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Inhibitory effect of membrane-binding drugs on excision repair of DNA damage in UV-irradiated Escherichia coli
Mutation Research, 112 (1983) 97-107 DNA Repair Reports
97
Elsevier Biomedical Press
Inhibitory effect of membrane-binding drugs on excision repair of DNA damage in UV-irradiated Escherichia coli Takeshi Todo and Shuji Yonei * Laboratory of Radiation Biology, Faculty of Science, Kyoto University, Kitashirakawa, Kyoto 606 (Japan)
(Received 26 August 1982) (Revision received20 October 1982) (Accepted 26 October 1982)
Summaff The effects of procaine and lidocaine on DNA-repair processes were investigated in UV-irradiated cells of E. coli with different DNA-repair capacities. The cells were irradiated with various doses of UV and then incubated at 37°C in M9 buffer (liquid-holding) or in EM9 medium in the presence or absence of membrane-binding drugs. The results obtained are as follows. (1) In strains H / r 3 0 (wild-type for D N A repair) and N G 3 0 (recA-), the increase in survival with increase in time of liquid-holding was almost completely inhibited by the addition of procaine and lidocaine. The same trends were observable under conditions of post-irradiation incubation in EM9 medium, more efficiently in r e c A - strain than in the wild-type strain. (2) The addition of these drugs gave an apparent enhancement of the frequency of UV-induced mutation to arginine prototrophy, corresponding to a decrease in survival. (3) There were negligible effects of the drugs on survival and mutation in the excision-repair-defective strain, Hs30 (uvrB-). (4) The removal of thymine dimers from D N A was actually reduced by the addition of procaine. From these results it is concluded that procaine and lidocaine inhibited excision-repair process in UV-irradiated E. coli cells. Procaine and lidocaine are typical local anesthetics and known to interact with cell membranes causing alterations in the structural and functional organization. Therefore, it is suggested that a disorganization of the membrane structure brought about by the drugs may result in an inhibition of excision repair of D N A damage in E. coli, assuming that at least a component of excision repair is associated with the cell membrane. Possible mechanisms involved in this process are discussed.
* To whom correspondence should be sent. 0167-8817/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
98 When UV-irradiated cells of certain E. coli strains are held in buffer in the dark before the plating on nutrient agar plates, a gradual increase occurs in the number of cells able to form colonies on the plates [4,15,21]. This recovery phenomenon is called liquid-holding recovery (LHR) [15]. The major repair process that occurs during liquid holding (LH) is believed to be excision repair of UV damage in DNA [2,17,18]. In our earlier paper [28], we reported that membrane-binding drugs such as chlorpromazine and procaine inhibit the L H R in UV-irradiated cells of E. coli B. These drugs are known to interact with cell membranes causing changes in the structural and functional organization [8,10,13,25,29]. Therefore, it was suggested that, possibly, the structural integrity of cell membrane is essential for the excisionrepair process in E. coli [28]. On the other hand, Tang et al. [19] found that both beneficial (excision repair) and deleterious (DNA turnover) processes take place in UV-irradiated and bufferheld E. coli cells. The authors suggested that L H R depends upon a delicate balance between excision repair and DNA turnover [19,21]. In addition, it has been reported that the extent of L H R depends upon not only excision repair but also the efficiency of growth-medium-dependent repair [21]. Accordingly, it was necessary to clarify whether our previous findings about the effect of membrane-binding drugs on L H R are indeed the result of their inhibitory action on excision repair. So, we examined the dark-repair kinetics [5] of E. coli with different DNA-repair capacities in the presence and absence of the drugs as well as the effect of the drugs on the removal of thymine dimers from DNA. Here, we report that procaine and lidocaine inhibit the excision repair of UV-induced thymine dimers in DNA in E. coli cells.
Materials and methods Bacteria Escheriehia coli H / r 3 0 (Uvr+Rec+Phr-), Hs30 ( U v r B - R e c + P h r ) and NG30
( U v r + R e c A - P h r -) were kindly supplied by Drs. T. Kato and S. Kondo, Osaka University. A thymine-requiring mutant of strain H / r 3 0 (E. coli H / r 3 0 t h y - ) was isolated according to Okada et al. [6]. All these strains carry an auxotrophic mutation that requires arginine for growth [5]. Media
Bacterial cells were grown at 37°C with shaking in M9 medium [1] supplemented, at 0.25 mg/ml, with casamino acids (EM9 medium). Nutrient broth agar (8 g Difco Nutrient Broth, 4 g NaC1 and 20 g agar in 1000 ml water, pH 7.2) was used for measurement of survival. Mutation from arginine auxotrophy to prototrophy was assayed on minimal medium E [5] supplemented with or without liquid nutrient broth (5%). U V irradiation
The bacterial cells in exponentially growing phase were washed twice, resus-
99 pended in M9 buffer (M9 medium without glucose) at the concentration of 2 x 108 celts/ml and then UV-irradiated (254 nm) by germicidal lamps (15 W x 2). The fluence rate was 1.5 or 0.15 j / m 2 / s e c , estimated with a UV intensity meter (Topcon UVR254, Tokyo Kogaku, Tokyo). Post-irradiation incubation UV-irradiated cells of E. coli were subsequently held in M9 buffer at 37°C for up to 10 h (LH) or incubated in EM9 medium at 37°C for up to 3 h, in the presence or absence of membrane-binding drugs. Survival assay After holding or incubation, the bacterial cell suspensions were serially diluted and plated on nutrient broth agar containing acriflavine at 2 # g / m l . Acriflavine was added to reduce the repair on agar plates, since it almost completely inhibits excision repair of UV damage by binding with DNA [5,31; our unpublished result]. After the incubation at 37°C for 20 h, the number of viable colonies was counted to estimate the survival. Mutation assay LH, 0.1-0.5 ml samples of cell suspensions (about 2 x 108 cells/ml) were plated on minimal medium E agar with 5% liquid nutrient broth and acriflavine (2/~g/ml) and then incubated at 37°C for about 48 h. Large colonies, easily distinguishable from a background film of arginine auxotrophs, were scored as true revertants. Incubation in EM9. The mutation assay was carried out by almost the same method as L H except for plating on minimal medium E agar. Induced mutation frequencies were calculated as previously described [5]. Measurement of thymine dimers in DNA The cells of E. coli H / r 3 0 t h y - were grown at 37°C for at least 3 generations in EM9 medium supplemented with [6-3H]thymidine (15/~Ci/2/~g/ml). The cultures were harvested, washed twice and resuspended in M9 buffer at 2 x 108 cells/ml. After UV-irradiation, the cell suspensions were incubated at 37°C in M9 buffer or in EM9 medium with or without membrane-binding drugs. The measurement of thymine dimers in acid-insoluble fractions was carried out essentially by the method of Setlow and Carrier [9] with a slight modification [26]. Chemicals Lidocaine hydrochloride was kindly supplied by Fujisawa Pharmaceutical Co. (Osaka). Procaine hydrochloride and acriflavine were purchased from Sigma Chemical Co. (St. Louis, U.S.A.). [6-3H]Thymidine (15 Ci/mmole) was the product of New England Nuclear (Boston, U.S.A.).
