FEMS Microbiology Letters 99 (1992) 31-36 © 1992 Federation of European MicrobiologiCal Societies 0378-1097/92/$05.00 Published by Elsevier

31

FEMSLE 05154

Effect of flash photoreactivation on Escherichia coli recA induction by ultraviolet light Kazuo Y a m a m o t o Biological Institute, Faculty of Science, Tohoku Unirersity, Sendai, Japan Received 23 June 1992 Revision received 20 August 1992 Accepted 31 August 1992

Key words: Photoreactivation; recA induction; Mutagenesis; Ultraviolet light; Escherichia coli

1. SUMMARY

2. INTRODUCTION

Excision-deficient Escherichia coli, carrying the gene for the photolyase on a muiticopy plasmid, were irradiated with ultraviolet (UV) light then photoreactivated by illumination delivered from a camera flash unit. Such instantaneous illumination monomerizes only cyclobutane pyrimidine dimers already bound by the photolyase. Whereas the lethal effect of UV light and the number of C-to-T transition-type mutations induced by UV irradiation were both significantly reduced by subsequent irradiation with a single flash of light, single-flash photoreactivation did not reverse the induction of the recA gene by UV light. The results indicate, therefore, that non-photoreactivable DNA lesions play a role in recA induction.

Irradiation of DNA with ultraviolet (UV) light produces a variety of photoproducts, of which the major ones are the cyclobutane pyrimidine dimers (dimers) and the pyrimidine-pyrimidone [6-4] adducts ([6-4] adducts) that are formed in the DNA at 1/10th the frequency of dimers [1]. Both lesions were assumed to be the photoproducts responsible for UV mutagenesis. UV mutagenesis of Escherichia coli requires photoproducts in the gene being mutated and also for the induction of the SOS response [2,3]. A number of recent photoreactivation experiments [4-7] have strongly suggested that a dimer is the lesion directly responsible for most transition mutations. The identity of the photoproducts for the induction of the SOS response has been uncertain. It was shown that photoreactivation followed by UV irradiation reversed UV-iight induction of the umuC gene, one of the SOS genes [8]. Since photoreactivation is known to efficiently remove dimers from DNA [9,10] but is not known to

Correspondence to: K. Yamamoto, Biological Institute, Faculty of Science, Tohoku University, Sendal 980, Japan.

32 affect [6-4] adducts [10], the above finding suggested that a dimer is the principal UV-light lesion inducing umuC and other SOS genes. On the other hand, we have shown that photoreactiration reversed the lethal damage more effectively than SOS-inducing damage, which suggests that a dimer is not the lesion chiefly involved in SOS induction [11,12]. The experiments presented in this paper were designed to investigate whether the dimer is truly the lesion that induces the recA induction, one of the SOS responses, in excision-deficient E. coli. Using a camera flash unit, we showed that photoreactivation by a single flash of light reverses the killing effect and the mutagenic effect of UV light, but has little effect upon the influence of UV light on the expression of the recA gene.

3. M A T E R I A L S AND M E T H O D S

3.1. Bacterial strain, plasmid and culture conditions The E. coli K12 strain KY946 (ut, rA6 recA-lacZ fusion) has been described previously [12]. KY1836 is an ABl157 derivative with relevant markers argE3 his-4 ul:rA6. Plasmid pKY1 is a pBR322 derivative and carries the phr gene of E. coli [13] which codes for D N A photolyase that specifically removes dimers from DNA but is not known to affect other UV photoadducts. KY946 and KY1836 were transformed with pKY1 and designated KY946(pKY1) and KY1836(pKY1), respectively. The minimal medium used was M9 salts supplemented with 1% glucose and 1% casamino acids (K medium). For the mutation and survival assay, we used SEM agar containing 0.004% casamino acids [14]. Ampicillin at 50 /~g/ml was included in the media when necessary. 3.2. UV irradiation and photoreactiz~ation Either overnight or exponentially growing cultures were washed three times by centrifugation and resuspended in M9 salts. Five milliliters of the suspension of cells, in 90-ram diameter glass dishes, were irradiated with principally 254-nm

