Mutagenicity of dichlorvos and methyl methane-sulphonate for Escherichia coli WP2 and some derivatives deficient in DNA repair

Mutation Research, 19 (1973) 295-303 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

295

M U T A G E N I C I T Y OF DICHLORVOS AND M E T H Y L M E T H A N E S U L P H O N A T E F O R E S C H E R I C H I A COLI WP2 AND SOME D E R I V A T I V E S D E F I C I E N T IN DNA R E P A I R

B. A. BRIDGES, R. P. MOTTERSHEAD, M. H. L. GREEN ANDW. J. H. GRAY MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BNz 9QH (Great Britain) (Received March 2oth, 1973)

SUMMARY

The mutagenic and lethal action of methyl methanesulphonate (MMS) and dichlorvos (DDVP) has been studied on Escherichia coli WP2 and some derivatives deficient in DNA repair genes. The exrA + and recA + alleles were necessai 3, for significant mutagenesis b y either compound, and the uvrA gene affected neither the lethal nor mutagenic responses. Increased sensitivity to both compounds was sho~ua b y the exrA and uvrAexrA strains and in a more pronounced way by the uvrApolA, recA, and uvrAexrApolA strains. Bacteria deficient at the polA locus were 2 and 3 times more mutable b y D D V P and MMS respectively, consistent with the hypothesis that the absence of the lholA system for the repair of single-strand gaps results in a greater proportion of the total repair being channelled through the error-prone exrA+/recA+-dependent system. Single-strand breaks were detectable b y alkaline sucrose gradient centrifugation after both MMS and DDVP treatment of polA bacteria. Thus in all the tests carried out, both compounds showed similar patterns of activity, and the results are consistent With their known ability to alkylate DNA. The chief differences were quantitative; sensitivity increases were far more pronounced with MMS which was also a far more potent mutagen than DDVP.

INTRODUCTION DDVP (2,2-dichlorovinyl dimethyl phosphate) is the active constituent of the insecticide Vapona. I t has been shown to alkylate DNA17, 2, but there are conflicting reports as to its mutagenicity. In bacteria, L/SFROTH, KIM AND HUSSAINTM briefly reported the induction of streptomycin-independent revertants in a streptomycindependent strain of Escherichia coli B but no quantitative data were given. More recently induction of mutations to prototrophy were reported for E. coil WP2 trp using a semi-quantitative spot test technique b y ASHWOOD-SMITHet al. 1. Using the Abbreviations: DDVP, dichlorvos; MMS, methyl methanesulphonate.

296

B.A. BRIDGES et al.

same system DEAN11 obtained a negative result although mutagenesis was observed in parallel tests with Serratia marcescens. Weakly positive results in a number of species have been reported by VOOGD, JACOBS AND VAN"DER STELsS. We have examined the mutagenicity of DDVP in a quantitative way, using treatment of bacteria in suspension rather than on agar plates. We had previously obtained (unpublished observations) rathei variable results using the agar plate spot test which has been very successful in characterizing the mutagenicity of the fungicide captan s. On some days weak mutagenesis of E. coli WP2 by DDVP was observed (confirming ASHWOOD-SMITH et al.1), whereas on others, particularly when plates were supplemented with a low level of nutrient broth rather than tryptophan, there was no significant induction (confirming DEAN11).We concluded that the agar plate method s is inappropriate for detecting a weak mutagen that is both volatile and rather toxic. In view of the known methylating ability of DDVp~7,L we have carried out comparative studies with DDVP and MMS, another methylating agent more extensively studied, using derivatives of E. coli WP2 deficient in various DNA repair pathways. The results are entirely consistent with DDVP exerting its mutagenic effect via an error-prone repair event following alkylation of DNA. Quantitatively, however, there were significant differences between the effects of the two substances. MATERIALS AND METHODS

