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B A S E - C H ~ G E ~ ~ A G ~ E S I S AND PROPHAGE I ~ U G ~ O ~ IN STRAINS OF ESCHERZCHZA COLZ WITH DIFFEXFt"

DNA REPAIR CAPACITIES

SOHEI KONDO, HARUKO ICHIKAWA, KAZUHIRO IWO AND TAKES1 KAT0

Department of Fundamental Radiohgy, Fuculty of Medicine, Osaku University, Kita-ku, Osaka 530, Japan

Received April 8, 1970

O U R knowledge of the process of mutation induction is largely limited to simple cases of chemical mutagenesis in phage (FREESE 1963; HOWAR~ and

TESSMAN 1964; BAKER and TESSMAN 1968). The present paper concerns base- change mutagenesis in bacteria. Since bacteria are essentially different from phage in their possession of complete machinery for self-reproduction, mutagenesis in the bacterium is a priori expected to be, at least partly, different from that in phage. In fact, UV (ultraviolet radiation) and X rays are very weak mutagens for phage but are highly or moderately mutagenic for bacteria, whereas analogues of normal nucleic acid bases are highly mutagenic in phage (FREESE 1963; HOWARD and TESSNAN 1964) but only moderately mutagenic in bacteria. It has been previously pointed out (KONDO 1964.) that living systems seem to have increased radiation mutability with increasing genome complexity from phage to higher forms. One of the reasons why we expect different mutagenesis for living systems with different genome complexity is that detection of mutation is usually done not directly at the DNA level but only indirectly through some change in phenotypic characteristics of surviving individuals. Also, living systems with different genome complexity have markedly different capacities to tolerate DNA lesions induced by ionizing radiation, UV and 32P (see reviews by TERZI 1961; VON BORSTEL 1966; KONDO 1964; SPARROW, UNDERBRINK and SPARROW 1967).

The method adopted in the present experiments to probe mutagenesis was to compare mutation frequencies with various agents in E. coli strains possessing different radiosensitivities due to different DNA repair capacities. The four types of strains used (Table 1) were wild type (normal DNA repair capacity), Exc- (unable to excise dimers; defective in the presumed first step in the excision repair mechanism [ SETLOW and CARRIER 1964; BOYCE and H O W A R D - F ~ N D ~ S 1964; KELLY et al. 19691, Res- (unable to resynthesize the excised portion; defective in the presumed second step of excision repair [PETTIJOHN and HANA- WALT 1964; KELLY et al. 1969 J ) and Rec- (defective in recombinational repair [CLARK and MARGULIES 1965; HOWARD-FLANDERS 1968l) . Similar, though not so extensive, comparative studies were made on prophage-inducibilities in the same set of strains with the hope of elucidating the process of mutagenesis. The mutations studied in the present experiments were phenotypic reversions from Genetics 66: 187-217 October, 1970

188 SOHEI KONDO et al.

nonsense type auxotrophy (Arg) to prototrophy (Arg+). They belong to the same category as those prototrophic mutations which are believed to be almost all base-change mutations (OSBORN et al. 1967; BRIDGES, DENNIS and MUNSON 1967; BRIDGES and M U N ~ O N 1968; PERSON and OsBoRN 1968).

Physico-chemical alteration of DNA is often assumed to be the principal step in mutagenesis in phage (FREESE 1963). Recently, however, BAKER and TESSMAN (1968) have obtained suggestive evidence that the molecular environment at the replicating point in DNA may be responsible for different mutagenic specificities of some chemicals in phages S13 and T4, It has already been reported that de- ficiencies in X-ray resistance, due to exr (WITKIN 1967) and recA (MIURA and TOMIZAWA 1968; WITKIN 1969a) mutations, suppress almost completely the mutagenicity of UV and that strains of Proteus (BOHME 1967) and yeast (VON

BORSTEL et al. 1968) with different radiosensitivities have different spontaneous mutation rates. It has been previously reported in a preliminary form (KONDO 1968) that all the three kinds of error, i.e., repair error, recombination error, and replication error, are responsible for induction of mutations in E. coli. This conclusion is more fully documented in the present paper which presents decisive evidence to support the view of AUERBACH (1966, 1967) that cellular processes play an important role in mutagenesis.

MATERIALS AND METHODS

Bacterial strains: The two parent strains used were obtained from WITKIN (1964). Strain H/r30 is an arginine-requiring ( A r g ) substrain of the radiation-resistant variant (B/r type) of HARM'S (1962) p h r (photoreactivating-enzyme deficient) derivative of Escherichia coli B, and strain H/r30R is a phr+ revertant of H/r30. I t has been previously shown (ICHIKAWA and KONDO 1969) that the A r g character is due to an amber nonsense mutation at the argF locus. From these parent strains various radiosensitive derivatives were isolated and their characteristics

TABLE 3

Releuant characteristics of the E. coli strains used

DNA repair Auxotrophic Other characters Strain Phenotypic Genehc' marker Hcr phr T1 Rec

H/r30 wild type uur+rec+ argF,, + s - s + H/r30R wild type uurfrec+ a&,, + + s + Hs30 Exc- uvrB a d a m --I - R + Hs30R Exc- uurA a&,, - + R + R11 Exc- uvrA argFam - + s + R15$ Res- res-I WgFam *-I + s (+)t NG30$ Rec- recA argFam + - s -t

* These loci uvrA, uurB, recA, and res-I were cotransduced by Pl phage with malB, gal, cysC, and metE at frequencies of 71, 16, 8, and 4%, respectively (ICHIKAWA, unpublished; KATO and K O N D ~ 1970). 5 Symbols +, - and k mean, respectively, Hcr positive for W- and X-rayed T1 (or T3)

phage, negative for UV-irradiated T1 (or T3) phage, and positive for UV-irradiated T1 phage but negative for X-rayed T1 phage (KATO and KONDO 1967).

-f When used as recipient for conjugation with an Hfr strain, R15 and NG30 gave about 10-fold and 1 O"-fold less Argf recombinants than H/r30, respectively (ICHIKAWA, unpublished).

$ Evidence for the res-I and recA mutations is given in KATO and KONW (1970).

BASE-CHANGE MUTAGENESIS IN E . coli 189

have been partly reported (KONDO and &TO 1966; KATO and KONDO 1967). Out of about twenty radiosensitive mutants (KATO and KONDO 1967), the following strains, belonging to one of the three phenotypically distinct types, were selected for the present experiments: strains Hs30 (uvrB), Rll(uvrA), and Hs30R( uvrA) are Exc- (lacking ability to excise dimers), R15(res) is Res- (defective in repair-resynthesis [the second step of the excision repair process] due to defective DNA polymerase activity (KATO and KONDO 1969, 1970), and NG30(recA) is Rec- (lacking recombination ability). Relevant characteiistics of the strains used are given in Table 1.