light by two 15-W germicidal lamps with constant shaking, The fluence rate was 0.05 J m 2 s ~. For photoreactivation using a camera flash unit, 0.5-1 ml of the M9 salts suspension of irradiated cells was incubated at 37°C in a water bath for 2 rain and challenged with photoreactirating light through the glass test tube and polyvinyl chloride plastic (0.2-ram layer) with a single flash from a National Electric flash unit, model PE-200S. A flash of light illuminates for only a very short time, and photoreactivation events occur only in pre-existing enzyme-substrate complexes. For 10 rain of photoreactivation, two closely spaced 20-W daylight fluorescent lamps at a 20-cm distance were used as the light source. Polyvinyl chloride plastic and a glass Petri dish lid were placed between the fluorescent lamps and the sample aliquots in M9 salts. The polyvinyl chloride plastic used was known to absorb wavelengths below 380 nm [12]. All experiments except for photoreactivating treatment were carried out under yellow light,

3.3. UV induction of" [3-galactosidase After UV irradiation and photoreactivation, KY946(pKYI) cells in M9 salts were added to the 1% glucose and 1% casamino acids, and the suspensions were incubated at 37°C with shaking. Forty-five minutes after UV irradiation and photoreactivation samples were removed and assayed for/3-galactosidase activity essentially by the procedure of Miller [15]. 3.5. UV lethality and mutagenesis After UV irradiation and photoreactivation, KY946(pKY1) cells were diluted and plated on L agar to measure their colony-forming ability, and KY1836(pKY1) cell diluents were plated on SEM agar to test for reversion of the arginine and histidine auxotrophic mutations.

4. RESULTS AND DISCUSSION The effect of photoreactivation on induction of the recA gene was determined by measuring the level of 3-galactosidase synthesized under control

33

2000t

g

c O

i o \2 0

0

r0

~t0001

2

cn c Zx

10-1

0

0

UV(Jim2 ) Fig. l. Photoreactivation of recA gene induction. KY946 (pKY1) cells were assayed for/3-galactosidase activity, 45 min after photoreactivation that followed irradiation with various doses of UV light. Experiments were carried out more than 5 times and were reproducible. The results represent the value of one representative experiment done in parallel. (©) no photoreactivation; ( [] ) flash of photoreactivation; ( zx) 10 rain of photoreactivation.

o f t h e recA p r o m o t e r (Fig. 1). Single-flash p h o t o r e a c t i v a t i o n did not significantly r e d u c e t h e U V l i g h t - i n d u c e d levels o f synthesis o f R e c A p r o t e i n . P h o t o r e a c t i v a t i o n for 10 min by t h e f l u o r e s c e n t l a m p s r e v e r s e d t h e level of t h e i n d u c t i o n of /3g a l a c t o s i d a s e by a p p r o x i m a t e l y 5 0 - 6 0 % . T h e unc h a n g e d level o f / 3 - g a l a c t o s i d a s e in K Y 9 4 6 ( p K Y 1 ) i r r a d i a t e d with the flash unit a l o n e o r 10 rain of fluorescent lamps alone showed that neither kind of p h o t o r e a c t i v a t i n g light u s e d in t h e s e c o n d i t i o n s a f f e c t e d t h e b a c k g r o u n d level of recA i n d u c t i o n (Fig. 1, 0 J m - 2 samples). F i g u r e 2 shows t h a t b o t h a single flash o f light a n d 10 min of p h o t o r e activating light i n c r e a s e d t h e c o l o n y - f o r m i n g ability o f U V - i r r a d i a t e d K Y 9 4 6 ( p K Y 1 ) strain. A flash light i r r a d i a t i o n b e f o r e 4 J m -2 U V d i d not a l t e r t h e level o f /3-galactosidase s y n t h e s i z e d u n d e r c o n t r o l of t h e recA p r o m o t e r a n d did not a l t e r c o l o n y - f o r m i n g ability ( T a b l e 1). T e n m i n u t e s of p h o t o r e a c t i v a t i o n b e f o r e 4 J m -2 U V a g a i n did not a l t e r the level o f / 3 - g a l a c t o s i d a s e s y n t h e s i z e d