IOO Results
Effect of procaine and lidocaine on L H R in UV-irradiated E. coli Procaine and lidocaine are typical local anesthetics k n o w n to b i n d to cell m e m b r a n e s of bacteria [8,13,22] as well as of m a m m a l i a n cells [10,25]. In the first series of experiments, we re-examined the effect of m e m b r a n e - b i n d i n g drugs like procaine o n L H R in UV-irradiated E. coli with different D N A repair capacities. The results are shown in Fig. 1. Procaine a n d lidocaine had no killing effect on u n i r r a d i a t e d E. coli cells. In strains H / r 3 0 (wild-type for D N A repair) and N G 3 0 ( r e c A - mutant), the increase in survival with time of L H was almost completely reduced by the a d d i t i o n of these drugs. The drugs showed almost complete inhibition o n L H R in both strains. The i n h i b i t o r y effect of lidocaine was more effective t h a n that of procaine. Fig. 2 - s h o w s ~that the U V - i n d u c c d mutation-£~equencyg r a d u a l ~ d e c r e a s e d w i t h time of L H in strain H / r 3 0 , a n d that procaine a n d lidocaine prevented it markedly. The addition of these drugs gave negligible effects on survival a n d m u t a t i o n in strain Hs30 ( u v r B - mutant), which is genetically defective in excision repair of D N A d a m a g e a n d shows no L H R (data not shown).
Effect of procaine on dark repair in E. coli with different D N A repair capacities We next attempted to examine the effect of m e m b r a n e - b i n d i n g drugs o n the repair of D N A u n d e r different conditions of post-irradiation i n c u b a t i o n from LH.
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Fig. 1. The effect of procaine and lidocaine on LHR in UV-irradiated cells of E. coli H/r30 and NG30. The cells of E. coli H/r30 (wild-type for DNA repair) and NG30 (recA-) were irradiated with 49.5 and 2.7 J/m 2, respectively, and then held in buffer at 37°C with or without the membrane-binding drugs. After holding, the cell suspensions were serially diluted with M9 buffer and plated on nutrient broth agar . . . . t . . containing acrlflavme (2 #g/ml). These drugs did not affect survwal of unirradiated control cells of these strains (data not shown). (a) E. coli H/r30, (b),E. coli NG30. C) C), without drugs; • 0, with procaine (12.5 raM); • . . . . . . 0, with procaine (25 mM); • •, with lidocaine (7.5 mM); • . . . . . . •, with lidocaine (15 mM).
101
We compared the dark-repair kinetics [5] of El" coil with different DNA repair capacities, wild-type for DNA repair, uvrB- and recA- strains, in the presence and absence of the drugs. UV-irradiated cells were incubated at 37°C for various times
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Fig. 2. The effect of procaine and lidocaine on the decrase in UV-induced mutation frequency with time of LH in E. coli H/r30. The bacterial cells were irradiated with 49.5 J / m 2 and then held in M9 buffer as described under Fig. I. Mutation assay to arginine prototrophy was carried out on minimal medium E agar containing both liquid nutrient broth (5%) and acriflavine (2 ~tg/ml). Symbols are the same as those for Fig. I.
in EM9 medium and subsequently plated on nutrient broth agar with acriflavine (for survival assay) or on minimal medium E agar (for mutation assay). Because acriflavine almost completely inhibits the repair on agar plates [5,31], its addition to the plates allowed us to observe the repair process(es) that occurs during incubation in EM9 medium just after irradiation. Fig. 3 shows the dark-repair kinetics of 3 strains in the presence and absence of procaine. The drug showed only a small killing effect on unirradiated control cells of the 3 strains when added at 25 mM. In wild-type and recA- cells, the survival gradually increased with time of post-irradiation incubation in EM9 medium. The addition of procaine resulted marked inhibition of the increase in survival. The effect increased with increase in concentration of the drug. On the other hand+ there was only a small extent of recovery, but no significant effect of the drug was observed on survival of the excision-repair-defective mutant, Hs30. Furthermore+ acriflavine added to the post-irradiation incubation medium exerted an effect similar to that of procaine (data not shown). The kinetics of mutation induction by UV in strains H / r 3 0 and Hs30 are shown in Fig. 4. UV-induced mutation to arginine prototrophy was expressed linearly with time of post-irradiation incubation and reached the maximal level 120 min after
102
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Fig. 3. Dark-repair kinetics of UV-irradiated E. coli with different DNA-repair capacities in the presence and absence of procaine. The cells of E, coli H/r30 (uvr + rec + ), Hs30 (uvrB ) and NG30 (recA-) were irradiated with 49.5, 3 and 2.7 J / m 2, respectively, and subsequently incubated at 37°C in EM9 medium with or without procaine. Survival assay was carried out on nutrient broth agar containing acriflavine (2 #g/ml). (a) H/r30, (b) Hs30 and (c) NG30. - - , unirradiated; . . . . . . , irradiated with UV; C), without procaine; L with procaine (12.5 mM); A, with procaine (25 mM). i r r a d i a t i o n . In strain H / r 3 0 , the a d d i t i o n o f p r o c a i n e r e s u l t e d in an a p p a r e n t e n h a n c e m e n t o f m u t a t i o n f r e q u e n c y , c o m p a r e d w i t h the c o n t r o l w i t h o u t p r o c a i n e . H o w e v e r , the m u t a t i o n f r e q u e n c y in strain H s 3 0 w a s n o t a f f e c t e d b y the a d d i t i o n o f procaine. (a)
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Fig. 5. Fluence-effect curves of UV-induced mutation in E. coil H / r 3 0 and Hs30 under various conditions of post-irradiation incubation. Closed symbols, E. coli H / r 3 0 ; open symbols, E. coli Hs30. Procaine was added to final concentrations of 0 m M (O, o), 6 m M (A, ,',), 12.5 m M (v, v) and 25 m M
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Fig. 6. The effect of procaine on the removal of thymine dimers from DNA. The cells of E. coil H / r 3 0 t h y - were labeled with [6-3H]thymidine (15 # C i / 2 # g / m l ) , irradiated with UV (49.5 J / m E) and then incubated at 37°C in M9 buffer (a) or in EM9 medium (b) in the presence (o . . . . . . O) or absence (© O ) of procaine (25 mM). Thymine dimers in acid-insoluble fractions were measured by essentially the method described by Setlow and Carrier [9].