Microorganisms

The strains used, their derivation and sources are shown in Table I. All the strains carry an ochre nonsense mutation*°, 4 blocking a stage before anthranilic acid in the tryptophan synthesis pathway 18. Mutations to prototrophy are due either to reversions at the stluctural gene (at least some of which are A : T to G:C transitions) or to ochre suppressor mutations 5. The uvrA + gene controls the excision from DNA of pyrimidine dimers TM and (probably) cross links and large adducts TM. The polA + gene controls DNA polymerase I which is responsible for the repair of many single-strand gaps in DNA, for example most of those remaining after pyrimidine dimer excision and X-irradiation ~5,s4. Such gaps, together with other gaps not reparable by DNA polymerase I, may also be repaired by another process, the details of which are largely unknown, which iequires the recA + (refs. 19, 14) and exrA + (ref. 22) genes. This process is error-prone, and many mutagens exert their mutagenic effect largely TABLE

I

STRAINS OF Escherichia coli USED IN THIS INVESTIGATION All s t r a i n s a r e t r y p t o p h a n

No.

WP2 W P 2 uvrA CM56I CM571 CM6II WP67 WPI2

a u x o t r o p h s d u e t o a n ochre m u t a t i o n

R e p a i r markers recA exrA

polA

+

+

+

+

+

+

+

--

-2_ + + +

+ _ -+ --

+ + + --

+ + ---

-

-

-

Derived f r o m

Source

B/r WP2 WP2 WP2 W P 2 uv r A W P 2 uv r A WP67

W I T H I N ( v i a DOUDNEY) H I L L 13 BRIDGES et al. s BRIDGES el al. s BRIDGES et al. s WITHIN WITHIN

uvrA

-

MUTAGENICITYOF DDVP AND MMS IN E. coli

297

or exclusively through this pathway, i.e., they do not mutate recA or exrA strains (refs. 26, 6, 16, 22, 8).

Chemicals D D V P (batch ACD/7I/3, 99°./0 pure) was kindly supplied b y Mr. B. J. Dean (Tunstall Laboratory, Shell Reseal ch Ltd., Sittingbourne, Kent). MMS was obtained from the Aldrich Chemical Company.

Mutation induction All the strains except WP67 were grown to about 2. lO 8 bacteria per ml in M9 glucose salts medium with IO ~ug per ml tryptophan at 37 ° with gentle aeration. Strains WP67 and W P I 2 were found to require citrate for good growth and so DAVIS AND MINGIOLI salts ,0 were used instead of M9. Bacteria were filtered or centrifuged and resuspended in phage buffer 3 at about 2. IO° bacteria per ml for treatment with D D V P or MMS. Mutants were assayed on glucose minimal medium of DAVIS AND MINGIOLI1° supplemented with 5 % v / v Oxoid Nutrient Broth and solidified with 1.5 % Davis New Zealand Agar. The broth supplement permits several generations of growth of the auxotrophic bacteria, sufficient to allow phenotypic expression of newly induced mutations. Viable bacteria m a y also be determined on the same medium after appropriate dilution. For the estimation of mutants, o.2 ml was plated on to a 2o-ml plate; preliminary experiments showed that negligible induction of mutations occurred after plating. Colonies were counted after 2 days' incubation at 37 °. The frequency of induced mutations was calculated according to the formula of SEDGWlCK AND BRIDGES ~2.

Toxicity determinations Bacteria were grown to about 2. lO 8 peI ml in Oxoid Nutrient Broth No. 2 at 37 ° with aeration and suspended in phage buffer at 2. i o ' per ml for treatment. After incubation for I h at 37 ° the suspensions were diluted as required (at least Ioo-fold) and plated on to Oxoid Nutrient Broth No. 2 solidified with 1.5% Davis New Zealand Agar. Colonies were counted after 24 h at 37 °.

Sucrose gradient sedimentation Bacteria were labelled by growing in M9 supplemented with 1°/0 Casamino acids, 25 jug/ml tryptophan, 0.5% glucose, o.1% citrate, 25 juCi/o.5 jug/ml [3H]thy midine and IOO/~g/ml deoxyadenosine. They were treated in phage buffer and the molecular weight of the DNA examined by alkaline sucrose gradient centrifugations as described elsewhere TM. RESULTS

Comparative mutagenesis of uvrA, exrA and recA strains Preliminaiy experiments showed that when lethal concentlations of D D V P were used, both the lethal and mutagenic effects were rather variable. We therefore selected a single treatment for all the strains that would, as far as possible, leave the majority of the population in a viable state. Bacteria were incubated for I h at 37 ° in phage buffer with and without MMS (0.04%) or D D V P (0.2%). The results (Table II)

298

BRIDGES et al.