Media: The media used for growth of cultures and for scoring survival or mutants were essentially the same as used previously (WITKIN 1963; KONDO and KATO 1966). The growth medium was nutrient broth (NB) containing 8 g Difco Nutrient Broth, 4 g NaC1, 1 liter water, at pH 7. Mutation and survival were assayed on the same plating medium, 5% SEM (semi- enriched medium) agar (minimal E agar supplemented with 0.4% glucose, 5% liquid nutrient broth and 0.5% NaCl) . The PA medium used for assay of prophage induction and survival was made of peptone agar (IO g Polypeptone, Daigo Chemicals, Osaka, 2.5 g NaCI, 15 g agar, 1 liter water, at pH 7) and 0.4% beef extract (Wako Pure Chemical Co.). The top agar used for plaque assay was 0.7% agar containing 1% Difco Tryptone and 0.5% NaC1. When it was necessafy to kill streptomycin-sensitive cells, the concentration of streptomycin (SM) added was 50 .ug/ml for the SM cover top agar and 100 pg/ml for either regular top agar or bottom agar. Detailed descriptions of the prophage assay were given previously (TAKEBE et al. 1967; Iwo 1968). Phw- phate buffer or 0.85% saline was used for dilution.

Treatments with mutagens: The mutagens used were UV, X rays, 4NQ0 (4-~tro-quinoline- 1-oxide) (obtained from Prof. H. ENDO, Kyushii Univ.), MMC (Mitomycin C) (Kyowa Hakko Co.), EMS (ethyl me~anesulfonate~, MMS (methyl me~anesu l fona~) and NTG (N-methyl- N'-nitro-N-nitrosoguanidine) (Aldrich Chemical Co.). Overnight cultures were washed twice, resuspended, and then starved in ni/l5 phosphate buffer (pH 6.8) for 1 hr at 37°C. The buffer suspensions were exposed to UV or X rays, or they were kept in the dark at 30°C or room temper- ature for 1 to 3 hr after addition of one of the above-mentioned chemicals. Mutagens were re- moved by washing in buffer with centrifugation three (or two) times for mutation (or prophage) induction experiments. An ethyl alcohol solution of 4NQO (2 mg/ml) (a powerful carcinogen: NARAHAR~ FUKUOKA and SUGIMURA, 1957) was stored in a refrigerator and added to culture suspensions for mutation or prophage induction. The MMC solution was prepared for each set of experiments by dissolving MMC powder in buffer solution. NTG solution (1 mg/ml) was prepared by dissolving in buffer solution and storing in a deep freezer until use. MMS and EMS to be added to culture suspensions were taken from bottled solutions purchased from Koch-Light Laboratories and stored in a refrigerator. UV irradiation was done with two 15 W low-pressure mercury Toshiba germicidal lamps, emitting prinlalily 2537 A. and X rays were given with a Toshiba X-ray generator operated at 180 kVp and 25 mA with a 1.0 mni eluminum filter. The UV-dose rates used were 10 erg/mmZ/sec for resistant strains and 0.8 erg/mmZ/sec for sensitive strains. These were measured by a ZnS meter (Toshiba Hoshasen Co.) calibrated against a standardized thermopile. The X-ray dose rate was 450 R/min measured by the Fricke dosimeter.

Assay for mutation and prophage induction: For mutation assay, usually 0.1 ml samples, diluted 10-fold [containing about 1 0 7 cells for control], were plated on SEN agar and incubated at 37°C for about 40 hr. For assaying low mutation yield, 0.1 to 0.5 ml samples of cell suspensions concentrated about 4-fold were plated. The number of large colonies, which were distin- guishable from a background film of auxotrwphs, was scored for each plate. Induced mutation frequency Fi was calculated, unless otherwise stated, as ( M ~ - ~ ) / ( l O ~ ~ ~ ) , where Mi and M are the number of mutant colonies per plate for the treated cells and the control, respectively, and Ni denotes the number of surviving colonies per plate for the treated cells (plated at 10" dilution in ~ / 1 5 phosphate buffer). The rationale for estimation of mutation frequency by this method is based on the fact that &l was almost independent of the initial surviving cell number per plate for the control (see Figure 1). Therefore, when M , after treatment with a putative mutagen did not become larger than M at doses around 37% survival (which usually give maximal values for M , as exemplified in Table 4), we conclude that the treatment is non-mutagenic or is negligibly mutagenic. For some purposes, minimal (MM) agar containing E medium and


0.4% glucose with or without I pg/ml arginine, was used for assaying mutation. Prophage induction was assayed by scoring the number of infective centers. In order to minimize the effect of plaques arising from cells spontaneously induced during incubation, plates covered with the ordinary top-agar layer were incubated for 3 hr, then covered with 2.0 ml soft agar (0.5% agar, 1% Difco Tryptone, 0.5% NaCl) containing streptomycin (50 pg/ml) and incubated further until the time of plaque counting.

Photoreactivation ireatment: Photoreactivation treatments were given as previously described (KONDO and K A ~ 1966) with a large Bausch and Lomb grating monochromator and a 500 W high pressure mercury arc lamp (Philips SP-500).

RESULTS

Spontaneous mutation: According to LURIA-DELBRUCK (1943) and NEW- COMBE ( 1 W8), the average number of prototrophic bacteria per liquid culture, m, and that of prototrophic colonies per plate, M , are given, respectively, by

m = (a N / l n 2) In (C a N/ln 2 )

M = M O 4- a(N - No)/ln 2 and

where a is mutation rate per bacterium per division cycle, N (or N o ) the average number of viable auxotrophic bacteria at the end of growth (or initially plated), C the number of cultures in an experiment, and M (or M O ) the number of prototrophic colonies per plate appearing after (or beforej incubation. NB- grown overnight cultures, without washing or starvation, were diluted or con- densed and then plated on SEM plates (Figure 1). As previously reported (DEMEREC and CAHN 1953), the number of mutants per plate, M , which was scored after 40 hr of incubation in the present experiment, was almost independent of the number No of viable cells initially plated. The 5% nutrient broth in SEM agar limits the final number N of auxotrophic bacteria to an almost constant value regardless of N o within a proper range (DEMEREC and CAHN 1953). This explains why M on SEM agar is almost independent of No. As previously de-

I N I T I A L N U M B E R O F CELLS PER P L A T E FIGURE 1.-Very weak correlation between the number of spontaneous mutants per plate and

the initial number of cells plated. Numbers of prototrophs in strains H/r30, R15, and NG30 spontaneously induced on SEM agar during incubation at 37°C for 2 days, are plotted against number of cells initially plated.