104
Fig. 5 shows the fluence-effect curves of mutation induction by UV under various conditions of post-irradiation incubation. An enhancing effect of procaine on the mutation frequencies was dependent upon the drug concentration, although the maximal level did not reach that observed in strain Hs30. Effect of procaine on the removal of thymine dimers from DNA The cells of E. coli H / r 3 0 t h y were labeled with [6-3H]thymidine and irradiated with 49.5 J / m z, followed by incubation at 37°C for various times. Thymine dimers in DNA were measured by the DNA-hydrolysis-paper-chromatography method originally described by Setlow and Carrier [9]. Fig. 6 shows the time course of the removal of thymine dimers from DNA in cells incubated in M9 buffer (a) or in EM9 medium (b) after UV-irradiation. Under both conditions, procaine reduced the removal of thymine dimers from DNA, significantly if not completely.
Discussion
Membrane-binding drugs such as chlorpromazine and procaine sensitize the killing effect of ionizing radiation on E. coli preferentially under hypoxic conditions of irradiation [11,12,14,27]. The radiosensitizing effect may be partly due to the inhibitory action of the drugs on the repair of radiation-induced damage to DNA [12,14,27]. The repair of DNA and ultimate survival in irradiated E. co// are modulated by the physical state (fluidity) of lipids of the cell membrane [23,24]. These characteristics are common in membrane-related phenomena. Therefore, these observations may indicate that DNA repair in E. coli following exposure to radiation is at least partly associated with the cell membrane. However, there are only few reports about possible involvement of the cell membrane in DNA repair processes in UV-irradiated E. coli. In our earlier papers [28,30], we reported that membrane-binding drugs like procaine inhibited L H R in UV-irradiated E. coli B. As L H R only occurs in excision-repair-proficient cells [2,14,17,18], it seemed possible that a part of the excision-repair process is associated with the cell membrane [28,30]. However, Tang et al. [19] have suggested that LHR depends upon a delicate balance between DNA turnover and excision repair of UV damage. Therefore, more direct evidence was required for an understanding of mechanisms of the action of membrane-binding drugs on excision repair in E. coli. Local anesthetics like procaine and tranquillizers like chlorpromazine are known to cause important effects on bacterial cell membranes, including structural disorganization and inhibition of membrane-bound enzymatic activities [13,22,29]. These effects are dependent upon the hydrophobic interactions of the drugs with membranes [8,13], which are indicated by measurement of octanol-water partition coefficients [13]. The logarithmic values of the partition coefficients of procaine, lidocaine and another local anesthetic, tetracaine, were 1.9, 2.3 and 3.3, respectively. As can be seen in Fig~ 1, the inhibitory effect of lidocaine on LHR was more effective than that of procaine. In addition, tetracaine was the most effective of the 3 drugs on L H R (our unpublished results). These findings corresponded well to the
105 measured octanol-water partition coefficients of the 3 drugs. Therefore, it is concluded that the above-mentioned effects result from a disturbance a n d / o r disorganization of the cell membrane induced by these drugs. From the present experimental results it is concluded that the addition of procaine and lidocaine to the post-irradiation incubation medium results in the inhibition of an excision-repair process operating on pyrimidine dimers in DNA in E. coli, in consideration of the following facts. (1) Ganesan and Smith [2], Tang and Patrick [20] and Smith [16] have shown that excision repair alone is sufficient for L H R in recA- strains. As is evident from Fig. 1, procaine and lidocaine almost completely inhibited L H R in strain NG30 (recA- mutant). The same trends were observed under conditions of post-irradiation incubation in EM9 medium, more efficiently in NG30 than in wild-type strain H / r 3 0 (Fig. 3). (2) There were negligible effects of the drugs on survival and UV-induced mutation in excision-repair-defective strain Hs30 (Figs. 3, 4 and 5). (3) The addition of procaine apparently enhanced the frequencies of UV-induced mutation, correspondingly to a decrease in survival (Figs. 1-5). The fluence-mutation induction curves (Fig. 5) also confirmed the effect of procaine. This observation is probably not due to a direct enhancement of error-prone DNA repair (SOS repair) [32], since there was no enhancing effect on mutation in strain Hs30 (Figs. 4 and 5). (4) The removal of thymine dimers from DNA was actually reduced by the addition of procaine (Fig. 6). Pyrimidine dimers produced by UV in DNA may be removed from the DNA by a multi-enzymatic mechanism, excision repair [3,7,9,16,32]. In E. coli, pyrimidine dimers are recognized by a UV endonuclease (a complex of uvrA, uvrB and uvrC gene products [7]), which makes a single-strand nick, or incision, on the 5' side of the dimer. Subsequently, an exonucleolytic excision releases the oligonucleotides containing the dimers, and the excision gaps are patched by repair replication, the intact region of the strand opposite the gaps serving as template for replacement of the missing nucleotides. Finally, the DNA strand is sealed by DNA ligation [7,9,16,32]. No doubt the addition of membrane-binding drugs like procaine results in an inhibition of the initial step, incision, a n d / o r the excision step in the repair process, because the removal of thymine dimers was inhibited by the drugs (Fig. 6). However, the molecular and biochemical mechanisms of the inhibition were not elucidated in this experiment. Some factor in the excision repair process may be bound to the cell membrane, which may be affected by a structural disorganization brought about by membrane-binding drugs. Alternatively, the excision-repair process is a co-ordinated series of biochemical reactions as mentioned above [3,32]. Although the manner in which all the steps are co-ordinated is not understood, the structural disorganization of the cell membrane may result in a disturbance of this co-ordination. The disturbance may lead to lethal double-strand breaks of DNA by overlapping of excision gaps. It is also possible that, if attachment of DNA to cell membrane is essential for DNA repair, this process may be susceptible to drugs affecting the structure of the cell membrane. Furthermore, there may be other possible mechanisms involved iri the inhibition of excision repair by membrane-binding drugs. Work along these lines is currently being undertaken in this laboratory.
106
Acknowledgements We express our gratitude to Dr. M.A. Shenoy of the Biology and Agriculture Division, Bhabha Atomic Research Center of India for helpful discussions. This work was partly supported by a grant for scientific research from the Ministry of Education, Science and Culture of Japan.