B.A.

TABLE

II

MUTAGENESIS

OF

WILD

recA-, exrA-

TYPE

AND

uurA-DEFICIENT

STRAINS

OF

Escherichia coli

BY

0 . 0 4 % ( v / v ) M M S AND 0 . 2 % (V/V) D D V P AT 37 ° FOR 60 r a i n E a c h i n d u c e d m u t a t i o n f r e q u e n c y is t h e m e a n of b e t w e e n 3 a n d 6 d e t e r m i n a t i o n s .

Mutagen

Strain

S u r v i v i n g fraction

Induced mutation frequency ( £ standard error)

MMS

WP2 W P 2 uvrA CM56I CM57I

1.o6 ± o . 4 3 . i o ° 1.o 7 j_ o . 6 6 . I O ° 9.91 ± 0.34" lO - I 2.84 • i o t (2 e x p e r i m e n t s o n l y ) 8.05 • IO -x (2 e x p e r i m e n t s o n l y )

4.07 ± 1.82. lO -.7 4.63 i 0.33" lO -7 7.37 ± 2.33" lO-9 o

9.02 ± O. l O - l O -1 8.o8 ± o . 5 8 - 1 o -1 1.o 3 ± 0.62. lO -1 3.89 :k o . 8 1 . lO -1 (2 e x p e r i m e n t s o n l y ) 7.06 :~ o . 6 8 . I O -1 (2 e x p e r i m e n t s o n l y )

I . I I ± 2 . 2 8 . 1 o -7 1.13 ± 2 . 7 6 . 1 o - 7 8.33 :k 1.37"IO " o

CM6I I DDVP

WP2 W P 2 uvrA CM56I CM57I CM6II

o

o

show that the pattern of response of the strains to the two compounds was qualitatively similar. Both were mutagenic and for each compound WP2 and WP2 uvrA were mutated to the same extent. Strains carrying the recA (CM57 I) or exrA (CM56I, CM6II) alleles were mutated either not detectably or at less than lO% of the fiequency for the isogenic exrA + strains WP2 and WP2 uvrA. It is noteworthy that both the mutagenesis of the latter strains and mutagen stability of the exrA strain CM56I were demonstrated without any detectable lethality in the populations. From these results we conclude that both MMS and DDVP give rise to base-

~c 1 l > O5 c. o

c

I

I

I

c. o

!

~ O

•- ; ~

O~

0

°I 3 o

.£g-"

-6o

~x

?g gg

z,

O

.4i'~ 2

z -

!

j

o

J

_ 8-.~'%

%× E-Z

/

2

•

®/

/

/

/

I

I

I

I

20

40

60

80

MMS

treatment

(min)

I-

i

60 DDVP

I 120 treatment

I 180 (min)

F i g . I. V i a b i l i t y a n d i n d u c e d m u t a t i o n f r e q u e n c y a s a f u n c t i o n of t i m e of i n c u b a t i o n in 0 . 0 4 % ( v / v ) MMS. Q, E. coli W P 2 ; o , E. coli W P 2 u v r A . F i g . 2. V i a b i l i t y a n d i n d u c e d m u t a t i o n f r e q u e n c y as a f u n c t i o n of t i m e of i n c u b a t i o n i n o . 2 % ( v / v ) D D V P . e , E. coli W P 2 ; O, E. coli W P 2 u v r A .

MUTAGENICITYOF DDVP AND MMS IN E. coli

299

pair substitution mutations wholly or almost wholly via the error-prone ezrA +/recA +dependent pathway for DNA repair. The irrelevance of the uvrA gene indicates that the premutational lesions are not excised from the DNA by the endonuclease responsible for the excision of pyrimidine dimers. Figs. I and 2 show that certain differences between MMS and DDVP are apparent in the kinetics of mutation induction in WP2 and WP2 uvrA. With MMS, induction was most extensive in the first 20 min after which a slower rate was seen. With DDVP the reverse was true. More variability from one run to another was seen with DDVP (cf. standard errors in Table II) and the two curves illustrated were selected because of their similarity. Nevertheless the slight lag before the establishment of a constant rate of induction was seen in all the experiments carried out. A further difference is that DDVP was a much weaker mutagen than MMS. The latter at 0.o4% induced about four times as many mutants in 60 min as DDVP at five times the concentration (or about 2.5 times the molarity).

