Kinetics of induction and decay of error-prone DNA repair activity in Escherichia coli after treatment with nalidixic acid

57

Mutation Research, 72 (1980) 57--62 © Elsevier/North-Holland Biomedical Press

KINETICS OF INDUCTION AND DECAY OF ERROR-PRONE DNA REPAIR ACTIVITY IN Escbe~icbia coli AFTER TREATMENT WITH NALIDIXIC ACID

S.N. S A R K A R

and R A M E N D R A

K. P O D D A R

Biophysics Section, Physics Department, University College of Science, Calcutta 700 009 (India) (Received 30 January 1979) (Revision received 4 January 1980) (Accepted 14 March 1980)

Summary Inducible error-prone D N A repair activity was detected by infecting nalidixic acid-pretreated E. coli cells with UV-irradiated phage ~X174. Induction and decay kinetics of reactivation very much resembled that of mutagenesis of the UV-damaged phage. Repair as well as mutagenic activity increased for about 30 rain. The maximal error-prone repair capacity, which was induced in the cell during the 30 rain nalidixic acid treatment, rapidly died out during subsequent cell growth in absence of nalidixic acid. Induction of this repair mode was not observed in a recA- mutant. In the presence of nalidixic acid plus rifampicin both repair and mutagenic effects were abolished.

Treatments such as exposure to ionizing and non-ionizing radiations [12], thymine starvation [15] or nalidixic acid (NAL) [3], cause premature termination or retardation [17] of DNA replication in E. coll. These disturbances seem to induce aberrant re-initiation of new replication forks [4] and also elicit a variety of other inducible responses, which have been termed "SOS functions" by Radman [16]. One of the SOS functions includes an extra capacity to promote the survival and mutagenesis of ultraviolet-irradiated infecting phages. This phenomenon is also termed Weigle or W reactivation after its discoverer [20]. Most investigations relating to such reactivation have been carried out with phage )~ [9]. SingleHtranded (SS) DNA phages, such as • X174, have, however, some distinctive advantages in this regard. The nonmetabolic photoenzymic repair process (photo-reactivation) is marginally effective in reactivating SS DNA phages [14]. These phages do not undergo host~ell reactivation of UV lesions on their DNA [22], nor is there any multiplicity reactivation [ 6,18]. There is also no possibility o f pro-phage reactivation which

58 is one of the repair modes of phage ~ [9]. Thus W reactivation appears to be the only repair m o d e b y which the intracellular repair of the abnormal replicative (RF) molecules formed out of the infecting SS DNA of the irradiated phage is possible. N o t only UV-irradiation b u t also thymine starvation triggers this kind of repair [19]. This repair m o d e for the SS DNA operates only in recA ÷ strains [6], requires protein synthesis de novo [7] and is accompanied b y mutation [2]. Hence, this mutagenic reactivation of UV-irradiated phage q~X174 can be used as a clear~ut indicator of the error-prone DNA repair activity in E. coli cells. This paper reports the results of our studies on the kinetics of induction and decay of this repair mode in E. coli cells pretreated with NAL. Materials and m e t h o d s

Bacteria and phage strains Bacteriophages, apX174 wild-type and its lysis
59 Thus the number of clear plaques produced by UV-irradiated ¢Xam3 on E. coli cells gives an estimate of the reverse mutation ¢PXam3 to wild-type during intracellular reactivation. Results

Dependence o f DNA-repair activity of E. coli cells on the concentration o f NAL Fig. 1 shows the effects of exposure of E. coli C cells to various concentrations of NAL before infection with UV-irradiated phage &X174 on the inducibility of their repair activity for SS DNA. NAL-pretreated cells showed pronounced induction of W reactivation, i.e. increased survival of UV-irradiated ¢ X 1 7 4 as compared with untreated cells. W reactivation was maximal at about 30 min of incubation for cells exposed to NAL at 50 or 75/~g/ml. Pretreatment with NAL at 25 #g/ml was only marginally effective. After 30 rain this DNA

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Fig. I . S u r v i v a l o f U V - i r r a d i a t e d w i l d - t y p e ( w t ) ~ X 1 7 4 in E. eoli C (recA + u v r A +) eells v e r s u s i n c u b a t i o n t i m e o f t h e s e cells p r / o r t o p h s g e i n f e c t i o n i n p r e s e n c e o f N A L a t 2 5 (o)0 5 0 (/~) o r 7 5 , g / m l (o). T h e v a l u e s o f i n f e c t i v e c e n t e r s a t z e r o t / m e i n d i c a t e t h o s e o f U V - i r r a d i a t e d p h a g e s a_-_~_yed o n u n t r e a t e d c o n t r o l cells. T h e s u r v i v ~ p h a ~ e f r a c t i o n w a s 6 . 6 × 1 0 - 4 w h e n t h e U V d o s e w a s a p p r o x . 1 0 0 0 e r g / m m 2 . Fig. 2. L y s l s p a t t e r n s o f H A L - t r e a t e d s n d c o n t r o l E. t o l l C cells i n f e c t e d w i t h n o r m a l ~ X 1 7 4 . Ceils g r o w n i n T B a t 37°(~ u p t o a b o u t 2 × 1 0 8 p e r m l w e r e d i v i d e d i n t o 2 part~. O n e p a r t w a s k e p t a s c o n t r o l and 50 ,g NAL/ml was added to the other part. Both were incubated at 37°C for 30 min. Bacteria from b o t h c u l t u r e s w e r e h a r v e s t e d s e p a r a t e l y o n M i l l / p o r e filters, r e s u s p e n d e d i n f r e s h T B a t 3 7 ° C a n d i n f e c t e d w i t h n o r m a l ~ X 1 7 4 a t m . o A . = 1 . 5 . T h e a b s o r b a n c e a t 5 4 0 n m w a s o b s e r v e d f o r S 0 r a i n . C o n t r o l ceils ( o ) ; N A L - t r e a t e d eells ( e ) . F i n a l p h a ~ e t i t e r s w e r e t h e n d e t e r m i n e d , t h e y i e l d b e i n g 1.5 × 1 0 1 0 p h a ~ e s / m l in c o n t r o l cells a n d 1 . 0 × I 0 I 0 p h a g e s / m l i n N A L - t r e a t e d cells.

