . M u t a t i o n a f t e r p o s t - e x p o s u r e to 2 5 4 - n m r a d i a t i o n at p l o t s t h e s a m e d a t a as r e p r e s e n t e d b y t h e closed circles ( 2 5 4 n m + 3 6 5 n m ) after 254-nm radiation alone.
AB 1 1 5 7 as a f u n c t i o n of t h e (o) p o s t - e x p o s u r e t o 2 5 4 - n m 30 J m - 2 (¢). T h e d o t t e d / / n e b u t c o r r e c t e d f o r t h e survival
31 corrected for survival after far-UV alone is depicted by the dotted line in Fig. 5. The 365-nm radiation seemed to have a maximum lethal sensitizing effect in the middle (0.8--1.4 J m -2) of the dose range used. To determine the influence of excision-repair on the mutational and lethal interaction, similar experiments were carried out with an excisionless strain. Fig. 6 illustrates that not only is there no mutational antagonism in a doubly irradiated uvrA strain but that there exists a marked positive interaction [20] between the long- and short-wavelength UV radiations. No lethal synergism was observed at the 365-nm radiation dose used. By analogy with the repair-proficient strain, the curve of far-UV mutation induction plotted as a function of the square of the dose used shows similar kinetics with or without 365-nm radiation pre-treatment (Fig. 3). When the frequency of mutation induced by far UV in the strain AB 1886 uvrA is plotted as a function of surviving fraction (see Fig. 4A), the curve coincides closely with that for the repair-proficient strain. This is an indication that the probability of occurrence of this type of mutation per lethal event is not modified by excision-repair and would also be expected if, at the same survivor level, the same number of error-prone repair events had occurred. However, the mutation probability more than doubled when the uvrA strain was pre-treated with 106 J m -2 at 365 nm. The change in far-UV-induced mutation frequency as a function of 365-nm dose in AB 1886 uvrA did not follow a simple pattern. Fig. 7 illustrates that, with a constant far-UV dose (1 J m-2), the mutation frequency rose sharply until a pre-exposure dose of 8 X 10 s J m-: at 365 nm ~vas reached and then
I00 ~ 8 8 6 u v r A
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Fig. 6. T h e e f f e c t o f p r e - e x p o s u r e o f t h e e x c i s i o n l e s s K 1 2 strain A B 1 8 8 6 t o 1 0 6 J m - 2 o f 3 6 5 - n m radiat i o n o n t h e l e t h a l a n d m u t a g e n i e r e s p o n s e s o f t h e cells t o 2 5 4 - n m r a d i a t i o n . Survival a f t e r 2 5 4 - n m radiat i o n o n l y ( o ) ; survival ,after 3 6 5 - n m + 2 5 4 - n m r a d i a t i o n ( e ) ; m u t a t i o n t o h i s t i d i n e i n d e p e n d e n c e a f t e r 2 5 4 - n m r a d i a t i o n o n l y ( ~ ) ; m u t a t i o n a f t e r 3 6 5 - n m + 2 5 4 - n m r a d i a t i o n (A). Fig. 7 . F a r - U V - i n d u c e d m u t a t i o n t o h i s t i d i n e i n d e p e n d e n c e a n d survival in A B 1 8 8 6 u u r A as a t h e p r e - e x p o s u r e d o s e o f r a d i a t i o n at 3 6 5 n m . Survival w i t h ( e ) a n d w i t h o u t ( o ) p o s t - e x p o s u r e r a d i a t i o n at 1 J m - 2 . M u t a t i o n a f t e r p o s t - e x p o s u r e t o 2 5 4 - n m r a d i a t i o n at 1 J m - 2 ( 0 ) . T h e p l o t s t h e s a m e d a t a as r e p r e s e n t e d b y t h e c l o s e d circles ( 2 5 4 n m + 3 6 5 n m ) b u t c o r r e c t e d f o r after 254-nm radiation alone.