Relative toxicity of MMS and DDVP BRIDGES, LAW AND MUNSON~ have postulated that the actual premutation lesion in exrA+-dependent mutagenesis is a single-strand DNA break or gap. If such breaks are produced directly or indirectly by MMS and DDVP one would predict that strains deficient in the repair of such breaks (exrA and recA but not uvrA) should be more sensitive to the lethal action of the compounds. This should also be true for polA bacteria which lack an alternative iepair pathway fol some types of single-strand breaks~5, 24. Figs. 3 and 4 show the survival of a number of E. coli strains after exposme for 60 rain at 37 ° to various concentrations of the mutagens. In general the above predictions are confirmed for both MMS and DDVP. With both compounds the wild-type and uvrA strains were equally resistant. The exrA and exrAuvrA strains were more sensitive, and the polAuvrA, polAuvrAexrA and recA strains the most sensitive of all. Qualitatively, therefore, the two compounds had similar effects on viability. Quantitatively, however, there were significant differences. Although MMS was less toxic than DDVP for the wild-type strains the reverse was true for the sensitive

1

• "--'@--.a~ ,k

u

n

f

0'~ •

"G

o.;

0.~

•

~o5 pot uvr exr~

0.0; 0001

I

I

i

I

\ !

0"01

MMS

concentration

"~" I} ,r-I \Qexr ......

pol u v r . 1 \

005

~pot uvr I

I

(>I ('/. v/v)

I

o.O o,

'

'

,

,

DDVP concentration (a/,v/v)

Fig. 3. Loss of v i a b i l i t y a f t e r 6o m i n i n c u b a t i o n a t 37 ° w i t h v a r i o u s c o n c e n t r a t i o n s of MMS. ©, E. coli W P 2 ; iX, W P 2 uvrA; V, CM56I; D, C M 6 I I ; O, W P 6 7 ; R, CM57I; V, W P I 2 . Fig. 4- Loss of v i a b i l i t y a f t e r 6o m i n i n c u b a t i o n at 37 ° w i t h v a r i o u s c o n c e n t r a t i o n s of D D V P . S y m b o l s as for Fig. 3.

300

B.A.

B R I D G E S et

al.

strains; the recA strain for example, was about IOO times more sensitive than the wild-type to MMS but only about 4 times more sensitive to DDVP. This suggests that MMS damage is less readily repaired by any one pathway than DDVP damage or, put another way, that MMS damage requires both the polA+ and recA ~/exrA pathways for maximum repair. A comparison with the data in Table II shows that bacteria grown in minimal medium and treated at a higher population density appeared to be slightly more resistant to both MMS and DDVP than the broth grown bacteria used for the toxicity expeliments.

Scission of D N A The sensitivity data presented above suggests that both mutagens should. produce single-strand breaks in DNA. We have examined the molecular weight of DNA from treated bacteria by alkaline sucrose gradient centrifugation. Since the polA-~-dependent process is known to be rapid (for example with X-ray-induced breaks 24) the polAuvrA strain was used to eliminate repaii during the exposure to the mutagen. The DDVP and MMS treatments were identical to those used to obtain the data in Table II, i.e., in the mutagenic and non-lethal range for E. coli WP2 uvrA

7

i

i

i

eo i

o

o x

5 -

Control

MMS

DDVP "~'O'~t

.

?

, Q-

/

3

o

1

o.