BASE-CHANGE MUTAGENESIS IN E. coli 191

scribed (DEMEREC and CAHN 1953), N values were estimated by assaying colony formers in phosphate buffer suspensions obtained by washing the plates after mutant colonies were cut out of the agar or by washing fractional pieces of agar bearing no visible mutant colonies. Cell growth stopped after about 12 hr incubation and the final number of viable bacteria per plate was about the same during 12 to 48 hr incubation for H/r30R and Hs30R as previously reported by DEMEREC and CAHN (1953) with B/r strains. However, R15 and NG30 gradually died during incubation. To reduce the effect of cell death, large amounts of bacteria (2 to 3 X lo9 cells per plate) were plated on minimal agar and SEM plates following the method described in the next paragraph, and MO and M were scored, respectively, after a 2-day incubation. Cells of strains R15 and NG30 reached their maximal N values of about 1.5 and 2 x loxo cells per plate, respectively, in about 7 hr incubation and kept almost the same N value up to 13 hr incubation, but only about half of them were viable when assayed after 48 hr incubation. Such gradual death cannot inhibit the colony forming ability of prototrophic cells because they grow very rapidly. Now even the last groups of mutations, which must have occurred before cell growth had stopped (around the 7th hr of incubation), had more than 5 hr to prepare for expression of ,their prototrophic character before the cell death started. Therefore, we assume that the maximal N value, N,,,, is to be used instead of N,, ( N value at the 443th hr of incubation) for estimation of mutation rates by Equation (2). N,, was estimated from the optical density of N,, multiplied with the ratio of an N,, to its optical density obtained in a separate experiment. Optical density was almost the same for N,, and N48. For H/r30R and Hs30R, we used N,, because there was no appreciable cell death. Table 2 summarizes the mutation rates estimated in this way. To demonstrate that mutation rate is independent of No, rates at normal No values (about lo7 cells/plate) are also shown for strain H/r30R.

TABLE 2

Spontaneous mutation rates estimated from the number of prototrophic clones appearing during growth on SEM agar

Strain * N' or N,,, Experiment

H/r30R

N/No* US( X lo-@) 1-2.3X loio

Hs30R 1.6-2.7X 10'0

N / N o a(X10-g)

R15 1.3-1.7X1010

N, , , /N~ U( x 10-9)

A B C D E F

average mutation rates ( x 10-9)

103 1.5 103 1.4 103 1.2 103 1.9 7 1.5 6 1.3

1.5 f 0.3

5 1.0 6 1.4 6 0.8 7 1.1 14 1.2 5 2.6

1.4 t 0.7

8 3.3 7 3.8 6 4.9 7 5.6 9 5.1 6 4.8

4.6 f 0.9

NG30 1.1-2.3X 101'

N,,,/N, 4 X 10-9

8 0.63 8 0.28 7 0.M 6 0.47 7 0.47 8 0.84

0.56 f 0.19

* No, N,, and N denote, respectively, the initial, the maximal, and the fmal number of viable

Mutation rate a was calculated by NEWCOMBE'S (1948) method 4 (see Equation (2) in text). auxotrophic bacteria per SEM plate.

1 92 SOHEI KONDO et al.

TABLE 3

Spontaneous mutation rates estimated from the average numbers of prototrophic bacteria per culture in series of similar cultures started from small inocula

Strain H/r30R Hs30R R15 NG30 Experiment A B A B A B A B C

Inoculum (number of bact.) 2x104 2x104 2 x i 0 4 2x104 3x103 1x10" 2x104 1x104 1x10"

Number of prototrophic bacteria

Culture No. ( X 5 )

1 49 2 88 3 39 4 29 5 53 6 . .

( X 10/3.G)

77 81.

21 0 31 72 61

( X5) ( X 10/3.6)

34 43 31 153 49 20 12 195 81 93

150 51

(X5 )

145 132 98 62 56 58

~

( X 10/3.6)

100 86

283 89 99

129

( X 5 ) ( X10/3.G)(X10/3.6)

5.5 39 16 23 17 22 7.5 34 61 10 24 13

27.5 . . . . 81 . . . .

Average number of prototrophic bacteria per culture 258 248 297 255 460 365 129 79 78

Bacteria per culture, X 1O1O 1.6 1.4 1.55 1.7 1.2 1.1 1.3 1.3 1.4

x i t 9 2.1 2.2 2.3 1.9 4.3 3.7 1.4 1.0 0.89 Mutation rate,*

* Mutation rates were calculated by LURIA-DELBRUCK'S (1943) method, i.e., NEWCOMBE'S ( 19 1.8) method 2 (see Equation ( 1 ) in text).

Mutation rates were estimated also by applying Equation (1). Two or three separate experiments were carried out, and for each, 4 to 6 NB cultures of 10 ml were grown for about 14 hr at 37°C with shaking. The inocula contained about IO4 bacteria. The grown cultures were washed once. starved in phosphate buffer for 1 hr at 37°C by shaking, washed again and then 0.3 (or 0.2) ml samples were plated on minimal agar after 4-fold (or IO-fold) concentration in buffer. The results are shown in Table 3. As will be seen from comparison of Tables 2 and 3, the mutation rates estimated by the two different methods are in a fairly good agreement, and we may conclude that the spontaneous mutability is high in R15, low in NG30, and normal in Hs30R when compared with the mutability in H/r30R.

Mutation and killing induced by UV and 4NQO: Figures 2 and 3 and Table 4 show a comparison of UV and 4NQ0 sensitivities to killing and mutation among strains H/r30R, Hs30R, R15, and NG30. Differential sensitivities of these four strains to killing and mutation by UV are almost the same as those by 4NQO. It should be noted that number of mutants per plate for strain NG30 did not significantly increase beyond that for the control after treatment with either UV or 4NQ0, whereas R15 gave rise to mutation yields significantly beyond the spontaneous value in the same dose ranges and both R15 and NG30 showed roughly similar killing sensitivity to UV and 4NQ0 (Table 4). Therefore, it is not very likely that high killing sensitivity masked appearance of UV- or 4NQO-induced miitations in NG30. All the negative results of mutation induction, partly re-

BASE-CHANGE MUTAGENESIS IN E. coli

10''

193

NG30 R15

4 NC@ DOSE pM-hr 1 2oo 5p I I I I I I

FIGURE 3.-Induced HdOR, and R15. See the

p ;'Wr 30 R

3 /

Z 91 6'- +

uv om^ 16'-

,I; 4NQO DOSE(pMhr1 5 / / 10 5 rpo 5 1070

10'. L ' 'r , ' 1 ,

10 5 100 5 1000 2 uv DOSE( erg/mm2)

mutation frequency versus dose of UV and 4NQ0 legend of Figure 2 for the 4NQ0 treatment.

for strains H/r30R,


TABLE 4

Typical cases of mutations to prototrophy in Rec- strain NG3O compared with those in R e d strain H/r30, H/r30R, or RI5

Survival (10-6 dil.) Mutations (undiluted) Plating Plating Induced volume Colouy volume Colony frequenciest

Strain Mutagen Dose (ml) counts’ (ml) counts‘ (10-6)

UV (erg/”*) R15 0

1.5 3

NG30 0 1.5 3

4NQO (M.hr)