2

4 uv (Jim 2)

6

Fig. 2. Photoreactivation of UV-induced killing. KY946(pKY1) cells were photoreactivated as described in the legend to Fig. 1, then plated on L agar to measure UV lethality. The results represent the value of one representative experiment done in parallel. Symbols as in Fig. 1.

u n d e r c o n t r o l o f t h e recA p r o m o t e r n o r colonyf o r m i n g ability ( T a b l e 1). It has b e e n r e p o r t e d that near-UV (300-400 nm) illumination induces growth d e l a y o f E. coli which i n c r e a s e s r e s i s t a n c e o f the cells to the l e t h a l action o f 254-nm r a d i a tion [16]. If this is t h e case, a flash o f light as well as 10-min p h o t o r e a c t i v a t i o n b e f o r e a n d after 4 J m - 2 U V i n c r e a s e s survival to the s a m e extent. T h e results shown in Figs. 1 a n d 2 a n d T a b l e 1 i n d i c a t e that the p h o t o r e a c t i v a t i o n devices we used i n d u c e little o r no growth delay. Thus, the p h o t o r e a c t i v a t i n g effect o n survival is t h e c o n s e q u e n c e o f r e m o v a l o f d i m e r s by e n z y m a t i c p h o -

Table 1 Photoreactivation of lethality and /3-galactosidase induction in E. co# strain KY946(pKY1) UV treatment

Relative survival

/3-Galactosidase units

0 J/m z 4 J/m 2 4 J/m z + flash 4 J/m 2 + 10 rain PR flash+4 J/m ~ 10 min PR+4 J/m 2

1 3.52× 10-~ 8.04 × 10-1 1 3.17×10 1 3.15× 10 1

313 1687 1477 622 1729 1601

34

f 7

10-~

[3 /

¢-

,~.~I 0 -5 c .O

J

~5 E T

[]

i

10-7 . . . . . . |

. . . . . . . .

5

i

|0

.

.

.

.

.

50

UV(J/m 2 ) Fig. 3. Photoreactivation of UV-induced argE3, his-4 to Arg +, His + mutations in strain KYI836(pKY1). Cells were photoreactivated as described in the legend to Fig. 1. Each point represents the average of 2 or 3 independent experiments. Symbols for photoreactivation are the same as in Fig. 1.

toreactivation. We further tested the effects of flash photoreactivation on the UV-induction of the umuC gene using umuC-lacZ fusion. Again single flash photoreactivation did not significantly reduce the UV-light-induced levels of synthesis of UmuC protein and increased colony-forming ability (data not shown). Thus, enzymatic photoreactivation repairs lethal lesions but does not repair recA and umuC inducing lesions. We then measured the UV-light-induced simultaneous reversion of argE3 (ochre) and his-4 (ochre) to Arg + and His + in E. coIi K12 KY1836(pKY1). Figure 3 shows that flash photoreactivation reverses the UV light-induced mutations. All the simultaneous reversions of argE3 and his-4 to Arg + and His + in KY1836, an ABl157 derivative must be due to the ochre-suppressing t R N A that maps in the supE-supB region [14]. These suppressor mutations have been shown to arise from C-to-T transitions at sites in D N A where a thymine-cytosine dimer may target mutation [6]. Thus, photoreactivation by a flash of light as well as 10 rain

