The mutagenic and lethal effects of monofunctional methylating agents in strains of Haemophilus influenzae defective in repair processes

Mutation Research 21

Elsevier Publishing Company, Amsterdam Printed in The Netherlands

T H E MUTAGENIC AND L E T H A L EFFECTS OF MONOFUNCTIONAL M E T H Y L A T I N G AGENTS IN STRAINS OF HAEMOPHILUS INFLUENZAE D E F E C T I V E IN R E P A I R PROCESSES*

R. F. KIMBALL, JANE K. SETLOW AND MINI LIU**

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. (U.S.A.) (Received December 7th, 197 o)

SUMMARY

4 m u t a n t strains of Haemophilus influenzae were tested for their response to the monofunctional methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS). 2 of these strains are unable to excise pyrimidine dimers, I is slow in rejoining breaks produced by excision, and I is unable to support recombination or rejoin the single-strand breaks produced in DNA by X-rays. The 3 strains with defects in the excision repair process are like wild-type in their sensitivity to killing by the 2 agents; the recombinationless strain is more sensitive to both agents b y a factor of 3-4. MMS does not induce a detectable number of mutations to cathomycin resistance at doses that give equal survival from mutagenic doses of MNNG and approximately equal numbers of induced single-strand breaks in the DNA. I t is suggested that the mutation induced by MNNG is due to a specific a l t e r a t i o n - perhaps alkylation of guanine on the oxygen at the 6 position--produced b y this agent but not by MMS. 3 of the strains, including the recombinationless one, give approximately the same mutation yield as wild-type with MNNG. i of the 2 excisionless strains gives a distinctly lower yield. This m u t a n t has been shown earlier to lack the endonuclease that makes the initial incision for the excision repair process in DNA containing pyrimidine dimers. It is suggested that this endonuclease can also act on the MNNG-specific mutagenic alteration to enhance the probability that it will cause mutation. It seems improbable that the complete excision repair process is involved in this enhancement, or indeed that excision repair of biologically significant alterations produced b y monofunctional alkylating agents occurs.

* Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. ** Present address: Lowell House A-52, Harvard University, Cambridge, Mass. (U.S.A.). Abbreviations: DMS, dimethyl sulfate; EMS, ethyl methanesulfonate; MMS, methyl methanesulfonate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine.

Mutation Res., 12 (1971) 21-28

22

R. F. K I M B A L L , J A N E K. S E T L O W , M I N I LIU

INTRODUCTION

Monofunctional alkylating agents have been widely used as mutagens, but it is not yet clear which of the alterations they produce in DNA is responsible for mutation (see e.g. DRAKEa). Moreover, little is known about what effect, if any, repair processes have on mutation induction by such compounds. Repair of single-strand breaks in DNA and repair synthesis have been reported for both MMS and MNNG~,",~2,14,~5. However, PRAKASHAND STRAUSS~3have been unable to find any evidence for excision of alkylated bases in B. subtilis, despite the evidence for an endonuclease in this species that attacks alkylated DNA (ref. 21). Mutations that produce defects in the excision of pyrimidine dimers have been reported to have little if any effect on mutation induction or killing of Escherichia coli by MNNG, MMS, or EMS (refs. 6, 22, 23). Mutants sensitive to killing by alkylating agents are known *.~6,2~,2a, and at least some of these are also sensitive to UV and X-rays and deficient in recombination. Indirect evidence suggests that repair can eliminate all or nearly all the MNNG-produced premutational damage leading to recessive lethal mutations in Paramecium aurelia ~. The present paper reports attempts to examine some of these problems in a set of 4 m u t a n t strains of Haemophilus influenzae with partially analyzed defects in excision repair or recombination ~%18. MNNG and MMS, 2 methylating agents with very different properties, are compared for mutation induction, killing, and production of single-strand breaks. Emphasis is placed on mutation, with sufficient information about killing and single-strand break production to put the mutation results in perspective. A subsequent paper will present more details on the other 2, especially on the formation and repair of single-strand breaks. Our results emphasize the uniqueness of the mutagenic alteration produced by MNNG and suggest that the endonuclease responsible for the initial incision step in the excision repair of pyrimidine dimers enhances mutagenesis by the MNNG-produced alteration. MATERIALS AND METHODS