5x10-6 RI5 0

10-5

10-5

NG30 0 5X1W

Xrays (kR) H/r30R 0

1 2

NG30 0 1 2

MMS ( m g m ” l ) RI5 0

0.2 0.4

0.2 0.4

NG30 0

NTG (pgml-1.h) H/r30 0

2 4 6

NG30 0 2 4 6

0.1 0.1 0.1 0.1 0.1 0.1

0.1 0.1 0.1 0.1 0.1 0.1

0.025 0.025 0.025 0.05 0.05 0.05

0.05 0.05 0.05 0.05 0.05 0.05

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

336 278 280

1255 41 5 103

41 7 121 57

309 156 69

91 73 56

21 3 171 54

260 182 157 323 199 147

213 230 175 200 195 110 77 54

0.2 0.2 0.2 0.2 0.2 0.2

0.1 0.1 0.1 0.1 0.1 0.1

0.2 0.2 0.2 0.5 0.5 0.5

0.2 0.2 0.2 0.2 0.2 0.2

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

77 97 95 12 11 6

73 84 95 9

10 Y

31 61 89 14 14 11

87 99

139 6 3 6

25 292

2423 -4000

9 21 63

106

. . 3.6 3.2 . .

< O < O

. .

9 39

. . 0.6 0

. . 5.1

13

0 < O

. .

. . 1.6 8.3 . .

< O 0

. . 29

340 -500

. . 2.7

18 45

* Each value is the average of duplicate or triplicate plates, f Frequencies were operationally calculated as described in text and not corrected for statistical

fluctuation. Therefore, “<O” or “0” means simply that number of mutants per plate for the treated cells happened to be smaller than or equal to that for the control.


ported in Table 4 or Figures 3 and 4, were confirmed by two or more separate experiments. Therefore, we may conclude that NG30 has no mutability or a very reduced mutability toward UV and 4NQO. This conclusion means that the Rec+ character is indispensable for efficient conversion of UV or 4NQO damage to a mutant base sequence in the daughter DNA.

The notion (KONDO and KATO 1968) that 4NQO lesions are repaired by the same mechanism as that effmtive for UV-induced pyrimidine dimers is supported by the following experimental results. 4NQO- or UV-treated cells of H/r30R and Hs30R were held in nutrient broth (NB) for various times at 37°C and then plated on SEM 4- AC agar (SEM agar with acriflavine added to a final concentration of 1 ,ug/ml). Acriflavine (AC) was added to reduce the recovery on agar plates, since WITKIN (1961) and SETLOW (1964) showed that acriflavine inhibits excision repair of dimers by binding with DNA. As shown in Figure 4, the rate of loss of susceptibility to AC-enhanced killing in H/r30R after 4NQ0 was as high as that after UV, whereas susceptibility to AC-enhanced killing was not lost with strain Hs30R after treatment with either UV or 4NQO.

As shown in Figure 5 , we were unable to detect any recovery effect of photo-

m

Hfr30R Hs30R I

s e m o sem+ACO m

." 0 10 20 30

N B H O L D I N G TIME (min.)

FIGURE 4.-Dark repair kinetics for killing caused by UV and 4NQO. Survival of UV- or 4NQO-treated cells of strains H/r30R and Hs30R are plotted against time of holding the cells in liquid NB (nutrient broth) at 37°C before plating on SEM or SEM + AC (SEM containing 1 pg acriflavine per ml) agar plates.


' O ' r

\ \

GNOO U V SURVIVAL 0

MUTATION

10

FIGURE 5.-Non-photoreactivahility of 4NQO-induced killing and mutation in strain H/r30R. Aliquots of cells treated with 4NQ0 or UV were illuminated with PR (photoreactivation) light, at 4047 or 3341 A, with various doses and then plated on SEM plates.

activation treatment on mutation and killing induced by 4NQ0 in H/r30R whereas the same photoreactivation treatment markedly reduced mutation and killing induced by UV in the same strain. These results mean that 4NQO lesions are chemically different from the photoreactivable UV lesions (pyrimidine dimers) although both are repaired by the same dark-repair mechanism with almost equal efficiency.

Mutation and killing induced by MMC: Figures 6 and 7 show kinetics of killing and mutation in strains H/r30, R15, NG30, and Hs30R after treatment with MMC. The yields of mutation were plotted not only as mutants per survivor (i.e., mutation frequency) but also in terms of the number of mutants per plate. Since the number of mutants per plate did not increase beyond the number of spontaneous mutants after MMC treatment for Hs30R and NG30, we can conclude (from the arguments in METHODS under Assay for mutation) that yields of MMC-induced mutants in these Exc- and Rec- strains are zero or negligibly small. It is difficult to rule out the possibility that high killing sensitivity masked


FIGURE 6.--Survival (in solid symbols) and number of mutants per SEM plate (in open symbols), a t various doses of mitomycin C, in strains H/r30, R15, and NG30. Mutability is also replotted in terms of frequency of induced prototrophs per survivor for strains H/r30 and R15. Cells were exposed to MMC of various concentrations for 2 hr at 30°C. The number of cells per plate for scoring mutations at zero dose was 1.5 x 108 for NG30, 1.1 x 10s for R15 and 2.9 X los for H/r30.

the appearance of MMC-induced mutations in NG30 and Hs30R after MMC treatment but this is not likely. As shown in Figure 7, MMC-induced mutations in H/r30 were detectable at 0.025 yml-l.hr where NG30 and Hs30R showed nearly 100 % and 40 % survival, respectively.

The finding that MMC mutation is nonexistent (or negligibly small) in the Exc- strain Hs30R but does appear in Exc+ strain H/r30 and Res- strain RI5 sug- gests that MMC mutations are not due to MMC lesions themselves but occur as an effect of the excision function on MMC lesions. If this is the case, then addition of acriflavine to SEM agar to suppress the excising function should reduce the yield of mutants in H/r30 but should not affect the mutation yield in Hs30R. This prediction was supported by the results shown in Figure 7; addition of acriflavine reduced both mutation yield and surviving fraction in strain H/r30 but gave negligible effects on mutation and survival in strain Hs30R.