References 1 Davis, R.W., D. Botstein and J.R. Roth, Media, drug concentration, and nutritional supplements, in: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, New York, 1980, pp. 201-207. 2 Ganesan, A.K., and K.C. Smith, Dark recovery processes in Escherichia coli irradiated with ultraviolet light, II. Effect of uvr genes on liquid holding recovery, J. Bacteriol., 97 (1969) 1129-1133. 3 Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith, DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48 (1979) 783-836. 4 Jagger, J., W.C. Wise and R.S. Stafford, Delay in growth and division induced by near ultraviolet radiation in Escherichia coli B and its role in photoprotection and liquid holding recovery, Photochem. Photobiol., 3 (1964) 11-24. 5 Kondo, S., H. Ichikawa, K. lwo and T. Kato, Base-change mutagenesis and prophage induction in strains of Escherichia coli with different DNA repair capacities, Genetics, 66 (1970) 187-217. 6 Okada, T., K. Yanagisawa and F.J. Ryan, Elective production of thymine-less mutants, Nature (London), 188 (1960) 340-341. 7 Seeberg, E., Multiprotein interactions in strand cleavage of DNA damaged by UV and chemicals, Prog. Nucleic Acid Res. Mol. Biol., 26 (1981) 217-225. 8 Seeman, P., The membrane actions of anesthetics and tranquillizers, Pharmacol. Rev., 24 (1972) 583-655. 9 Setlow, R.B., and W.L. Carrier, The disappearance of thymine dimers from DNA: an error-correcting mechanism, Proc. Natl. Acad. Sci. (U.S.A.), 51 (1964) 226-231. 10 Sheetz, M.P., and S.J. Singer, Biological membranes as bilayer couples, A molecular mechanism of drug-erythrocyte interactions, Proc. Natl. Acad. Sci. (U.S.A.), 71 (1974) 4457-4461. 11 Shenoy, M.A., K.C. George, B.B. Singh and A.R. Gopal-Ayengar, Modification of radiation effects in single-cell systems by membrane-binding agents, Int. J. Radiat. Biol., 28 (1975) 519-526. 12 Shenoy, M.A., and K. Gopalakrishna, Biochemical aspects of radiation sensitization of E. coli B / r by chlorpromazine, Int. J. Radiat. Biol., 33 (1978) 587-593. 13 Silva, M.T., J.C.F. Sousa, J.J. Polbnia and P.M. Macedo, Effects of local anesthetics on bacterial cells, J. Bacteriol., 137 (1979) 461-468. 14 Singh, B.B., M.A. Shenoy and K.C. George, Nature of radiation and chemically induced lesions and role of cellular mechanisms in cell survival and mutagenesis, I. Membrane and cellular repair, Adv. Biol. Med. Phys., 17 (1980) 109-113. 15 Smith K.C., Ultraviolet radiation effect on molecules and cells, in: K.C. Smith (Ed.), The Science of Photobiology, Plenum, New York, 1977, pp. 113-142. 16 Smith, K.C., Multiple pathways of DNA repair in bacteria and their roles in mutagenesis, Photochem. Photobiol., 28 (1978) 121-129. 17 Swenson, P.A., Physiological responses of Escherichia coli to far-ultraviolet radiation, in: K.C. Smith (Ed.), Photochemical and Photobiological Review, Vol. I, Plenum, New York, 1976, pp. 269-385. 18 Tang, M., and M.H. Patrick, The role of DNA polymerase I in liquid holding recovery of UV-irradiated E. coli, Photochem. Photobiol., 26 (1977) 257-262. 19 Tang, M., T.V. Wang and M.H. Patrick, DNA turnover in buffer-held E. coil and its effect on repair of UV damage, Photochem. Photobiol., 29 (1979) 511-520. 20 Tang, M., and M.H. Patrick, Repair of UV damage in Escherichia coli under non-growth conditions, Photochem. Photobiol., 26 (1977) 247-255.
107 21 Tang, M., and K.C. Smith, The expression o$ liquid holding recovery in ultraviolet-irradiated Escherichia coli requires a deficiency in growth medium-dependent DNA repair, Photochem. Photobiol., 32 (1980) 763-769. 22 Tribhuwan, R.C., and D.S. Pradhan, Induction of alkaline phosphatase in Escherichia coil: Effect of procaine hydrochloride, J. Bacteriol., 131 (1977) 431-437. 23 Yatvin, M.B., Evidence that survival of gamma-irradiated Escherichia coli is influenced by membrane fluidity, Int. J. Radiat. Biol., 30 (1976) 571-574. 24 Yatvin, M.B., J.J. Gipp and W.H. Dennis, Influence of unsaturated fatty acids, membrane fluidity and oxygenation on the survival of an E. coli fatty acid auxotroph following 7-irradiation, Int. J. Radiat. Biol., 35 (1979) 539-548. 25 Yau, T.M., Procaine-mediated modification of membranes and of the response to X-irradiation and hyperthermia in mammalian cells, Radiat. Res., 80 (1979) 523-541. 26 Yonei, S., and K. Nozu, Change in the base composition of messenger RNA of Escherichia coli by ultraviolet irradiation and its recovery, Photochem. Photobiol., 9 (1969) 537-548. 27 Yonei, S., Modification of radiation effects on E. coil B/r and a radiosensitive mutant B~_ 1 by membrane-binding drugs, Int. J. Radiat. Biol., 36 (1979) 547-551. 28 Yonei, S., Inhibitory effect of membrane-specificdrugs on liquid-holding recovery in UV-irradiated E. coli cells, Int. J. Radiat. Biol., 37 (1980) 685-689. 29 Yonei, S., and T. Todo, Enhanced sensitivity to the lethal and mutagenic effects of photosensitizing action of chlorpromazine in ethylenediaminetetraacetate-treated Escherichia coil, Photochem. Photobiol., 35 (1982) 591-592. 30 Yonei, S., The physical and chemical nature of membrane and radiosensitivity, J. Radiat. Res., 23 0982) 3 (abstract). 31 Witkin, E.M., Modification of mutagenesis initiated by ultraviolet light through post-treatment of bacteria with basic dyes, J. Cell. Comp. Physiol., 58 (Suppl. l) (1961) 135-144. 32 Witkin, E.M., Ultraviolet mutagenesis and inducible DNA repair in Escherichia coil, Bacteriol. Rev., 40 (1976) 869-907.