Strains. The 6 strains of H. influenzae used in this work have been described previously~V, 18. Rd is the wild-type. The mutants D B I I 2 and D B I I 6 lack the ability to excise pyrimidine dimers and to carry out host-cell reactivation, but the mutations involved are in different cistrons. D B I I 2 appears to lack the endonuclease required for the initial incision of the DNA strand 2°. The defect in D B I I 6 could involve a later step in excisionlL but this is not yet established. D B I I 2 is probably a multiple mutant, but strain Rd(DBII2), which was derived by transformation of Rd with D B I I 2 DNA, seems to have the same defect in excision and is more probably a single m u t a n t t~. D B I I 5 can excise dimers but rejoins the resulting strand breaks more slowly than Rd. D B I I 7 is defective in recombination and in the rejoining of the single-strand breaks produced by ionizing radiation. All strains are sensitive to UV, but only D B I I 7 is sensitive to ionizing radiation. Media. The growth, dilution, and plating media have been described previously ~7. Exposure to alkylating agents. MNNG (Aldrich) or MMS (Aldrich) were dissolved in Tris-malate buffer 4 adjusted to p H 6.0 for MNNG or to p H 6. 9 for MMS. MNNG was dissolved by vigorous stirring at 37 ° for a few minutes prior to use. MMS was 31utatio,n l?cs., i2 (1971) 2t- 2<~;

EFFECTS OF MNNG AND MMS IN H. influenzae

23

made up just before use. The bacteria were grown to about lO 9 cells per ml (exponential phase), centrifuged, and resuspended in the buffer at 37 °. As soon thereafter as possible, the initial MMS or MNNG solution was diluted (from 1:6 to I :IO) into the bacterial suspensions to give the desired final concentration. Survival curves. For the determination of survival, o.I-ml samples of the exposure mixture were withdrawn at intervals, put into IO ml of ice-cold dilution medium, and diluted further as needed in cold dilution medium. Either o.I ml or I.O ml of the diluted suspensions was put into a petri dish, and liquid agar at 45 ° was added. Once the first layer had solidified, additional agar was layered on top to give a total of about 20 ml. In this procedure, the termination of the exposure to the agent depended on successive dilution by medium and agar. The lowest dilution used was 2ooo-fold, and for most of the points on the survival curve the dilution was greater by one to several orders of magnitude. Mutation frequency. For the determination of mutation, exposure to the agent was terminated by diluting i : io with cold dilution medium, centrifuging and resuspending in cold dilution medium twice, and finally centrifuging again and resuspending in liquid growth medium at a concentration between lO 7 and lO 8 bacteria per ml. The bacteria were grown in a shaking bath at 37 ° to about lO 9 per ml, 15% of sterile glycerol was added, and aliquots were frozen and stored at --80 °. For the mutation test, an aliquot was thawed and subsurface plated at appropriate dilutions in regular agar to determine the number of viable bacteria per ml, and in agar containing 2.5 /~g/ml of cathomycin (novobiocin) to determine the number of bacteria per ml resistant to this concentration of the drug. All experiments involved simultaneous determinations in Rd and one or more of the mutant strains. The growth and freezing allowed tests for mutation to be made after the bacteria had divided several times, thus largely eliminating problems of mutation fixation, expression, and mosaic segregation. This method also made it possible to carry out repeat tests on the same treated sample to confirm the results. Regrowth of frozen material showed that the frequency of cathomycin-resistant bacteria per lO 6 viable bacteria remained essentially constant after several additional generations of growth. Thus there is no detectable selection for or against cathomycin-resistant bacteria during a few generations of growth in liquid medium. Single-strand breaks. The method of McGRATH AND WILLIAMS11 as applied to H. influenzae 18 was used. The treated bacteria were prelabeled with either E3HITdR Ol~ E14C]TdR. The I3H]labeled bacteria were treated with the alkylating agent in the appropriate buffer, washed, and subsequently mixed with the [14Cllabeled controls. A o.o3-ml sample of the mixture was lysed in o.I ml of 0.5 N NaOH on top of a 4.9-ml, 5-20% alkaline (pH 12) sucrose gradient. The gradients were centrifuged at 30 ooo rev./min for 9 ° min in an SW39 or SWso.I rotor in a Spinco model L. The tubes were punctured, and successive 2-drop samples were collected on filter paper discs. The discs were washed in cold 5% TCA, alcohol, and acetone, and the 3H and 14C activities of the discs were measured in a scintillation counter, with 16 g of naphthalene-2,5bisE2-(5-tert-butylbenzoxazoly)]thiophene per gallon of toluene as the scintillation fluid.