of photoreactivation repaired the TC dimer which could be C-to-T mutated. Using P1 phage transduction, we introduced the phr-19 allele into KY946 and KY1836, and named the transduced strains KY949 and KY1839, respectively. Using these photoreactivation-deficient strains, we determined the effects of flash and fluorescent lamp irradiation. A single flash of photoreactivation and 10 min of photoreactivation did not reverse the induction of the recA gene by UV light, nor did it reduce the lethal and mutagenic effects of UV light (data not shown). The results again suggest that the observed phenomena are not the effects of near UV. In summary, Figs. 1 to 3 show that enzymatic removal of dimers from D N A by a single flash of light can reduce the frequency of dimer targeting mutations and increase colony-forming ability, but cannot reduce the induction of the recA and umuC genes. These data indicate that photoadducts other than dimers play a role in SOS induction. Ten minutes of photoreactivation repaired more than 90% of the lethal effect (Fig. 2). This finding is consistent with our previous data in which more than 90% of thymine-containing dimers disappeared from E. coli DNA within 10 min of fluorescent lamp irradiation [12]. On the other hand, 10 rain of photoreactivation did not reverse 90% of UV-induced recA induction (Fig. 1). Thus, these data again support the previous statement that non-photoreactivable photoadducts play a role in recA induction. Ten minutes of photoreactivation, however, reversed 60% of the UV-induced recA induction, suggesting that dimers surely play a significant role in recA induction. The above conclusion is different from that of Brash and Haseltine [8], who used the umuC gene as one of the representative SOS genes and an E. coli uL,rA host that lacked phr plasmid, and therefore had a wild-type (lower) level of photolyase, so needed longer time for completion of photoreactivation. Such longer illumination of light might induce growth delay [16]. They claim that dimers are the major SOS-inducing lesion since photoreactivation reversed umuC induction. Their results are consistent with part of the

35

observations reported here; 10 min of photoreactivation reversed approximately 60% of the recA and u m u C induction. In addition to these results, the results of single-flash photoreactivation indicate that non-photoreactivable photoadducts play an important role in recA induction.

ACKNOWLEDGEMENTS The financial support of the Ministry of Education, Science and Culture, Japan, is gratefully acknowledged.

REFERENCES [1] Patrick, M.H. and Rahn, R.O. (1976) In: Photochemistry and Photobiology of Nucleic Acids (Wang, S.-Y., Ed.) Vol. 2, pp. 35-95. Academic Press, London. [2] Radman, M. (1977) In: Molecular and Environmental Aspects of Mutagenesis (Prakash, L., Sherman, F., Miller, M, Lawrence, C. and Tabor, H.W., Eds.) pp.128-142, Charles C. Thomas, Springfield, IL. [3] Witkin, E.M. (1976) Bacteriol. Rev. 40, 869-907.

[4] Lawrence, C.W., Christensen, R.B. and Christensen, J.R. (1985) J. Bacteriol. 161, 767-768, [5l Protic-Sabljic, M., Tuteja, N., Munson, P.J., Hauser, J., Kraemer, K.H. and Dixon, K. (1986) Mol. Cell. Biol. 6, 3349-3356. [6] Bockrath, R., Ruiz-Rubio, M. and Bridges, B.A. (1987) J. Bacteriol. 169, 1410-1416. [7] Hutchinson, F., Yamamoto, K., Stein, J. and Wood, R.D. (1988) J. Mol. Biol. 202, 593-601. [8] Brash, D.E. and Haseltine, W.A. (1985) J. Bacteriol. 163, 460-463. [9l Harm, W. (1976) In: Photochemistry and Photobiology of Nucleic Acids (Wang, S.-Y., Ed.) Vol. 2, pp. 219-263, Academic Press, London. [10l Brash, D.E., Franklin, W.A., Sanear, G.B., Sancar, A. and Haseltine, W.A. (1985) J. Biol. Chem. 260, 1143811441. [11] Yamamoto, K. (1985) Mol. Gen. Genet. 201, 141-145. [12] Yamamoto, K., Shinagawa, H. and Ohnishi, T. (1985) Mutat. Res. 146, 33-42. [13] Yamamoto, K., Satake, M., Shinagawa, H. and Fujiwara, Y. (1983) Mol. Gen. Genet. 190, 511-515. [14l Kato, T., Shinoura, Y., Templin, A. and Clark, A.J. (1980) Mol. Gen. Genet. 180, 283-29l. [15] Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [16] Ramabhadran, T.V. and Jagger, J. (1976) Proc. Natl. Acad. Sci. USA 73, 59-63.