97
Elsevier Biomedical Press
Inhibitory effect of membrane-binding drugs on excision repair of DNA damage in UV-irradiated Escherichia coli Takeshi Todo and Shuji Yonei * Laboratory of Radiation Biology, Faculty of Science, Kyoto University, Kitashirakawa, Kyoto 606 (Japan)
(Received 26 August 1982) (Revision received20 October 1982) (Accepted 26 October 1982)
Summaff The effects of procaine and lidocaine on DNA-repair processes were investigated in UV-irradiated cells of E. coli with different DNA-repair capacities. The cells were irradiated with various doses of UV and then incubated at 37°C in M9 buffer (liquid-holding) or in EM9 medium in the presence or absence of membrane-binding drugs. The results obtained are as follows. (1) In strains H / r 3 0 (wild-type for D N A repair) and N G 3 0 (recA-), the increase in survival with increase in time of liquid-holding was almost completely inhibited by the addition of procaine and lidocaine. The same trends were observable under conditions of post-irradiation incubation in EM9 medium, more efficiently in r e c A - strain than in the wild-type strain. (2) The addition of these drugs gave an apparent enhancement of the frequency of UV-induced mutation to arginine prototrophy, corresponding to a decrease in survival. (3) There were negligible effects of the drugs on survival and mutation in the excision-repair-defective strain, Hs30 (uvrB-). (4) The removal of thymine dimers from D N A was actually reduced by the addition of procaine. From these results it is concluded that procaine and lidocaine inhibited excision-repair process in UV-irradiated E. coli cells. Procaine and lidocaine are typical local anesthetics and known to interact with cell membranes causing alterations in the structural and functional organization. Therefore, it is suggested that a disorganization of the membrane structure brought about by the drugs may result in an inhibition of excision repair of D N A damage in E. coli, assuming that at least a component of excision repair is associated with the cell membrane. Possible mechanisms involved in this process are discussed.
* To whom correspondence should be sent. 0167-8817/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
98 When UV-irradiated cells of certain E. coli strains are held in buffer in the dark before the plating on nutrient agar plates, a gradual increase occurs in the number of cells able to form colonies on the plates [4,15,21]. This recovery phenomenon is called liquid-holding recovery (LHR) [15]. The major repair process that occurs during liquid holding (LH) is believed to be excision repair of UV damage in DNA [2,17,18]. In our earlier paper [28], we reported that membrane-binding drugs such as chlorpromazine and procaine inhibit the L H R in UV-irradiated cells of E. coli B. These drugs are known to interact with cell membranes causing changes in the structural and functional organization [8,10,13,25,29]. Therefore, it was suggested that, possibly, the structural integrity of cell membrane is essential for the excisionrepair process in E. coli [28]. On the other hand, Tang et al. [19] found that both beneficial (excision repair) and deleterious (DNA turnover) processes take place in UV-irradiated and bufferheld E. coli cells. The authors suggested that L H R depends upon a delicate balance between excision repair and DNA turnover [19,21]. In addition, it has been reported that the extent of L H R depends upon not only excision repair but also the efficiency of growth-medium-dependent repair [21]. Accordingly, it was necessary to clarify whether our previous findings about the effect of membrane-binding drugs on L H R are indeed the result of their inhibitory action on excision repair. So, we examined the dark-repair kinetics [5] of E. coli with different DNA-repair capacities in the presence and absence of the drugs as well as the effect of the drugs on the removal of thymine dimers from DNA. Here, we report that procaine and lidocaine inhibit the excision repair of UV-induced thymine dimers in DNA in E. coli cells.
Materials and methods Bacteria Escheriehia coli H / r 3 0 (Uvr+Rec+Phr-), Hs30 ( U v r B - R e c + P h r ) and NG30
( U v r + R e c A - P h r -) were kindly supplied by Drs. T. Kato and S. Kondo, Osaka University. A thymine-requiring mutant of strain H / r 3 0 (E. coli H / r 3 0 t h y - ) was isolated according to Okada et al. [6]. All these strains carry an auxotrophic mutation that requires arginine for growth [5]. Media
Bacterial cells were grown at 37°C with shaking in M9 medium [1] supplemented, at 0.25 mg/ml, with casamino acids (EM9 medium). Nutrient broth agar (8 g Difco Nutrient Broth, 4 g NaC1 and 20 g agar in 1000 ml water, pH 7.2) was used for measurement of survival. Mutation from arginine auxotrophy to prototrophy was assayed on minimal medium E [5] supplemented with or without liquid nutrient broth (5%). U V irradiation
The bacterial cells in exponentially growing phase were washed twice, resus-
99 pended in M9 buffer (M9 medium without glucose) at the concentration of 2 x 108 celts/ml and then UV-irradiated (254 nm) by germicidal lamps (15 W x 2). The fluence rate was 1.5 or 0.15 j / m 2 / s e c , estimated with a UV intensity meter (Topcon UVR254, Tokyo Kogaku, Tokyo). Post-irradiation incubation UV-irradiated cells of E. coli were subsequently held in M9 buffer at 37°C for up to 10 h (LH) or incubated in EM9 medium at 37°C for up to 3 h, in the presence or absence of membrane-binding drugs. Survival assay After holding or incubation, the bacterial cell suspensions were serially diluted and plated on nutrient broth agar containing acriflavine at 2 # g / m l . Acriflavine was added to reduce the repair on agar plates, since it almost completely inhibits excision repair of UV damage by binding with DNA [5,31; our unpublished result]. After the incubation at 37°C for 20 h, the number of viable colonies was counted to estimate the survival. Mutation assay LH, 0.1-0.5 ml samples of cell suspensions (about 2 x 108 cells/ml) were plated on minimal medium E agar with 5% liquid nutrient broth and acriflavine (2/~g/ml) and then incubated at 37°C for about 48 h. Large colonies, easily distinguishable from a background film of arginine auxotrophs, were scored as true revertants. Incubation in EM9. The mutation assay was carried out by almost the same method as L H except for plating on minimal medium E agar. Induced mutation frequencies were calculated as previously described [5]. Measurement of thymine dimers in DNA The cells of E. coli H / r 3 0 t h y - were grown at 37°C for at least 3 generations in EM9 medium supplemented with [6-3H]thymidine (15/~Ci/2/~g/ml). The cultures were harvested, washed twice and resuspended in M9 buffer at 2 x 108 cells/ml. After UV-irradiation, the cell suspensions were incubated at 37°C in M9 buffer or in EM9 medium with or without membrane-binding drugs. The measurement of thymine dimers in acid-insoluble fractions was carried out essentially by the method of Setlow and Carrier [9] with a slight modification [26]. Chemicals Lidocaine hydrochloride was kindly supplied by Fujisawa Pharmaceutical Co. (Osaka). Procaine hydrochloride and acriflavine were purchased from Sigma Chemical Co. (St. Louis, U.S.A.). [6-3H]Thymidine (15 Ci/mmole) was the product of New England Nuclear (Boston, U.S.A.).