Mutation Res., 12 (1971) 2 1 - 2 8

24

R . F . KIMBALL, JANE K. SETLOW, MINI LIU

RESULTS

Survival. Figs. I and 2 show survival data for MNNG and MMS, respectively. The wild-type and the 3 mutant strains D B I I 2 , D B I I 5 , and D B I I 6 do not differ significantly from each other in their reaction to either agent, whereas D B I I 7 is distinctly more sensitive to both. The MNNG data show a greater spread, possibly because the low solubility in the buffer made it difficult to assure complete solution. 1'0-~

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Fig. i. P e r c e n t s u r v i v a l for 5 s t r a i n s of H. influenzae e x p o s e d t o o.38 m2l//MNNG for v a r i o u s t i me s . The e x p o s u r e s were m a d e a t a c o n c e n t r a t i o n of a b o u t io~ cells pe r ml in p H 6.o T r i s - m a l a t e buffer. S a m p l e s of o.i ml were r e m o v e d from t h e m i x t u r e a t t h e t i m e s shown, d i l u t e d i m m e d i a t e l y in cold d i l u t i o n m e d i u m b y a f a c t o r of lOO, a n d p l a t e d i m m e d i a t e l y a t still g r e a t e r d i l u t i o n s , e , R d ; O, D B I I 2 ; LJ, D B I I 5 ; A, D B I I 6 ; A, DBII7. Fig. 2. P e r c e n t s u r v i v a l for 5 s t r a i n s of H. influenzae e x p o s e d t o 4.6 m M MMS in p H 6.9 T r i s - m a l a t e buffer for v a r i o u s times. E x p o s u r e p r o c e d u r e a n d s y m b o l s s a m e as for Fig. i.

Repeat experiments failed to confirm any differences among the strains except for that between D B I I 7 and the others. The more repeatable MMS data suggest that this strain is some 3-4 times more sensitive than wild-type. The killing curves for D B I I 7 also differ in shape from those for the other strains, having slopes that decrease as the dose increases. With MMS the curves for the other strains resemble the general multihit type with a fairly high extrapolation number, but with MNNG they resemble a simple exponential. The data are too variable, however, to establish the exact form of the relationship. Mutation. The data for strain Rd are plotted in Fig. 3. The variation is quite great; consequently, comparison between strains is difficult if the data are plotted in the same way for all strains. To partially reduce this difficulty, the values for the mutant strains have been plotted as a ratio with those for Rd in the same experiment. These ratios are shown in Fig. 4. There is still considerable scatter, but examination shows that strain D B I I 2 and its derivative strain Rd(DBII2) have a consistently lower frequency than Rd, whereas none of the other 3 strains shows such a consistent pattern. In 13 of the 14 comparisons between Rd and D B I I 2 , Rd has the higher mutation frequency. The Mutation Nes., 12 (1971) 21 2,~

EFFECTS OF MNNG AND MMS IN H. influenzae

25

average ratio of D B I I 2 to Rd is 0.35. DBII7, which is more sensitive to killing, appears to be like D B I I 5 and D B I I 6 in showing no consistent difference from Rd. t00-

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F i g . 3- C a t h o m y c i n - r e s i s t a n t bacteria per lO 6 viable bacteria at 4 different exposure t i m e s to 1.9 m M M N N G . Strain R d . i i experiments. F i g . 4- M u t a t i o n frequencies in t h e m u t a n t strains expressed as a ratio w i t h the m u t a t i o n f r e q u e n c y in t h e c o r r e s p o n d i n g R d group f r o m t h e s a m e experiment. I I experiments. S y m b o l s as in F i g . i

w i t h the a d d i t i o n of • for strain t~d(DB112).