A tempting hypothesis to account for these findings is that MMC mutation is due to excision repair errors. If this is the case, then we may assume that a con- dition favorable for excision repair will enhance the yield of MMC mutations in H/r30. MMC-treated cells of H/r30 were incubated in phosphate buffer with glucose (0.4%) at 37"C, and at intervals aliquots were withdrawn and plated on SEM agar. Survival of MMC-treated cells increased with increase in time of


10 0 0.2 0.L 0.6 0.8

M M C DOSE ( d-mi.’hrJ

FIGURE 7.-Effects of acriflavine on survival and number of mutants per plate for strains H/r30 and Hs30R treated with mitomycin C. Cells were exposed to MMC in various concentrations at 30°C for 2 hr and then plated on SEM (0,O) or SEM + AC (e,.) (2 pg acriflavine per ml) plates. The number of cells per plate for scoring mutations at zerc dose was 1.4 x IO9 for H/r30 and 1.0 x IO9 for Hs30R.

liquid holding, as expected, but this liquid holding pretreatment decreased the MMC mutation yields on SEM agar (Figure 8). To elucidate these puzzling findings, the following experiment was carried out. MMC-treated cells of H/r30 were plated on MM agar plates with arginine enrichment (1 pg/ml), incubated at 37”C, and at intervals subjected to respreading with 0.1 ml of buffer with or without acriflavine (300 pg/ml) . As will be seen from Figure 9, which is based on duplicate experiments, acriflavine suppressed the mutation yields only when it was given at an early stage (up to about 1.5 hr) post incubation where cell division (i.e., increase in the survival in Figure 9) occurred at around 2.5 hr incubation. These suggest that MMC mutations are induced by unknown events ac- companying only those excision repairs occurring shortly before DNA replication.

Mutation and killing induced by X rcys and NTG: Figures 10 and 11 and Table 3 show comparative survivals and mutation frequencies of strains H/r30, Hs30R, R15, and NG30 after treatment with X rays and NTG. The Rec- strain NG30 did not show any appreciable yield of induced mutations after X-ray treatment, but did show appreciable yields of induced mutation after NTG treatment.

Mutation and killing induced by MMS and EMS: As shown in Figure 13 and


HOLDING TIME ( M I N . )

SURVIVAL

3x1 0' t CONT

'0 30 HOLDING TIME (MIN.)

FIGURE 8.-Effects of liquid holding the MMC-treated cells on their survival and mutation. Cells of H/r30 exposed to mitomycin C (0.3 pg/ml) at 30°C for 2 hr, were held in PO, buffer at 37°C for the various times indicated before plating OE SEM + AC (2 ug acriflavine per ml) agar. The number of surviving cells per plate for scoring mutations was 2.1 x 109 for the control and 1.4 x 109 after MMC treatment.

Table 4, EMS induced mutation in NG30 with high frequency but MMS did not. It is unlikely that high killing sensitivity masked the appearance of mutations in NG30 after MMS treatment; for R15, having similar killing sensitivity, showed high mutation yields after MMS treatment (Figure 12 and Table 4). Survivals and mutation frequencies of H/r30, Hs3OR, R15, and NG30 after treatment with MMS and those of H/r30, R15, and NG30 after treatment with EMS are shown in Figures 12 and 13, respectively. Figure 14 shows a detailed comparison of re- sponses of H/r30R and Hs30R to EMS.

Prophage induction: Strains H/r30R, R l l , R15, and NG30 were lysogenized with phage 480 and the lysogenized strains H/r30R(80), Rll(80) [an Exc- strain corresponding to Hs30R; the latter cannot be lysogenized with 480 because of its T1 resistance character], RI5 (80), and NG30 (80) were treated with UV, MMC, X rays, NTG, or EMS. Prophage induction frequencies per treated cell were calculated from the observed numbers of infective centers (see Iwo 1968 for detailed techniques). Results are summarized in Figure 15. There is a good, though not perfect, correlation in susceptibility between prophage induction and mutation induction. Interesting uncorrelated phenomena are that ENIS did not


A C TREATMENT(ht.1 FIGURE 9.-Effective period of acriflavine suppression of survival and mutation in strain

H/r30 treated with mitomycin C. Cells exposed to 0.3 pg/ml MMC at 30°C for 2 hr were plated on minimal agar medium enriched with arginine (1 pg/ml) and incubated at 37°C. At the intervals indicated, 0.1 ml sample of PO, buffer or AC solution (PO, buffer containing 300 gg acriflavine per ml) was added and then respreading treatment was given to each of a proper fraction of plates withdrawn from the incubated plates. The number of surviving cells per plate for scoring mutations at zero incubation time was 1.4 x l o 9 for the control and was 9.8 x 108 after MMC treatment.

induce prophage in strains H/r30R(80) and RI1 (80) but did induce significant amounts of prophage in NG30(80), and that R15 was highly susceptible to W induction of prophage though it was not so sensitive to UV mutation.

Patterns of mutagenicity and prophage induction: Essential parts of the results, mostly reported in the foregoing sections, are summarized in Tables 5 and 6. Dif- ferential sensitivities of the strains to mutagens are expressed in terms of the reciprocal of D,, (dose required for 37% survival) for killing and D, (dose required for frequency of lo-,) for mutation (Table 5), and in terms of pma, (maximal fraction of prophage-induced cells) and D, (dose required for maximal induction) for prophage induction (Table 6). D,, is adopted to measure comparative sensitivity by its reciprocal.

4NQ0 and MMS are the best UV mimetic and the best X-ray mimetic among the tested mutagens, respectively. NTG and EMS are the most powerful mutagens, because the least mutable strain, NG30 with the recA gene, is mutable

SOHEI KONDO et al. 202


I I I

NTG Dd%(J.ml--'hr)

X RAY DOSE (kR)

50

I I I 1

I O

1 5 I O I 5 1 0 1 6

FIGURE 10.-Fraction of colony formers at various doses of X rays and NTG in strains H/r30, Hs30R, R15, and NG30. Cells were exposed to NTG in various concentrations for 1 hr at 30°C.

toward these chemicals and because the maximal mutant yield per plate (which is given roughly by the mutation yield at D,, dose) is markedly higher for these chemicals than for other mutagens. This means that mutagenesis by NTG and EMS must be different in an essential step from that of other mutagens.

Qualitatively speaking, as argued by HILL (1963) , there is a striking similarity between mutability and prophage-inducibility: i) Spontaneous mutation rate and spontannous prophage-induction rate are both abnormally high for the Res- strain R15, abnormally low for the Rec-. strain NG30, and almost normal for the Exc- strains (Hs30R and Rl l (80) ) when compared with the normal strain H/r30R. ii) Exc- strain Rll(80) is highly sensitive to UV induction and Exc- strain Hs30R is also highly sensitive to UV-induced mutation. iii) Rec- strain NG30 is least susceptible to UV and X rays for induction of mutation and praphage, but rather sensitive to NTG for induction of mutation and prophage. Quantitatively speaking, however, some of these similarities do not hold: 1) Induction in Rec- strain NG30 is not detectable for mutation but significantly detectable for prophage


I Hit-30,'

- d H s 3 0 R

NG30 I

P e ' : I

p' I I I I

H/r30 Hs30R I x

X RAY DOSE(kR1 'I 2 4 6 810 I I I I 1

NTG DOSE (ImC;lhr) FIGURE 11 .-Induced mutation frequency of prototrophs versus doses of X rays and NTG for

strains H/r30 (.,0), Hs30R (.,U), R15 (A,A), and NG30 (V) . See the legend of Figure 10 for the NTG treatment.

after UV, X rays, and MMC. 2) EMS can induce prophage in NG30(80) but cannot in the wild type strain H/r30R(80) and Exc- strain RI 1 (80) whereas EMS is almost equally mutagenic for all strains of the wild, Exc-, Res-, and Rec- types. 3 ) Res- strain RI5 is highly sensitive to UV-induced induction of prophage but not so sensitive to UV-induced mutation.