IOO Results
Effect of procaine and lidocaine on L H R in UV-irradiated E. coli Procaine and lidocaine are typical local anesthetics k n o w n to b i n d to cell m e m b r a n e s of bacteria [8,13,22] as well as of m a m m a l i a n cells [10,25]. In the first series of experiments, we re-examined the effect of m e m b r a n e - b i n d i n g drugs like procaine o n L H R in UV-irradiated E. coli with different D N A repair capacities. The results are shown in Fig. 1. Procaine a n d lidocaine had no killing effect on u n i r r a d i a t e d E. coli cells. In strains H / r 3 0 (wild-type for D N A repair) and N G 3 0 ( r e c A - mutant), the increase in survival with time of L H was almost completely reduced by the a d d i t i o n of these drugs. The drugs showed almost complete inhibition o n L H R in both strains. The i n h i b i t o r y effect of lidocaine was more effective t h a n that of procaine. Fig. 2 - s h o w s ~that the U V - i n d u c c d mutation-£~equencyg r a d u a l ~ d e c r e a s e d w i t h time of L H in strain H / r 3 0 , a n d that procaine a n d lidocaine prevented it markedly. The addition of these drugs gave negligible effects on survival a n d m u t a t i o n in strain Hs30 ( u v r B - mutant), which is genetically defective in excision repair of D N A d a m a g e a n d shows no L H R (data not shown).
Effect of procaine on dark repair in E. coli with different D N A repair capacities We next attempted to examine the effect of m e m b r a n e - b i n d i n g drugs o n the repair of D N A u n d e r different conditions of post-irradiation i n c u b a t i o n from LH.
(b)
(o)
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O
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Fig. 1. The effect of procaine and lidocaine on LHR in UV-irradiated cells of E. coli H/r30 and NG30. The cells of E. coli H/r30 (wild-type for DNA repair) and NG30 (recA-) were irradiated with 49.5 and 2.7 J/m 2, respectively, and then held in buffer at 37°C with or without the membrane-binding drugs. After holding, the cell suspensions were serially diluted with M9 buffer and plated on nutrient broth agar . . . . t . . containing acrlflavme (2 #g/ml). These drugs did not affect survwal of unirradiated control cells of these strains (data not shown). (a) E. coli H/r30, (b),E. coli NG30. C) C), without drugs; • 0, with procaine (12.5 raM); • . . . . . . 0, with procaine (25 mM); • •, with lidocaine (7.5 mM); • . . . . . . •, with lidocaine (15 mM).
101
We compared the dark-repair kinetics [5] of El" coil with different DNA repair capacities, wild-type for DNA repair, uvrB- and recA- strains, in the presence and absence of the drugs. UV-irradiated cells were incubated at 37°C for various times
10-4 I
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~ o l
L
5
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Fig. 2. The effect of procaine and lidocaine on the decrase in UV-induced mutation frequency with time of LH in E. coli H/r30. The bacterial cells were irradiated with 49.5 J / m 2 and then held in M9 buffer as described under Fig. I. Mutation assay to arginine prototrophy was carried out on minimal medium E agar containing both liquid nutrient broth (5%) and acriflavine (2 ~tg/ml). Symbols are the same as those for Fig. I.
in EM9 medium and subsequently plated on nutrient broth agar with acriflavine (for survival assay) or on minimal medium E agar (for mutation assay). Because acriflavine almost completely inhibits the repair on agar plates [5,31], its addition to the plates allowed us to observe the repair process(es) that occurs during incubation in EM9 medium just after irradiation. Fig. 3 shows the dark-repair kinetics of 3 strains in the presence and absence of procaine. The drug showed only a small killing effect on unirradiated control cells of the 3 strains when added at 25 mM. In wild-type and recA- cells, the survival gradually increased with time of post-irradiation incubation in EM9 medium. The addition of procaine resulted marked inhibition of the increase in survival. The effect increased with increase in concentration of the drug. On the other hand+ there was only a small extent of recovery, but no significant effect of the drug was observed on survival of the excision-repair-defective mutant, Hs30. Furthermore+ acriflavine added to the post-irradiation incubation medium exerted an effect similar to that of procaine (data not shown). The kinetics of mutation induction by UV in strains H / r 3 0 and Hs30 are shown in Fig. 4. UV-induced mutation to arginine prototrophy was expressed linearly with time of post-irradiation incubation and reached the maximal level 120 min after
102
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I
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2
3
Fig. 3. Dark-repair kinetics of UV-irradiated E. coli with different DNA-repair capacities in the presence and absence of procaine. The cells of E, coli H/r30 (uvr + rec + ), Hs30 (uvrB ) and NG30 (recA-) were irradiated with 49.5, 3 and 2.7 J / m 2, respectively, and subsequently incubated at 37°C in EM9 medium with or without procaine. Survival assay was carried out on nutrient broth agar containing acriflavine (2 #g/ml). (a) H/r30, (b) Hs30 and (c) NG30. - - , unirradiated; . . . . . . , irradiated with UV; C), without procaine; L with procaine (12.5 mM); A, with procaine (25 mM). i r r a d i a t i o n . In strain H / r 3 0 , the a d d i t i o n o f p r o c a i n e r e s u l t e d in an a p p a r e n t e n h a n c e m e n t o f m u t a t i o n f r e q u e n c y , c o m p a r e d w i t h the c o n t r o l w i t h o u t p r o c a i n e . H o w e v e r , the m u t a t i o n f r e q u e n c y in strain H s 3 0 w a s n o t a f f e c t e d b y the a d d i t i o n o f procaine. (a)
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2 30 1 2 Post-irradiation incubation (h)
1
3
F i g . 4. The effect of procaine on the frequencies of UV-induced mutation to arginine prototrophy in E. H / r 3 0 and Hs30. Post-irradiation incubation was carried out as described below Fig. 3. Symbols are the same as those for Fig. 3.
coli
103
/ 10 -4
b
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100
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Fig. 5. Fluence-effect curves of UV-induced mutation in E. coil H / r 3 0 and Hs30 under various conditions of post-irradiation incubation. Closed symbols, E. coli H / r 3 0 ; open symbols, E. coli Hs30. Procaine was added to final concentrations of 0 m M (O, o), 6 m M (A, ,',), 12.5 m M (v, v) and 25 m M
(a, D).