Attempts to induce mutation to cathomycin resistance with doses of MMS that give survivals comparable to those given by MNNG were uniformly unsuccessful with Rd, DBII2, and DBII5, the only strains tried. We can say that the frequency of detectable cathomycin-resistant mutants induced with this agent, if it is not zero, is one or probably more orders of magnitude lower than with doses of MNNG that produce comparable survivals. Single-strand breaks in DNA. The number of single-strand breaks induced in the DNA of various strains of H. influenzae has been measured for both MNNG and MMS at various times after treatment. We have been unable to find evidence for repair of these breaks that is as clear as that reported by others1,12,1~. Instead we find a picture dominated by breakdown of DNA, with a relatively minor amount of repair superimposed. These findings will be discussed in more detail in a subsequent paper. Suffice it to say here that the numbers of breaks found both immediately and after various kinds and durations of post-treatment incubation, are approximately the same for the different strains, including the sensitive strain D B I I 7. In the survival range above IO% - - t h e range of interest for mutation--comparison of the number of breaks produced by MNNG and MMS showed that MMS produced, if anything, more breaks than MNNG, for a given level of survival. Thus the failure to detect mutation with MMS could not have been a result of a low level of effect on DNA, at least as measured by the number of single-strand breaks. Mutation Res., 12 ( 1 9 7 1 ) 2 1 - 2 8

20

R.F.

K I M B A L L , J A N E K. S E T L O W , M I N I L I U

DISCUSSION

MNNG is nmtagenic while MMS, at doses that give comparable levels of survival and single-strand breakage, is not. Therefore, it is unlikely that the major cause of mutations produced by MNNG is one of the alterations produced in common by the 2 agents. These common alterations include single-strand breaks and methylations of the 7-nitrogen of guanine and the 3-nitrogen of adenine. LAWLE¥ AND THATCHER7 have shown that MNNG, in the presence of concentrations of thiols comparable to those occurring in cells, alkylates the 6-oxygen of guanine; and they suggest that it may have other as yet undetermined actions that are different from those of DMS and presumably of the very similar MMS. LOVELESS9 has shown that EMS also alkylates the 6-oxygen of guanine, but that MMS does not. He had shown earlier 8 that EMS but not MMS produces mutations in T2 coliphage. Both LOVELESS9 and LAWLE',r AND THATCHER7 suggest that it may be the alkylation of the 6-oxygen of guanine that is mainly responsible for mutation induction by MNNG and EMS. Our results would fit nicely with this view, and we will assume that the mutations we are studying are due either to this or to some other special alteration, rather than to the alterations which are common to all methylating agents. These common alterations also may be mutagenie in our system but with such a low probability that their effect is not detected. MMS is mutagenic in Neurospora but produces mainly noncomplementing mutants and mutants with polarized complementation patterns, whereas MNNG produces mainly nmtants with nonpolarized patterns 1°. It is possible that the MMS mutants, either because they are lethal or for some other reason, cannot be detected by LOVELESS'ST2 system s or by our Haemophilus system. In any case, the Neurospora results also show that the bulk of the MNNG mutants must be produced by alterations in the DNA that are different from those produced by MMS. We will now turn to the problem of repair. It has been suggested',2,15 that there is an excision repair system for alkylated DNA comparable to that found for DNA containing pyrimidine dimers induced by UV, but possibly utilizing an initial endonuclease specific for alkylated DNA. PRAKASHA N D STRAUSSla, however, were unsuccessful in an attempt to demonstrate the removal of alkylated bases from the DNA of B. subtilis treated with MMS, although this organism has an endonucleolytic activity that can attack alkylated DNA (ref. 15). They seriously question, therefore, whether an excision repair mechanism really exists for monofunctionally alkylated DNA. Our own results are also not encouraging for the view that such a mechanism exists. D B I I 5 , which is slow to rejoin breaks resulting from excision of pyrimidine dimers and presumably is sensitive to UV for this reason, is no more sensitive than wild-type to killing or mutation induction by alkylating agents. If excision of alkylated bases occurs and, except for the initial endonucleolytic step, depends on the same mechanism that excises pyrimidine dimers, D B I 15 should be sensitive to these agents. On the other hand, D B I I 2 , which is missing the initial UV-endonuelease and consequently on the excision repair hypothesis should be like Rd in its response to alkylating agents, is the only strain with altered sensitivity to mutation induction by MNNG. It is resistant, however, whereas a defect in excision of alkylated bases should make it sensitive. The obvious conclusion is that no biologically significant effect can be 3,Iz#ation Res., i 2 (1971) 21 2,~