~

10 Fract;0n

I

I

20

30

number

Fig. 5. Alkaline sucrose gradient centrifugation of DNA from E. coli WP67 treated at 37° for 60 min with o.02% (v/v) MMS or o.1% (v/v) DDVP. Fractions were collected from the bottom of the gradient. Fig. 5 shows that a substantial lowering of the molecular weight was produced by both mutagens. Little or no DNA breakage was observed with the isogenic polA + strain at these doses (data not shown). In view of the rapidity of repair by DNA polymerase I z*, we conclude that most of the DNA breaks are repaired in the polA + strain during the treatment. This also makes it very unlikely that the lowering of molecular weight in the polA strain is due to the production of alkali-labile bonds rather than to strand breaks. At higher doses of both MMS and DDVP pronounced

MUTAGENICITY OF DDVP

AND MMS

IN

E . coli

3Ol

lowering of the molecular weight of the DNA was also shown by polA + bacteria. Studies are continuing, and will be reported elsewhere, on the kinetics of DNA degradation and its relation to survival and mutation induction.

Mutagenesis of polA bacteria There is considerable circumstantial evidence, both here and elsewhere, that single-strand DNA gaps or breaks are potential premutagenic lesions. What is not yet clear, however, is the relative mutagenicity of the various hypothetical types of break. It has been postulate& that at least one operational category of break is repairable either by the recA+/exrA + pathway or the potA + pathway and that in a polA strain all the effective repair should be channelled through the former pathway. Since this is known to be error-prone2e,L a higher induced mutation frequency should result. This was found to be so for y-irradiated logarithmic phase bacteria 7, although the effect was fairly modest, between 1.5- and 2-fold. We thought that MMS might enable a rather cleaner test of this hypothesis than ionizing radiation for two reasons: (a) it should be possible to choose a dose where there is little or no lethality, and (b) most of the breaks produced at such a dose should be polA+-reparable whereas about 20% of breaks produced by ionizing radiation are not 24. We chose lower doses than were used in the earlier experiment to minimise lethality and the production of breaks not reparable by the polA + pathway. The results (Table III) show clearly that polA bacteria yield about three times more mutants than polA + bacteria, thus confirming our prediction. With DDVP, to which the polA strain shows a smaller increase in sensitivity over the pol + strain, there was a correspondingly smaller increase in mutability (about 2-fold). Since exrA strains smvive at comparable doses but do not mutate, we can confirm the conclusion of BRIDGES AND MOTFERSHEAD~ that it is the repair system (i.e., a function of the organism) rather than the primary lesion (i.e., a function of the mutagen) that determines mutagenicity. We conclude that at least one type of single-strand break may be repaired accurately by one pathway but inaccurately (i.e., mutagenically by another. TABLE

III

MUTAGENESIS OF W P 2 u v r A AND W P 6 7 AT 37 ° FOR 6O m i D

uvrA polA BY 0 . 0 2 % ( v / v ) M M S AND O . 1 % ( v / v ) D D V P

The values are the mean of three determinations.

Strain

WP2 WP67

uvrA

Pol

+ --

Pol-/Pol + ratio

MMS Surviving fraction ( . I o -1)

Induced mutant frequency (4- standard error)

DD V P Surviving fraction (.lO -1)

Induced mutant frequency (4- standard error)

8.54 8.48

3 . 5 5 ± 1 . 4 2 " IO-T 1.18 4- o . I 9 . I O -6

8.38 8.59

1 . 7 9 4- 0 . 3 9 . IO -~ 3 . 8 4 4- o . 6 6 . 1 o -~

--

3-o4

--

2.14

DISCUSSION

Our results show clearly that DDVP is mutagenic to Escherichia coli at nontoxic concentrations. The response of strains deficient at four different repair loci

302

B.A. BRIDGES et al.