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P o s t - i r r a d i a t i o n incubation (h)
Fig. 6. The effect of procaine on the removal of thymine dimers from DNA. The cells of E. coil H / r 3 0 t h y - were labeled with [6-3H]thymidine (15 # C i / 2 # g / m l ) , irradiated with UV (49.5 J / m E) and then incubated at 37°C in M9 buffer (a) or in EM9 medium (b) in the presence (o . . . . . . O) or absence (© O ) of procaine (25 mM). Thymine dimers in acid-insoluble fractions were measured by essentially the method described by Setlow and Carrier [9].
104
Fig. 5 shows the fluence-effect curves of mutation induction by UV under various conditions of post-irradiation incubation. An enhancing effect of procaine on the mutation frequencies was dependent upon the drug concentration, although the maximal level did not reach that observed in strain Hs30. Effect of procaine on the removal of thymine dimers from DNA The cells of E. coli H / r 3 0 t h y were labeled with [6-3H]thymidine and irradiated with 49.5 J / m z, followed by incubation at 37°C for various times. Thymine dimers in DNA were measured by the DNA-hydrolysis-paper-chromatography method originally described by Setlow and Carrier [9]. Fig. 6 shows the time course of the removal of thymine dimers from DNA in cells incubated in M9 buffer (a) or in EM9 medium (b) after UV-irradiation. Under both conditions, procaine reduced the removal of thymine dimers from DNA, significantly if not completely.
Discussion
Membrane-binding drugs such as chlorpromazine and procaine sensitize the killing effect of ionizing radiation on E. coli preferentially under hypoxic conditions of irradiation [11,12,14,27]. The radiosensitizing effect may be partly due to the inhibitory action of the drugs on the repair of radiation-induced damage to DNA [12,14,27]. The repair of DNA and ultimate survival in irradiated E. co// are modulated by the physical state (fluidity) of lipids of the cell membrane [23,24]. These characteristics are common in membrane-related phenomena. Therefore, these observations may indicate that DNA repair in E. coli following exposure to radiation is at least partly associated with the cell membrane. However, there are only few reports about possible involvement of the cell membrane in DNA repair processes in UV-irradiated E. coli. In our earlier papers [28,30], we reported that membrane-binding drugs like procaine inhibited L H R in UV-irradiated E. coli B. As L H R only occurs in excision-repair-proficient cells [2,14,17,18], it seemed possible that a part of the excision-repair process is associated with the cell membrane [28,30]. However, Tang et al. [19] have suggested that LHR depends upon a delicate balance between DNA turnover and excision repair of UV damage. Therefore, more direct evidence was required for an understanding of mechanisms of the action of membrane-binding drugs on excision repair in E. coli. Local anesthetics like procaine and tranquillizers like chlorpromazine are known to cause important effects on bacterial cell membranes, including structural disorganization and inhibition of membrane-bound enzymatic activities [13,22,29]. These effects are dependent upon the hydrophobic interactions of the drugs with membranes [8,13], which are indicated by measurement of octanol-water partition coefficients [13]. The logarithmic values of the partition coefficients of procaine, lidocaine and another local anesthetic, tetracaine, were 1.9, 2.3 and 3.3, respectively. As can be seen in Fig~ 1, the inhibitory effect of lidocaine on LHR was more effective than that of procaine. In addition, tetracaine was the most effective of the 3 drugs on L H R (our unpublished results). These findings corresponded well to the
105 measured octanol-water partition coefficients of the 3 drugs. Therefore, it is concluded that the above-mentioned effects result from a disturbance a n d / o r disorganization of the cell membrane induced by these drugs. From the present experimental results it is concluded that the addition of procaine and lidocaine to the post-irradiation incubation medium results in the inhibition of an excision-repair process operating on pyrimidine dimers in DNA in E. coli, in consideration of the following facts. (1) Ganesan and Smith [2], Tang and Patrick [20] and Smith [16] have shown that excision repair alone is sufficient for L H R in recA- strains. As is evident from Fig. 1, procaine and lidocaine almost completely inhibited L H R in strain NG30 (recA- mutant). The same trends were observed under conditions of post-irradiation incubation in EM9 medium, more efficiently in NG30 than in wild-type strain H / r 3 0 (Fig. 3). (2) There were negligible effects of the drugs on survival and UV-induced mutation in excision-repair-defective strain Hs30 (Figs. 3, 4 and 5). (3) The addition of procaine apparently enhanced the frequencies of UV-induced mutation, correspondingly to a decrease in survival (Figs. 1-5). The fluence-mutation induction curves (Fig. 5) also confirmed the effect of procaine. This observation is probably not due to a direct enhancement of error-prone DNA repair (SOS repair) [32], since there was no enhancing effect on mutation in strain Hs30 (Figs. 4 and 5). (4) The removal of thymine dimers from DNA was actually reduced by the addition of procaine (Fig. 6). Pyrimidine dimers produced by UV in DNA may be removed from the DNA by a multi-enzymatic mechanism, excision repair [3,7,9,16,32]. In E. coli, pyrimidine dimers are recognized by a UV endonuclease (a complex of uvrA, uvrB and uvrC gene products [7]), which makes a single-strand nick, or incision, on the 5' side of the dimer. Subsequently, an exonucleolytic excision releases the oligonucleotides containing the dimers, and the excision gaps are patched by repair replication, the intact region of the strand opposite the gaps serving as template for replacement of the missing nucleotides. Finally, the DNA strand is sealed by DNA ligation [7,9,16,32]. No doubt the addition of membrane-binding drugs like procaine results in an inhibition of the initial step, incision, a n d / o r the excision step in the repair process, because the removal of thymine dimers was inhibited by the drugs (Fig. 6). However, the molecular and biochemical mechanisms of the inhibition were not elucidated in this experiment. Some factor in the excision repair process may be bound to the cell membrane, which may be affected by a structural disorganization brought about by membrane-binding drugs. Alternatively, the excision-repair process is a co-ordinated series of biochemical reactions as mentioned above [3,32]. Although the manner in which all the steps are co-ordinated is not understood, the structural disorganization of the cell membrane may result in a disturbance of this co-ordination. The disturbance may lead to lethal double-strand breaks of DNA by overlapping of excision gaps. It is also possible that, if attachment of DNA to cell membrane is essential for DNA repair, this process may be susceptible to drugs affecting the structure of the cell membrane. Furthermore, there may be other possible mechanisms involved iri the inhibition of excision repair by membrane-binding drugs. Work along these lines is currently being undertaken in this laboratory.