EFFECTS OF MNNG AND MMS IN H. influenzae

27

attributed at present to the excision of alkylated bases; and in light of PRAKASH AND GTRAUSS'S18 finding it seems probable that such excision does not occur. What, then, is the reason for the low mutation frequency in D B I I 2 ? The fact that the low frequency is also found in strain Rd(DBII2), which was derived from Rd b y transformation with D B I I 2 DNA, suggests that it is a consequence of the lack of the UV-endonuclease, though the possibility of some other defect caused by a mutation in a closely linked locus cannot be entirely excluded. The simplest suggestion is that the UV-endonuclease in wild-type cells can produce a single-strand break in the vicinity of the MNNG-specific mutagenic alteration, and that this break greatly increases the probability that the alteration will lead to a mutation. According to this view other steps in the dimer excision and repair process controlled by the loci that are m u t a n t in D B I I 5 and D B I I 6 would be irrelevant, since it is not the excision repair process but the specific initial incision by the UV-endonuclease that is involved. It m a y well be that this action is confined to the specific mutagenic alteration produced b y MNNG since neither lethality nor the overall number of strand breaks in D B I I 2 is detectably different from that in the wild-type. This hypothesis leaves open the possibility that the action of the UV-endonuclease could be reversed under favorable conditions by subsequent repair. All that we have shown is that the initial specific alteration produced by MNNG is not inevitably mutagenic. In any case it seems improbable that the single-strand break, if such it is, that enhances the chances for mutagenic conversion, acts by way of an error in recombination r e p a i r - - a mechanism suggested by WITKIN23for UV-induced mutations. If it does, we would expect MNNGinduced mutation to be greatly reduced, as UV-induced mutations are, in certain of the strains that are defective for recombination. WITKIN.2-~4 and KONDO~ find, on the contrary, that the level of mutation induction by MNNG in such strains of E. coli is about the same as in wild-type, and we have made the same finding for the recombination-defective strain (DBII7) of H. influenzae. REFERENCES I BOYCE, R. P., AND J. W. FARLEY, Production of single-strand breaks in covalent circular phage DNA in superinfected lysogens by monoalkylating agents and the joining of broken DNA strands, Virology, 35 (1968) 6Ol-6O9. 2 CERDA-OLMEDA, E., AND P. C. HANAWALT, Repair of DNA damaged by N-methyl-N'-nitroN-nitrosoguanidine in Escherichia coli, Mutation Res., 4 (1967) 369-371. 3 DRAKE, J. V~., The Molecular Basis of Mutation, Holden-Day, San Francisco, 197 o, 273 pp. 4 GOMORI, G., Preparation of buffers for use in enzyme studies, in S. P. COLOWlCK AND N. O. KAPLAN (Eds.), Methods in Enzymology, Vol. I, Academic, New York, 1955, pp. 138-146. 5 KIMBALL, R. F., Studies on the mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine in Paramecium aurelia with emphasis on repair processes, Mutation Res., 9 (197o) 261-271. 6 KONDO, S., Mutagenicity versus radiosensitivity in Escherichia coli, Proc. ::2th Intern. Congr. Genet., 2 (I968) 126-127. 7 LAWLEY, P. D., AND C. J. THATCHER, Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N'-nitro-N-nitrosoguanidine, The influence of cellular thiol concentrations on the extent of methylation and the 6-oxygen atom of guanine as a site of methylation, Biochem. J., 116 (197 o) 693-707 . 8 LOVELESS, A., The influence of radiomimetic substances on deoxyribonucleic acid synthesis and function studied in Escherichia coil/phage systems, III. Mutation of T2 bacteriophage as a consequence of alkylation in vitro; the uniqueness of ethylation, Proc. Roy. Soc. (London), Ser. B, 15o (1959) 497-508. 9 LOVELESS, A., Possible relevance of 0 - 6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides, Nature, 223 (1969) 2o6-207. io MALLING, I~. V., AND V. J. DE SERRES, Mutagenicity of alkylating carcinogens, Ann. N. Y. Acad. Sci., 163 (1969) 788-800. Mutation Res., I2 (1971) 21-28