(polA, uvrA, recA, exrA), moreover, is qualitatively similar for DDVP and the methylating agent MMS, with regard to both survival and mutagenesis. There is little doubt in our minds that the mutagenic action of DDVP is a result of its known ability to alkylate DNA17, ~. The irrelevance of the uvrA gene to the toxic and nmtagenic effects of both compounds shows that the lesions responsible for these effects are not susceptible to excision and confirm previous results with MMSg, 16. PRAKASH AND STRAUSS 21 have shown that 14C-methyl groups transferred to DNA from MMS persist for well over an hour in both Bacillus subtilis and Escherichia coll. Like MMS, D D V P requires a functional recA ~/exrA + repair system for mutagenesis to occur. The methylations (or the ensuing DNA strand breaks) aie therefore not mutagenic either in their own right or when repaired b y the polA ~ pathway, but only when dealt with by the error-prone recA+/exrA + pathway. The enhanced mutagenicity of both compounds for the polA strain illustrates what happens when repair is channelled obligatorily through the error-prone pathway. The demonstration of the production of single-strand DNA breaks by D D V P and MMS is consistent with the hypothesis that such breaks are the raw material for error-prone repair 8. We emphasize, however, that the present data do not enable any conclusion to be drawn regarding possible differences in the probability of an error being made when different types of break are repaired by the recA+/exrA -~ pathway, nor are they inconsistent with the hypothesis that mutations in Rec+/Exr + strains m a y also arise from gaps formed during post-replication repair (there is some evidence for this type of repair in Bacillus subtilis following MMS treatment2~). Although there are m a n y similarities between the action of MMS and DDVP there are also quantitative differences. MMS is much more mutagenic than DDVP whether comparison is made at comparable concentrations or at doses producing similar DNA breakage. In addition MIVIS lesions are more likely to be lethal than D D V P lesions when either the p o l A - or the recA-~/exrA + repair system alone is operative, as shown b y the relatively greater sensitivity of single deficient mutants. The cause of the lowering of molecular weight of DNA in bacteria treated with MMS and D D V P has not been determined. In Micrococcus lysodeikticus there is a nuclease that is specific for methylated DNA ~3 but its existence has not been demonstrated in E. coli. It is interesting that in neither B. subtilis nor E. coli do methylations appear to be removed from the DNA during incubation ~. Depurination leading to strand breaks in the alkaline gradient m a y also be a cause of the lowering of molecular weight at higher doses in polA ~ strains; nothing is known about the way in which depulination is repaired in vivo. In addition to the methylation of purines, there appears to be the possibility that D D V P and MMS m a y attack the phosphate groups in DNA 2. This m a y lead directly to a scission of the backbone resulting in a particularly "clean" and easily repaired type of break. The genetic effects of such lesions, if they occur, deserve investigation. ACKNOWLEDGEMENTS

The authors are grateful to Dr. EVELYN M. WITKIN for supplying them with strains W P I 2 and WP67.