function of to 254-nm d o t t e d line t h e survival
32 declined, at least over the dose range used. No lethal synergism was observed in the doubly irradiated excisionless strain. Discussion An explanation of the preceding results must take into account n~)t only the mutational antagonism observed in the repair-proficient strain AB 1157 and the synergism in AB 1886 uvrA in doubly irradiated cells but also the lethal interaction in AB 1157 and lack of it in AB 1886. The widely accepted hypothesis that most UV mutations arise as a result of errors in post-replication repair [26] and that this error-prone repair system is probably inducible [9, for review see 27] should also be taken into account. Near-UV has been shown temporarily to suppress induced enzyme synthesis [15,23] and this almost certainly relates to the inhibition of protein synthesis seen at these wavelengths [10,15]. Radiation at 365 nm also leads to a strong, dose-dependent but reversible inhibition of DNA synthesis (author's unpublished results). The following hypothesis will take into account the probability that the induction of error-prone repair will be temporarily inhibited by 365-nm radiation and that DNA replication will also be transiently slowed or halted. The model has been developed primarily to explain the mutation data on reversion to p r o t o t r o p h y but is n o t incompatible with the data on m u t a t i o n to T5 resistance. Irradiation of the repair-proficient strain AB 1157 (and AB 1886 uvrA) with far-UV radiation is presumed to induce an error-prone repair pathway that acts upon unexcised lesions with a probability of error that is reflected by the m u t a t i o n frequency in Figs. 2, 3, 4 and 6. Excision-repair is probably involved in both the mutational antagonism and lethal synergism, since neither effect is seen in the excisionless strain (Fig. 6). The antagonistic effect of 365-nm radiation on 254-nm mutation frequency could be explained as an increase in the ratio of error-free (excision) to error-prone repair as a result of the near-UV-induced growth and macromolecular synthesis delay [6,13]. However, there is no apparent reason why this should also lead to a lethal synergism. It is known that dimer excision is slowed by 365-nm radiation [21], and the strong lethal interaction probably reflects damage to (or resulting from) excision-repair. One way this could occur is if a less balanced excision-repair process leads to enlarged gaps and a consequent higher probability of abortive excision because of a gap overlapping a dimer in the opposite strand. Sedgwick [ 12] has suggested that closely spaced lesions (in opposite strands) that reach the replication point interfere with constitutive post-replication repair and that the resulting formation of overlapping daughter-strand gaps is the inducing signal for synthesis of error-prone repair systems. The longer time available for excision after 365-nm radiation, possibly combined with an imbalance in the process itself (see above), may well lead to a higher probability of formation of a lethal double-strand break from closely spaced lesions [ 1,5,8 ] or to a dimer overlapping a gap (which would eventually lead to a double-strand break at the replication point). As a consequence, cells that contain potential inducing signals for error-prone repair are inactivated with a much higher probability in populations treated with a combination of 365- and 254-nm radia-