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R. F. KIMBALL, JANE K. SETLOW, MINI LIU

11 MCGRATH, R. A., AND R. W. WILLIAMS, R e c o n s t r u c t i o n in vivo of irradiated Escherichia coli deoxyribonucleic acid; The rejoining of b r o k e n pieces, Nature, 212 (1966) 534-535. 12 OLSON, A. O., AND K. M. BAIRD, Single-strand breaks in Escherichia coli D N A caused hy treatm e n t w i t h nitrosoguanidine, Biochim. Biophys. Acta, 179 (1969) 513-514. 13 PRAKASH, L., AND B. STRAUSS, Repair of alkylation d a m a g e : Stability of m e t h y l g r o u p s in Bacillus subtilis treated with m e t h y l m e t h a n e sulfonate, J. Bacteriol., lO2 (197 o) 760 766. 14 ~{EITER, H., AND B. STRAUSS, Repair of danlage induced b y a m o n o f u n c t i o n a l alkylating agent in a transformable, ultraviolet-sensitive strain of Bacillus subtilis, J. Mol. Biol., 14 (1965) 179194. 15 RE1TER, H., B. STRAUSS, M. ROBBINS AND R. MARONE, N a t u r e of the repair of methyl m e t h a n e sulfonate induced d a m a g e in Bacillus subtilis, J. Bacteriol., 93 (1967) lO36-1o62. 16 SEARASHI, T., AND B. STRAUSS, Relation of repair of d a m a g e induced b y a m o n o f u n c t i o n a l alkylating agent to the repair of d a m a g e induced b y ultraviolet light in Bacillus subtilis, Biochem. Biophys. Res. Commun., 20 (1965) 680-687. 17 SETLOW, J. K., D. C. BROWN, M. E. BOLING, A. MATTINGLY AND M. P. (;ORDON, Repair of deoxyribonucleic acid in Haemophilus influenzae, I. X - r a y sensitivity of ultraviolet-sensitive m u t a n t s a n d their b e h a v i o r as h o s t s to ultraviolet-irradiated bacteriophage and t r a n s f o r m i n g deoxyribonucleic acid, J. Bacteriol., 95 (1968) 546-558. 18 SETLOW, J. K., M. L. RANDOLPH, M. E. BOLING, A. MATTINGLY, G. PRICE AND M. P. GORDON, Repair of D N A in Haemophilus influenzae, II. Excision, repair of single-strand breaks, defects in t r a n s f o r m a t i o n , a n d h o s t cell modification in UV-sensitive m u t a n t s , Cold Spring Harbor Symp. Quant. Biol., 33 (1968) 209 218. 19 SETLOW, J. K., M. E. BOLING AND K. L. BEATTIE, Repair of D N A in Haemophilus influenzae, I I I . Excision and r e c o m b i n a t i o n defects and the site of repair of ultraviolet-irradiated transforming DNA, in Genetic Concepts and Neoplasia, Williams and Wilkins, Baltimore, Md. 197 o, pp. 555-568. 20 SETLO~,V, R. B., J. K. SETLOW AND ~¥. L. CARRIER, Endonuclease from Micrococcus l~tleus which has activity t o w a r d ultraviolet-irradiated deoxyribonucleic acid; I t s action on transforming deoxyribonucleic acid, J. Bacteriol., lO2 (197 o) 187 192. 2i STRAUSS, B., M. COYLE AND M. ROBBINS, Consequences of alkylation for the behavior of DN:\, Ann. N. Y. Acid. Sci., 163 (1969) 765-787 . 22 WITKIN, E. M., Mutation-proof and m u t a t i o n - p r o n e modes of survival in derivatives of Escherichia coli B differing in sensitivity to ultraviolet light, Brookhaven Syrup. Biol., 20 (i907) 17-55. 23 VqlTKIN, E. M., The role of D N A repair and recombination in mutagenesis, Proc. 12th Intern. Congr. Genet., 3 (1969) 225-245. 24 V~rlTKIN, f?.. ~'I., The m u t a b i l i t y t o w a r d ultraviolet light of recombination-deficient strains of Escherichia coli, Mutation Res., 8 (1969) 9 14-

Mutation Res., 12 (1971) 21 28