CONCLUSIONS A N D DISCUSSION

STRAUSS (1968) and WITKIN (1969b, 1969~) have recently published excellent reviews on DNA repair and mutagenesis. Therefore, we shall confine ourselves to discussion of the topics directly related with the conclusions deducible from the present experiments.

First, we need to make clear two points: the nature of the Arg+ mutations studied and the characteristics of the different DNA repair capacities introduced


t o

16 -I a 2 > K 3 c n -

10

16

I b NG30

!

\. \ \. ! ! '*

I I \

1 NG30 .02 .04

M.M.S. DOSE ( M.hr.1

1 o3

z l o 2 =

2 Z -I in 71

;o m

lo1 7 s m

1 oo

FIGURE 12.-Survivals (open symbols) and numbers of mutants per plate (solid symbols), at various doses of methyl methanesulfonate, in strains H/r30, R15, and NG30. Cells were exposed to MMS in various concentrations at 30°C for 2 hr. Into these data were incorporated the data of HdOR and H/r30 obtained from separate experiments after applying a dose adjusting factor of 0.6 which makes the two mutation data of H/r30 in agreement. The number of cells per plate for scoring mutants at zero dose was 6.5 x 108 for H/i-30, 6.2 x 108 for Hs30R, 5.2 x 108 for R15, and 3.2 x 108 for NG30.

10

1 0 .A Q > > 2 v)

- a

16

10-

-0 c-- --

'A

, , \

\

V

1 I

0.2 0.4 t E.M.S. DOSE (M.hr.1

FIGURE 13.-Survivals (open symbols) and numbers of mutants per plate (solid symbols), at various doses of ethyl methanesulfonate, in strains H/r30, R15, and NG30. Cells were exposed for 2 hr at 30°C to EMS in various concentrations. The number of cells per plate for scoring mutants at zero dose was 2.2 x 108 for H,/r30, 1.2 x 108 for R15, and 1.2 x 108 for NG30.

206

I I I 1 0.1 0.2 0.3 0.4

EMS CONCENTRATION (M) FIGURE 14.-Survivals and numbers of mutants per plate, at various concentrations of ethyl

methanesulfonate, in strains H/r30R and Hs30R. Cells were exposed to EMS at 37°C for 30 min. The batch of EMS used in this experiment was different from that used for the data given in Figures 13 and 15. The number of cells per plate for scoring mutants at zero dose was 2 x l o 7

for H/r30R and 1.9 x IO' for Hs30R.

into the genome background of the strains in which the Arg-l- mutations were studied. The mutations studied include (1 ) non-suppressor mutations (probably true reversions) and (2) Sup+ mutations at the supE locus which can suppress various nonsense auxotrophic markers of the amber type including the marker argF,, used (ICHIKAWA and KONDO 1969). These mutations belong to the same category as the prototrophic reversions of WITKIN'S other auxotrophic mutants reported by OSBORN et al. (1967), BRIDGES, DENNIS and MUNSON (1967), BRIDGES and MUNSON (1968), and PERSON and OSBORN (1968). As argued by these authors and by WITKIN (196913) , evidence supports the view that these Sup+ mutations are mostly due to base-change type mutations. This conclusion is also supported by our finding that NTG is one of the most powerful mutagens for the Argf mutations among the tested mutagens (Table 5 ) ; NTG is well known as a powerful mutagen to induce base-change mutations but not frameshift mutations ( AMES and WHITFIELD 1966).

The Exc- strains Hs30 and Hs30R used possess, respectively, uvrB and uvrA


10-4 Y

i o + L 300 6;o do uv DOSE (erg/-*)

10-4: 1 1 0.5 1 .o 1.5 .1.7

M M C DOSE (Fm1-l. hr)

I I I 10 20 30 40

X-RAY DOSE (kR)

V

> CL 3 YI

I I 1 80 160 240

N.T.G. DOSE ( 6 n l - t hr )

c

Y 10-3 NG30(80 ) n

'V Y

z 4

10-4 - > PI 3 YI Rll(80)

I 1 1

0.2 0.4 0.6

E . M . S . DOSE (M-hr)

FIGURE 15.-Survivals and fractions of prophage-induced cells in strains H/r30R(80), RI1 (80), R15(80), and NG30(80) plotted against doses of UV, MMC, X rays, NTG, and EMS.

208 SOHEI KONDO et a2.

4 h a a 4 " P 2 '8 E "

I

+ .

+ + + + + + + +

+

I I tI + +I

+ T + + + + t +

+ I + + +

+ + + + +


genes either of which is known to make the cell unable to induce nicks in vivo in UV-irradiated DNA molecules (HOWARD-FLANDERS 1968). Recent evidence supports the view that incision is the initial step in excision repair mechanism and that the second step is governed by DNA polymerase to excise dimers from DNA and resynthesize the excised portion (KELLY, ATKINSON, HUBERMAN and KORN- BERG 1969). Res- strain R15, which lacks DNA polymerase activity in vitro, like the strain recently isolated by DE LUCIA and CAIRNS (1969), is assumed to be defective in the second step in excision repair. Two other strains R13 and 016 previously isolated possess phenotypic characters very similar to those of R15 (&TO

and KONDO 1967), and show elevated spontaneous mutabilities as high as that in R15 (KONDO, ICHIKAWA and KATO in preparation). These three Res- strains lack DNA polymerase activity and all three strains have the res genes closely linked to metE as the polA mutant (GROSS and GROSS 1969), and show normal host cell reactivation (Hcr+) for infectious Plkc phage but show H c r for trans- ducing DNA of Plkc phage. These and other evidence that the res gene is responsible for repair resynthesis have been published elsewhere (KATO and KONDO 1969, 1970). According to WITKIN (personal communication), not all but a ma- jority of pol mutants including the polA mutant also possess elevated spontaneous mutability. Rec- strain NG30 with the recA gene has normal excision ability but it is UV sensitive because of deficient recombination ability. According to RUPP and HOWARD-FLANDERS (1968), UV-irradiated cells of a uvrA r e d + strain initially give rise to daughter stxands with gaps opposite the pyrimidine dimers in the parental template strands but these gaps are slowly repaired during further incubation. Deficiency in this post-replication repair or so-called “recmbina- tional repair” may be responsible for the high UV sensitivity of recA strains (RUPP, WILDE and HOWARD-FLANDERS 1970).

In order to facilitate the following argument, essential parts extracted from the mutagenesis patterns summarized in Table 5 are rearranged in Table 7 in simplified qualitative terms. We shall discuss mutagenesis following the types classified in Table 7.