33 tions. This would not only lead to the lethal synergism observed (Figs. 1 and 2) but would also be expected to reduce the probability of m u t a t i o n in the survivors because a much smaller fraction of the surviving cells will be capable of inducing mutagenic DNA repair. A far greater lethal synergism was observed in the strain B/r than in the repair-proficient K12 strain (Fig. 1). According to the above hypothesis, this would mean that an even greater number of closely spaced lesions would be converted to lethal events. Since this will selectively inactivate a still higher fraction of the potentially m u t a n t cells, an even lower mutation frequency should be observed. This was indeed so, since no mutation was detectable in E. coli B/r after the combined radiation treatments. Because the technique used for detecting mutations involves post-irradiation growth of the treated cells before scoring for mutation, it may be argued that 365-nm radiation specifically reduces the growth of m u t a n t wild-type cells and thereby results in the reduced frequency of mutations induced by 254-nm radiation. Although this possibility cannot be eliminated by current data, such an interpretation is not consistent with the results obtained with similar nearUV doses with the excisionless strain. In an excision-defective strain, the closely spaced lesions will presumably arrive at the replication point intact. The lack of lethal synergism in the uvrA strain (Fig. 6) suggests that at any given far-UV dose, there was an equal chance of making a lethal mistake with or without 365-nm-radiation pre-treatment. The chance of an error leading to a mutation, however, was apparently increased (Figs. 3, 4 and 6) by 365-nm radiation so that, even though the induction of error-prone repair may be delayed (see above), it is evident that in the excisionless strain, the lesions remained stable enough to allow its eventual operation. At least two possibilities may be suggested to explain the apparent mutational synergism between the two types of radiation. The first is based on the proposal [11] that at least two types of post-replication repair occur in E. coli, one being constitutive and error-free and the other being an inducible error-prone process. There is evidence (see Introduction) that certain constitutive "rec"-related repair systems are inhibited by near-UV. If the cellular changes induced by 365-nm radiation favour operation of the error-prone post-replication repair system, but result in the same total magnitude of repair, a higher m u t a t i o n rate will result. No lethal synergism would be expected. These considerations will not apply to the excision-proficient strain since the error-prone repair-inducing lesion itself will have been selected out of the population. At higher doses of 365-nm radiation, the inhibition of error-prone repair induction m a y become effectively irreversible and lead to the observed decline in 254-nm radiationinduced mutation (Fig. 7). One factor that is not simply explained by this first suggestion is the lack of 365-nm mutagenesis in the uvrA strain under these experimental conditions. The uvrA strain was inactivated by a dose of 365-nm radiation very close to 106 times greater than the dose necessary to give the same surviving fraction after 254-nm radiation (over the dose range tested; see Figs. 6 and 7). This factor of 106 approximately coincides with the ratio of total lesions known to be induced at the two wavelengths: dimers at 254 nm and dimers and single-
34 strand breaks at 365 nm [18]. So, if the 365-nm lesions are pre-mutagenic, considerable mutagenesis would be expected after 106 J m -2 at 365 nm (equivalent to 1 J m -2 at 254 nm; see Fig. 6), particularly if this radiation does indeed increase the proportion of error-prone compared with error-free repair. It has been suggested by Witkin [27] that near-UV lesions are cryptic, that is, they are susceptible to error-prone repair (but see below) although they are not capable of inducing it. This would then explain why 365-nm radiation alone is poorly mutagenic. Furthermore, when a mixture of 254- and 365-nm radiations are used (in doses equivalent in terms of lesions), the 254-nm-induced error-prone repair would act on the " c r y p t i c " 365-nm lesions and be expected to lead to an increase in the mutation rate, as observed in Fig. 6. Mutation would increase above the 254-nm background as a function of 365-nm dose and be expected to decline at higher doses if the induction of error-prone repair is eventually irreversibly inhibited (Fig. 