106
Acknowledgements We express our gratitude to Dr. M.A. Shenoy of the Biology and Agriculture Division, Bhabha Atomic Research Center of India for helpful discussions. This work was partly supported by a grant for scientific research from the Ministry of Education, Science and Culture of Japan.
References 1 Davis, R.W., D. Botstein and J.R. Roth, Media, drug concentration, and nutritional supplements, in: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, New York, 1980, pp. 201-207. 2 Ganesan, A.K., and K.C. Smith, Dark recovery processes in Escherichia coli irradiated with ultraviolet light, II. Effect of uvr genes on liquid holding recovery, J. Bacteriol., 97 (1969) 1129-1133. 3 Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith, DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48 (1979) 783-836. 4 Jagger, J., W.C. Wise and R.S. Stafford, Delay in growth and division induced by near ultraviolet radiation in Escherichia coli B and its role in photoprotection and liquid holding recovery, Photochem. Photobiol., 3 (1964) 11-24. 5 Kondo, S., H. Ichikawa, K. lwo and T. Kato, Base-change mutagenesis and prophage induction in strains of Escherichia coli with different DNA repair capacities, Genetics, 66 (1970) 187-217. 6 Okada, T., K. Yanagisawa and F.J. Ryan, Elective production of thymine-less mutants, Nature (London), 188 (1960) 340-341. 7 Seeberg, E., Multiprotein interactions in strand cleavage of DNA damaged by UV and chemicals, Prog. Nucleic Acid Res. Mol. Biol., 26 (1981) 217-225. 8 Seeman, P., The membrane actions of anesthetics and tranquillizers, Pharmacol. Rev., 24 (1972) 583-655. 9 Setlow, R.B., and W.L. Carrier, The disappearance of thymine dimers from DNA: an error-correcting mechanism, Proc. Natl. Acad. Sci. (U.S.A.), 51 (1964) 226-231. 10 Sheetz, M.P., and S.J. Singer, Biological membranes as bilayer couples, A molecular mechanism of drug-erythrocyte interactions, Proc. Natl. Acad. Sci. (U.S.A.), 71 (1974) 4457-4461. 11 Shenoy, M.A., K.C. George, B.B. Singh and A.R. Gopal-Ayengar, Modification of radiation effects in single-cell systems by membrane-binding agents, Int. J. Radiat. Biol., 28 (1975) 519-526. 12 Shenoy, M.A., and K. Gopalakrishna, Biochemical aspects of radiation sensitization of E. coli B / r by chlorpromazine, Int. J. Radiat. Biol., 33 (1978) 587-593. 13 Silva, M.T., J.C.F. Sousa, J.J. Polbnia and P.M. Macedo, Effects of local anesthetics on bacterial cells, J. Bacteriol., 137 (1979) 461-468. 14 Singh, B.B., M.A. Shenoy and K.C. George, Nature of radiation and chemically induced lesions and role of cellular mechanisms in cell survival and mutagenesis, I. Membrane and cellular repair, Adv. Biol. Med. Phys., 17 (1980) 109-113. 15 Smith K.C., Ultraviolet radiation effect on molecules and cells, in: K.C. Smith (Ed.), The Science of Photobiology, Plenum, New York, 1977, pp. 113-142. 16 Smith, K.C., Multiple pathways of DNA repair in bacteria and their roles in mutagenesis, Photochem. Photobiol., 28 (1978) 121-129. 17 Swenson, P.A., Physiological responses of Escherichia coli to far-ultraviolet radiation, in: K.C. Smith (Ed.), Photochemical and Photobiological Review, Vol. I, Plenum, New York, 1976, pp. 269-385. 18 Tang, M., and M.H. Patrick, The role of DNA polymerase I in liquid holding recovery of UV-irradiated E. coli, Photochem. Photobiol., 26 (1977) 257-262. 19 Tang, M., T.V. Wang and M.H. Patrick, DNA turnover in buffer-held E. coil and its effect on repair of UV damage, Photochem. Photobiol., 29 (1979) 511-520. 20 Tang, M., and M.H. Patrick, Repair of UV damage in Escherichia coli under non-growth conditions, Photochem. Photobiol., 26 (1977) 247-255.
107 21 Tang, M., and K.C. Smith, The expression o$ liquid holding recovery in ultraviolet-irradiated Escherichia coli requires a deficiency in growth medium-dependent DNA repair, Photochem. Photobiol., 32 (1980) 763-769. 22 Tribhuwan, R.C., and D.S. Pradhan, Induction of alkaline phosphatase in Escherichia coil: Effect of procaine hydrochloride, J. Bacteriol., 131 (1977) 431-437. 23 Yatvin, M.B., Evidence that survival of gamma-irradiated Escherichia coli is influenced by membrane fluidity, Int. J. Radiat. Biol., 30 (1976) 571-574. 24 Yatvin, M.B., J.J. Gipp and W.H. Dennis, Influence of unsaturated fatty acids, membrane fluidity and oxygenation on the survival of an E. coli fatty acid auxotroph following 7-irradiation, Int. J. Radiat. Biol., 35 (1979) 539-548. 25 Yau, T.M., Procaine-mediated modification of membranes and of the response to X-irradiation and hyperthermia in mammalian cells, Radiat. Res., 80 (1979) 523-541. 26 Yonei, S., and K. Nozu, Change in the base composition of messenger RNA of Escherichia coli by ultraviolet irradiation and its recovery, Photochem. Photobiol., 9 (1969) 537-548. 27 Yonei, S., Modification of radiation effects on E. coil B/r and a radiosensitive mutant B~_ 1 by membrane-binding drugs, Int. J. Radiat. Biol., 36 (1979) 547-551. 28 Yonei, S., Inhibitory effect of membrane-specificdrugs on liquid-holding recovery in UV-irradiated E. coli cells, Int. J. Radiat. Biol., 37 (1980) 685-689. 29 Yonei, S., and T. Todo, Enhanced sensitivity to the lethal and mutagenic effects of photosensitizing action of chlorpromazine in ethylenediaminetetraacetate-treated Escherichia coil, Photochem. Photobiol., 35 (1982) 591-592. 30 Yonei, S., The physical and chemical nature of membrane and radiosensitivity, J. Radiat. Res., 23 0982) 3 (abstract). 31 Witkin, E.M., Modification of mutagenesis initiated by ultraviolet light through post-treatment of bacteria with basic dyes, J. Cell. Comp. Physiol., 58 (Suppl. l) (1961) 135-144. 32 Witkin, E.M., Ultraviolet mutagenesis and inducible DNA repair in Escherichia coil, Bacteriol. Rev., 40 (1976) 869-907.