Type I . Mutation due to errors in recombination provoked by excisable DNA damage: As argued previously by WITKIN (1969a, 1969b, 1969c), MIURA and TOMIZAWA (1968), and KONDO (1968), we conclude that this type of mutagenesis requires the existence of not only unrepaired excisable damage (either dimers or 4NQ0 damage) but also a functional recombination mechanism, because mutations ‘after UV or 4NQ0 did not occur in the Rec- strain but did occur in the other three strains with the r e d + gene, and because the highest mutability was found with the Exc- strain. Since the differential sensitivity to mutation between wild type (denoting the wild-type DNA repair capacity) and Exc- paralleled that to killing, we conclude that the same excisable damage is responsible for both killing and mutation induction by UV or 4NQO. Therefore, we propose the model given in the heading of this paragraph. A similar model has been previously proposed by WITKIN (1 969b) with a model of mutational recombination-error.

4NQ0 damages are not pyrimidine dimers (Figure 5). Recently, ISHIZAWA and ENDO (1 969) showed that 4NQ0 induces, in intnacellular phage T4, transition


mutations of the GC -+ AT type but not AT + GC or frameshift mutations. Type I I . Mutation due to errors in recombination provoked by unexcisable

DNA damage: If X-ray or MMS damage contains an excisable component as a large fraction and if the excisable component is mutagenic, then the Exc- strain should be far more X-ray or MMS mutable than wild type. Since this was not the case, we must conclude that unexcisable damage is responsible for the X-ray and MMS induced mutations of base-change type. Since the differential sensitivity between the Res- and the wild-type strain to X-ray or MMS killing did not parallel that to X-ray or MMS mutation, we conclude that the major cause of killing is different from that of mutation. Therefore, double-strand breakage assumed ‘as the major cause of X-ray killing (FREIFELDER 1966; KAPLAN 1966) would not lead to base-change mutation. These conclusions are compatible with WITKIN’S (1969b, c) model that a small fraction of single-strand gaps are repaired via recombination instead of by repair synthesis and that these give rise to mutations. Depurination or strand breakage induced by MMS may lead, partially, to lethality as usually assumed (BROOKES and LAWLEY 1961; STRAUSS 1968) but it may also produce WITKIN’S premutional single-strand gaps. Alternatively, base modification such as alkylation of purines may lead to mutation ( STRAWS 1968) by provoking recombination indirectly (e.g., via action of restriction or similar en- zymes).

Type I l l . Mutution due to errors in recombination provoked by excision-repair errors: MMC-induced mutants were detected with a low but significant frequency in wild-type and Res- strains but not in either the Exc- or the Rec- strain. This means that MMC mutation requires at least two mechanisms: excision repair and recombination. Since it is known that MMC induces cross-links between inter- strands of DNA molecules ( IYER and SZYBALSKI 1963), unrepaired MMC damage may be almost all lethal and hence nonmutagenic. This may be the case in the Exc- strain. Then since MMC damage is reparable by excision repair (BOYCE and HOWARD-FLANDERS 1964b; HANAWALT and HAYNES 1965), secondary damage induced in DNA via action of an excision repair mechanism on MMC damage should be responsible for the MMC mutation. The nature of the secondary damage can be speculated from the experimental results. Liquid holding recovery treatments enhanced the survival of MMC-treated cells but reduced the yield of MMC mutation in the wild-type strain (Figure 8). Furthermore, acriflavine post- treatments given to suppress the excision-repair of MMC damage showed that only the excision repair events occurring shortly before or during the first post- MMC DNA replication are mutagenic (Figure 9). From these emerge the following two models. Repeated repair model: Excision repairs are accompanied by errors but the first-round errors are subjected to second-round repair, leaving a reduced amount of errors, and so forth. Thus, only the repair errors made shortly before DNA replication remain unexcised and induce mutation in the daughter DNA strands. Repair-intermediate model: The DNA replicating fork will en- counter various forms of DNA alteration produced in the intermediate steps of excision repair of DNA damage. The repair-intermediate of MMC damage may be half-repaired, cross-linking with one arm excised, or single-stranded portion of

BASE-CHANGE MUTAGENESIS IN E. coli 21 1

DNA due to an extensive DNA degnadation like that known to occur accompany- ing excision repair of dimers ( BILLEN 1968).

Type IV. Mutation due to errors in recombination spontaneously provoked: Since the spontaneous mutation rate is almost equal between wild-type and the Exc- strain, as previously suggested with the same or similar pairs of strains (KONDO and KATO 1966; HILL 1968), spontaneous mutation cannot be ascribed to such external mutagens that produce excisable damage to DNA. If recombination is spontaneously provoked, it will give rise to mutagenic errors as is the case of recombination provoked by external mutagens. The working hypothesis that a cause of spontaneous mutations in wild type is errors in spontaneous recombination nicely fits the fact that the spontaneous frequency was higher in the wild- type, Exc-, and Res- strains (i.e., Rec+ types) than in the Rec- strain. This differential mutability may not be due to high lethality in the Rec- strain on agar because a similar difference was observed either by incubation of bacteria on SEM agar (Table 2) or by assaying mutants in overnight liquid culture with minimal agar (Table 3). In diploid yeast, not base-change but frameshift mutants have an elevated spontaneous mutability during meiosis (MAGNI and VON BORSTEL 1962). Therefore. recombination errors in yeast are assumed to produce not base-change but frameshift mutations ( MAGNI 1963). This discrepancy may suggest that recombination mechanisms in haploid E. co2i are different, at least partly, from those in diploid yeast. One attractive explanation for the elevated spontaneous mutability in the Res- strain is that it has a reduced repair capacity for spontaneous recombination errors.

Type V . Mutation due to replication error: As reported previously with phage (LOVELESS 1966) and E. coli ( STRAUSS 1962), the two chemicals, MMS and EMS, which are usually classified as the same type of alkylating agent concerning their lethality (LOVELESS 1966; STRAUSS 1968), showed different mutagenicity patterns. In the present experiments, we found that the Rec- strain was almost as highly mutable as the other three strains toward EMS but not at all to MMS, to which all the other three strains were almost equally mutable. Furthermore, the Rec- strain was not mutable either to UV, 4NQ0, X rays, or MMC which are all known to produce damage to DNA. Thus we propose that EMS produces damage in non-DNA material which damage leads to production of progeny DNA full of errors even from intact DNA. Plausible candidates for the assumed non-DNA material are membrane factors, DNA replication polymerase, or other relevant components of the DNA replication machinery. Another possibility is that EMS produces error-promoting damage in DNA which in turn induces errors in daughter DNA. Available evidence mentioned below strongly supports the former, although the latter possibility (STRAUSS 1962) cannot yet be ruled out. The prophage induction pattern obtained after EMS treatment was as follows (Figure 15) : the Rec- strain showed a slight but significant increase in prophage induction frequency beyond the spontaneous one at intermediate doses but the wild-type and Exc- strains showed monotonic decreases with increasing EMS dose. This suggeslts that EMS slightly provokes prophage induction but the induction is SO

low that it is masked in the wild-type strain by the loss of the bacterial capacity


to produce mature phage after spontaneous induction due to damage to DNA replication machinery. It has already been reported by LOVELESS (1966) that EMS is more effective in inactivation of bacterial capacity than inactivation of phage T2 in uitro and that the reverse is the case with MMS.