MUTAGENICITY OF D D V P

AND M M S IN E. coli

303

REFERENCES I ASHWOOD-SMITH, M. J., J. TREVINO AND R. RING, Mutagenicity of dichlorvos, Nature, 240 (1972) 418-42o. 2 BEDFORD,C. T., AND J. ROBINSON, The alkylating properties of organophosphates, Xenobiotica, 2 (1972) 3o7-337. 3 BOYLE, J. M., AND N. SYMONDS, Radiation sensitive mutants of T4D.I. T4y: a new radiationsensitive mutant; effect of the mutation on radiation survival, growth and recombination, Mutation Res., 8 (1969) 431-439 • 4 BRIDGES, B. A., R. E. DENNIS AND R. J. MUNSON, Mutation in Escherichia coli B/r WP2 tryby reversion or suppression of a chain termination codon, Mutation Res., 4 (1967) 5o2-5o4 • 5 BRIDGES, B. A., R. E. DENNIS AND R. J. MUNSON, Differential induction and repair of ultraviolet damage leading to true reversions and external suppressor mutations of an ochre codon in Escherichia coli B/r WP2, Genetics, 57 (1967) 897-908. 6 BRIDGES, B. A., J. LAW AND R. J. MUNSON, Mutagenesis in Escherichia coli, II. Evidence for a common pathway for mutagenesis by ultraviolet light, ionizing radiation and thymine deprivation, Mol. Gen. Genet., IO3 (1968) 266-273. 7 BRIDGES, B. A., AND R. P. MOTTERSHEAD, Gamma ray mutagenesis in a strain of Escherichia toll, deficient in DNA polymerase I, Heredity, 29 (1972) 2o3-211. 8 BRIDGES, B. A., R. P. MOTTERSHEAD, M. ANNE ROTHWELL AND M. H. L. GREEN, Repairdeficient bacterial strains suitable for mutagenicity screening: Tests with the fungicide captan, Chem.-Biol. Interactions, 5 (1972) 77-84. 9 BRIDGES, B. A., AND R. J. MUNSON, Excision-repair of DNA damage in an auxotrophic strain of Escherichia coli, Biochem. Biophys. Res. Commun., 22 (1966) 268-273. io DAVIS, B. D., AND E. S. MINGIOLI, Mutants of Escherichia coli requiring methionine or vitamin Blz, J. Bacteriol., 6o (195 o) 17-28. II DEAN, B. J., The mutagenic effects of organophosphorus pesticides on microorganisms, Arch. Toxikol., 3° (I972) 67-74. I2 GREEN, M. H. L., W. J. H. GRAY, S. G. SEDGWICKAND B. A. BRIDGES, Repair of DNA damage produced by gamma radiation in Escherichia coli K-I2 and a radiation sensitive exrA derivative during inhibition of protein synthesis and normal DNA replication by chloramphenicol, J. Gen. Microbiol., in press. 13 HILL, R. F., Dose-mutation relationships in ultraviolet-induced reversion from auxotrophy in Escherichia coli, J. Gen. Microbiol., 3° (1963) 281-288. 14 KAPP, D. S., AND K. C. SMITH, Repair of radiation-induced damage in Escherichia coli, II. Effect of rec and uvr mutations on radiosensitivity, and repair of X-ray induced single-strand breaks in deoxyribonucleic acid, J. Bacteriol., lO 3 (197 o) 49-54. 15 KATO, T., AND S. KONDO, Genetic and molecular characters of X-ray sensitive mutants of Escherichia coli defective in repair synthesis, J. Bacteriol., lO4 (197o) 871-881. 16 KONDO, S., H. ICHIKAWA,K. Iwo AND T. KATO, Base change mutagenesis and prophage induction in strains of Escherichia coli with different repair capacities, Genetics, 66 (197 o) 187-217. 17 L6FROTH, G., Alkylation of DNA by dichlorvos, Naturwissenschaften, 57 (197 o) 393-394. 18 L6FROTH, G., C. KIM AND S. HUSSAIN, Alkylating property of 2,2-dichlorovinyl dimethyl phosphate: a disregarded hazard, E M S Newsletter, 2 (1969) 21-27. 19 MORIMYO, M., Z. I. HORII AND K. SUZUKI, Appearance of low molecular weight DNA in a Recm u t a n t of Escherickia coli K-IZ irradiated with X-rays, J. Radiation Res., 9 (1968) 19-25. 20 OSBORN, M., AND S. PERSON, Characterization of revertants of E. coli WU36-IO and WP2 using amber mutants and an ochre mutant of bacteriophage T4, Mutation Res., 4 (1967) 504-507. 21 PRAKASH, L., AND B. STRAUSS, Repair of alkylation damage: stability of methyl groups in Bacillus subtilis treated with methyl methanesulphonate, J. Bacteriol., lO2 (197 o) 76o-766. 22 SEDGWlCK, S. G., AND B. A. BRIDGES, Survival, mutation and capacity to repair single-strand DNA breaks after gamma-irradiation in different E x r - strains of Escherichia coli, Mol. Gen. Genet., 119 (I972) 93-1o2. 23 STRAUSS,B., AND M. ROBBINS, DNA methylated in vitro by a monofunctional alkylating agent as a substrate for a specific nuclease from Micrococcus lysodeikticus, Biochim. Biophys. Acta, 161 (1968) 66-75. 24 TowN, C. D., K. C. SMITH AND H. S. KAPLAN, DNA polymerase required for the rapid rejoining of X-ray induced DNA strand breaks in vivo, Science, 172 (1971) 851-853. 25 VOOGD, C. E., J. J. J. A. A. JACOBS AND J. J. VAN DER STEL, On the mutagenic action of dichlorvos, Mutation Res., 16 (1972) 413-416. 26 WITKIN, E. M., Mutation-proof and mutation-prone modes of survival in derivatives of Escherichia coli B differing in sensitivity to ultraviolet light, Brookhaven Syrup. Biol., 20 (1967) I7-55.