7). Alternatively, the complete lack of mutagenesis may simply mean that the lesions are not susceptible to errorprone repair. This latter possibility is favoured by experiments (this laboratory) that have shown that 365-nm-induced lesions in phage are not susceptible to Weigle reactivation [ 24] in host cells (repair-proficient or uvrA ) preirradiated at 254 nm. Further evidence is being sought to distinguish between these possible explanations of the mutational interaction in the excisionless strains. The anti-mutagenic action of near-UV radiation in repair-proficient cells and the synergistic interaction in excisionless strains may have significance b e y o n d these fundamental considerations. It has been pointed out [19] that, although dependent on many factors, the daily terrestrial sunlight dose of wavelengths in the range 334--405 nm will often exceed the near-UV doses used in this study. A possible mutagenic interaction between natural sunlight and the growing list of natural or artificially produced mutagens should be considered. Acknowledgements This work was supported by the following Brazilian granting agents: CNPq (National Research Council), CNEN (National Nuclear Energy Council), C P E G / U F R J (University Council for Post-Graduate studies) and FINEP/ FNDCT-375/CT (Study and Project Grants/National Fund for Scientific and Technological Development). The author thanks Dr. L.R. Caldas for his interest and encouragement and Dr. R.D. Ley for helpful discussion and comments on the manuscript. References 1 Bonura, T., and K.C. Smith, Enzymatic production o f deoxyribonucleic acid double-strand breaks after ultraviolet irradiation of Escherichia coli, J. Bacteriol., 121 (1975) 511--517. 2 Bridges, B.A., R.J. M u n s o n , C.F. Arlett and D.R. Davies, Interaction between ultraviolet light and X-radiation d a m a g e in the induction of mutants in Escherichia coli: The effect of s o m e modifying treatments, J. Gen. Microbiol., 46 (1967) 339--346. 3 Davies, D.R., C.F. Arlett, R~I. M u n s o n and B.A. Bridges, Interaction between ultraviolet light and X-radiation d a m a g e in the induction of mutants in Escherichia coli: The response in strains with normal and reduced ability to repair ultraviolet damage, J. Gen. Microbiol., 46 (1967) 329--338. 4 Eisenstark, A., Mutagenic and lethal effects of visible and near-ultraviolet light on bacterial cells, in:
35 E.W. Caspari (Ed.), A d v a n c e s in Genetics, Vol. 12, A c a d e m i c Press, New Y o r k , 1 9 7 1 , p p . 1 6 7 - - 1 9 8 . 5 H a r m , W., Effects of d o s e - f r a c t i o n a t i o n o n ultraviolet survival of E s c h e r i c h i a coli, P h o t o c h e m . P h o t o biol., 7 ( 1 9 6 8 ) 7 3 - - 8 6 . 6 Jagger, J., W. Curtis-Wise a n d R.S. S t a f f o r d , Delay in g r o w t h a n d division i n d u c e d b y near-ultraviolet r a d i a t i o n in Escherichia coli B a n d its role in p h o t o p r o t e c t i o n a n d l i q u i d - h o l d i n g r e c o v e r y , P h o t o chem. Photobiol., 3 (1964) 11--24. 7 K a d a , T., C.O. D o u d n e y a n d F.L. Hass, M u t a t i o n f r e q u e n c y response of i r r a d i a t e d b a c t e r i a to a s e c o n d r a d i a t i o n e x p o s u r e , M u t a t i o n Res., 3 ( 1 9 6 6 ) 1 1 8 - - 1 2 8 . 8 Moss, S.H., a n d D.J.G. Davies, I n t e r r e l a t i o n s h i p of repair m e c h a n i s m 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. Bacteriol., 1 2 0 ( 1 9 7 4 ) 1 5 - - 2 3 . 9 R a d m a n , M., SOS repair h y p o t h e s i s : p h e n o m e n o l o g y of a n inducible D N A repair 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. Setlow (Eds.), Molecular Mechanisms for repair of D N A , p a r t A, P l e n u m , New Y o r k , 1 9 7 5 , p p . 3 4 7 - - 3 5 5 . 10 R a m a b h a d r a n , T.V., Effects o f n e a r - u l t r a v i o l e t a n d violet r a d i a t i o n s (313---405 n m ) o n D N A , R N A , a n d p r o t e i n s y n t h e s i s in E. coli B/r: I m p l i c a t i o n s f o r g r o w t h d e l a y , P h o t o c h e m . P h o t o b i o l . , 22 ( 1 9 7 5 ) 117--123. 