Type V f II. Mutation due to mixture of replication and recombination errors: NTG is one of the most powerful mutagenic and carcinogenic agents (MANDELL and GREENBERG 1960; SUGIMURA and FUJIMURA 1967). The mutagenicity pattern of NTG is qualitatively similbar to that of EMS but reduction of mutagenicity in the Rec- strain compared with that in wild type was more pronounced for NTG than for EMS (Table 5). Furthermore, in contrast to EMS, NTG was as powerful as MMC for prophage induction in the wild-type strain (Figure 15). Therefore, we conclude that NTG mutations occur through two processes, Type I1 (MMS-type mutagenesis) and Type V. CERDA-OLMEDO, HANAWALT and GUEROLA (1968) have recently reported evidence to show that NTG induces mutations through reaction with the DNA-replicating point. This fits our model if we assume that NTG-induced damage to DNA replication machinery is re- pairable or that the damaged portion can be replaced by an intact component. However, their hypothesis that NTG reacts specifically with replicating regions of DNA is not compatible with our data because our cells were treated with NTG when their DNA’s were not replicating (i.e., stationary phase culture suspended in phosphate buffer). Our model is compatible with the hypothesis of BAKER and TESSMAN (1968) that NTG affects the molecular environment of the DNA replicating point rather than the DNA itself.

In summary, we reach the conclusion that base-change mutation in E. coli can be induced in at least two ways; via recombination error and replication error. Recombination errors are assumed to result from spontaneous recombination or recombination provoked by DNA damage induced directly (with external agents such as UV, 4NQ0, X rays, MMS, NTG) cyr indirectly (as secoiidary damage due to errors in excision-repair of primary DNA damage such as that caused by MMC) . The replication error is assumed to occur due to damage to DNA replication machinery after treatment with chemicals (such as EMS or NTG) or spontaneously.

NISHIOKA and DOUDNEY (1969) claimed that a fraction of streptomycin resistance mutations induced by UV in a B/r type strain are due to excision-repair errors. If so, the claimed fraction of mutations belong to Type 111. We may, in general, assume that mutations induced in the wild-type strain by conventional mutagens usually contain a minor component of repair-error mutations of Type 111 in addition to the major component of mutations of Type I or 11.

The Res- strain had elevated spontaneous mutability but normal mutabilities toward all the mutagens tested except MMS. This is a puzzling contrast. It was noticed, however, during the course of the present experiments that cells of the Res- strain treated with UV, 4NQ0, or MMC (but not with EMS) give rise to many Arg+ colonies of various small sizes in addition to large Arg+ colonies. These petit Arg+ colonies were neglected in the scoring of Arg+ mutants during the present experiments because the petit mutations were usually rare in the


other three strains used. Detailed studies of mutations in Res- are in progress and will be published elsewhere. A preliminary conclusion is that the true induced mutation frequencies in the Res- strain will become considerably higher than those reported in this paper if we add the petit mutations. Therefore, the increased recombination-error model (Type IV) seems promising. The data on prophage induction also support this model as follows. UV induction of prophage was almost equally high in both the Res- and Exc- strains but extremely low in the Rec- strain (Figure 15 and Table 6). This means that prophage induction requires recombination mechanisms as previously reported ( FUERST and SIMINO- VICH 1965; BENGURION and CLARK 1967; HERTMAN and LURIA 1967) and that the Res- strain may be error-prone in the recombinational functiorz and hence highly susceptible to induction. SPEYER’S (1966) finding that a T4 mutant with defective DNA polymerase activity has a high spontaneous mutability seems to correspond to the high spontaneous mutability of the Res- strain.

The Rec- strain lysogenized with phage 080 was inducible by UV, MMC, or X rays although its maximal frequency was about 100- to 1000-fold lower than the maxima in other strains of Rec+ type. These low frequencies were detectable when spontaneous frequencies were minimized by applying the streptomycin- top-agar-cover technique to bacteria in stationary phase as previously described by TAKEBE et al. (1967). Since there is parallel between mutation and prophage induction (HILL 1963), these results suggest that mutabilities of the Rec- strain to these agents may exist, but be too low to be detected. If this is the case, it means that physicochemical alterations in the DNA of E. coli can induce, in principle, base-change mutations bypassing recombination, as is supposed the case with phage (FREESE 1963) but the frequency is usually too low to be detected in E. coli.

Actual mutagenesis will be much more complicated than argued above. Most of the above-mentioned models have been proposed not as conclusive ones but rather as working hypotheses for future studies of mutagenesis.

One of the authors (S.K.) became more interested in mutagenesis than physics thanks to the guidance of DR. C. AUEHBACH during her 3 months’ visit to Japan as a visiting professor in 1961 and he started chemical mutagenesis work with the encouragement of DRS. Y. TAZIMA and H. ENW. The authors wish to express their gratitude to DR. H. ENDO for the generous gift of 4NQ0, to DR. H. TAKEBE for his guidance of prophage induction experiments, to N. MURAOKA for her technical help in EMS experiments and their deep appreciation to DRS. E M. WITKIN, JOHN JAOGER, B. S . STRAUSS, and R. F. KIMBALL for their valuable suggestions and comments during the preparation of this manuscript. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Japan.

SUMMARY

Rase-pair reversion (from argF,, to prototrophy) and prophage induction ($80) by various mutagens were compared in E. coli strains that differ in DNA repair capacity. These include wild type, exc (unable to excise pyrimidine dimers) , res (polymeraseless) and reCA (recombinationless). Mutability and prophage-induction patterns were strikingly similar. Reversion patterns lead to


the conclusion that base-change mutations require not only premutational DNA damage (an excisable one after UV and 4-nitroquinoline-l-oxide, an unexcisable one after X rays and methyl methanesulfonate, and repair-errors after mitomycin C ) but also recombinational events; premutational damage only provokes recombination which in turn induces mutation probably through recombinational errors. Mutations also can be induced, bypassing recombination mechanisms, by ethyl methanesulfonate. N-methyl-N’-nitro-N-nitrosoguanidine induces mutations by a combination of these two mechanisms. Since spontaneous mutation and spontaneous prophage-induction are lower in the recA strain but higher in the res strain than in wild type, both phenomena seem to require, at least partly, the action of recombination machinery; KORNBERG’S DNA polymerase, which is defective in the res strain. may participate in repair of spontaneous recombination errors.

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- ,

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i~ug~o~ escherzchza colz with diffexft · base-ch~ge ~~ag~esis and prophage i~ug~o~ in strains of...

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