11 Sedgwick, S.G., I n d u c i b l e e r r o r - p r o n e repair in Escherichia coli, Proc. Natl. A c a d . Sci. (U.S.A.), 72 (1975) 2753--2757. 12 S e d g w i c k , S.G., Misrepair of o v e r l a p p i n g d a u g h t e r s t r a n d gaps as a possible m e c h a n i s m for UV i n d u c e d m u t a g e n e s i s in U V R strains o f Escherichia coli: A general m o d e l for i n d u c e d m u t a g e n e s i s b y m i s r e p a i r (SOS repair) of closely s p a c e d D N A lesions, M u t a t i o n Res., 41 ( 1 9 7 6 ) 1 8 5 - - 2 0 0 . 13 S w e n s o n , P.A., Physiological r e s p o n s e s of E s c h e r i c h i a coli to far-ultraviolet r a d i a t i o n , in: K.C. S m i t h (Ed.), P h o t o c h e m . P h o t o b i o l . Reviews, Vol. 1, P l e n u m , New Y o r k , 1 9 7 6 , p p . 2 6 8 - - 3 8 7 . 14 S w e n s o n , P.A., a n d R.B. Setlow, I n h i b i t i o n of the i n d u c e d f o r m a t i o n of t r y p t o p h a n a s e in E s c h e r i c h i a coU b y near-ultraviolet r a d i a t i o n , J. Bacteriol., 102 ( 1 9 7 0 ) 8 1 5 - - 8 1 9 . 15 S w e n s o n , P.A., J.E. Ives a n d R.L. S c h e n l e y , P h o t o p r o t e c t i o n of E. coli B/r: R e s p i r a t i o n , g r o w t h , m a c r o m o l e c u l a r s y n t h e s i s a n d repair o f D N A , P h o t o c h e m . P h o t o b i o l . , 21 ( 1 9 7 5 ) 2 3 5 - - 2 4 1 . 16 Tyrrel], R.M., The i n t e r a c t i o n o f near-UV ( 3 6 5 n m ) a n d X-irradiations on w i l d - t y p e a n d repair-defic i e n t strains o f Escherichia coli K 1 2 : Physical a n d biological m e a s u r e m e n t s , Int. J. R a d i a t . Biol., 25 (1974) 373--390. 17 Tyrrell, R.M., Synergistic lethal a c t i o n of ultraviolet r a d i a t i o n s a n d mild h e a t in E s c h e r i c h i a coli, P h o t o c h e m . P h o t o b i o l . , 24 ( 1 9 7 6 ) 345---351. 18 Tyrrell, R.M., L e t h a l cellular changes i n d u c e d b y near-ultraviolet r a d i a t i o n , in: Proceedings of V I t h UV S y m p o s i u m , Ultraviolet R a d i a t i o n in Biology a n d Medicine, K i i h l u n g s b o r n , 1 9 7 7 . 19 Tyrrell, R.M., Solar d o s i m e t r y w i t h r e p a i r d e f i c i e n t b a c t e r i a l spores: A c t i o n s p e c t r a , p h o t o p r o d u c t m e a s u r e m e n t s a n d a c o m p a r i s o n w i t h o t h e r biological systems, P h o t o c h e m . P h o t o b i o l . , (in press). 20 Tyrrell, R.M., R a d i a t i o n synergism a n d a n t a g o n i s m , in: K.C. S m i t h (Ed.), P h o t o c h e m . P h o t o b i o l . Reviews, Vol. 3, P l e n u m , New Y o r k , 1 9 7 8 , pp. 3 5 - - 1 1 3 . 21 TyrreU, R.M., a n d R.B. Webb, R e d u c e d d i m e r excision in b a c t e r i a following n e a r u l t r a v i o l e t ( 3 6 5 n m ) r a d i a t i o n , M u t a t i o n Res., 19 ( 1 9 7 3 ) 3 6 1 - - 3 6 4 . 22 Webb, R.B., Lethal a n d m u t a g e n i c effects o f near-ultraviolet r a d i a t i o n , in: K.C. S m i t h (Ed.), P h o t o c h e m . P h o t o b i o l . Reviews, Vol. 2, P l e n u m , New Y o r k , 1 9 7 7 , p p . 1 6 9 - - 2 6 3 . 23 Webb, S.J., a n d J.S. Bhorjee, The effect of 3 0 0 0 t o 4 0 0 0 A light o n t h e synthesis of ~-galactosidases a n d b a c t e r i o p h a g e s b y Escherichia coli B, Can. J. Microbiol., 13 ( 1 9 6 7 ) 6 9 - - 7 9 . 24 Weigie, J., I n d u c t i o n of m u t a t i o n in a b a c t e r i a l virus, Proc. Natl. Acad. Sci. (U.S.A.), 3 9 ( 1 9 5 3 ) 6 2 8 - 636. 25 Witkin, E.M., The effect o f acriflavine o n p h o t o r e v e r s a l of lethal a n d m u t a g e n i e d a m a g e p r o d u c e d in b e c t e r i a b y u l t r a v i o l e t light, Proc. Natl. A c a d . Sci. (U.S.A.), 50 ( 1 9 6 3 ) 4 2 5 - - 4 3 0 . 26 Witkin, E.M., U l t r a v i o l e t - i n d u c e d m u t a t i o n a n d D N A repair, A n n u . Rev. Microbiol., 23 ( 1 9 6 9 ) 4 8 7 - 514. 27 Witkin, E.M., Ultraviolet m u t a g e n e s i s a n d i n d u c i b l e D N A repair in Escherichia coli, Bacteriol. Rev., 4 0 (1976) 869--907.