60 repair activity remained more or less constant for a b o u t another 30 min and then decreased (not shown in the figure).

Effect o f N A L pretreatment on phage growth The effect o f NAL pretreatment on phage growth was studied. C cells from E. coli were infected separately with normal ~ X 1 7 4 , then either treated with N A L at 50 ttg/ml or left untreated. The rates of the lysis were observed optically (Fig. 2). The slopes of the lysis curves as well as the final phage yields (see legend) were more or less similar. Thus the intracellular phage growth did n o t appear to be much affected by exposure of the host cells to 50 gg NAL/ml before infection. Kinetics o f induction and decay of mutagenic phage reactivation in NALtreated E. coli Fig. 3 shows the kinetics of induction of W reactivation of UV-irradiated wild-type (wt) phage ~ X 1 7 4 as well as that of reverse mutation of UV-irradiated ~pXam3 -~ wt in E. coli cells pretreated with 50 tzg NAL/ml. The SS DNA repair (W reactivation) as well as the mutagenic activities of the cells increased almost pari passu with the time of pre-incubation in NAL and reached maximal values b y a b o u t 30 min, in c o n f o r m i t y with the earlier finding (Fig. 1). The maximally induced cells (30 min pretreatment with 50 #g NAL/ml) were then freed of NAL by filtration on Millipore filters and allowed to grow in fresh pre-warmed (37°C) TB in absence of NAL. After various intervals, samples were withdrawn, separately infected with UV-irradiated phage ¢PX174 and ~pXam3, and infective centers were assayed as before. The results are also 1"5

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F i g . 3. I n d u c t i o n and d e c a y o f e r r o r - p r o n e SS D N A r e p a i r ( m u t a g e n i c r e a c t i v a t i o n o f (I)X174) in N A L t r e a t e d E. coli cells. I n f e c t i v e c e n t e r s o f U V - i r r a d i a t e d w t ¢ X 1 7 4 (o) and f ~ e q u e n c y o f r e v e r t a n t s , ~ P X a r n 3 --~ w t ( 4 ) , b o t h a s s a y e d o n E. coil (recA +, u v r A +) cells p r e t r e a t e d w i t h N A L a t 5 0 D g / m l . T h e v a l u e s at z e r o t i m e i n d i c a t e t h e c o r r e s p o n d i n g v a l u e s o b t a i n e d b y assays o n u n t r e a t e d c o n t r o l cells. F i g . 4. S a m e e x p e r i m e n t s , as in Fig. 3, c a r r i e d o u t w i t h (a) s t r a i n H F 4 7 1 2 ( r e c A - uvrA +) p r e t r e a t e d w i t h N A L at 5 0 # g / m 1 ; and ( b ) E. coli C (recA + u v r A +) p r e t r e a t e d w i t h 5 0 / ~ g N A L / r n l p l u s 5 0 / ~ g r i f a m p i c i n / m L A s b e f o r e , o i n d i c a t e s i n f e c t i v e c e n t e r s , a n d ~ f r e q u e n c y o f r e v e r t e n t s i n (b) b u t ~ i n (a) is i n f e c t i v e c e n t e r s o f U V - i r r a d i a t e d ~)X~rn3.

61

shown in Fig. 3. As time went on, the capacity to W-reactivate the irradiated w t phage, as well as the capacity to reverse mutate UV-irradiated ~ X a m 3 -~ wt , decayed gradually and after about 60 rain both capacities were almost completely abolished. These induced capacities were not observed at all when similar experiments were carried out either with strain HF4712 carrying the recA mutation, or with the E. coli C if rifampicin at 50/~g/ml was present in addition to NAL at 50/~g/ml during pre-incubation (Fig. 4). Concluding remarks The experiments described here provide evidence that treatment of E. coli cells with NAL induced the repair activity responsible for increased survival of UV-irradiated ~X174. This repair was also error-prone because,~ among the reactivated survivors, mutants were always found in proportion t o the extent of reactivation. This repair activity was not observed in NAL-treated recAcells, or in NAL- plus rifampicin-treated recA ÷ wild-type cells. The pattern of kinetics here was similar to the 3 sets of kinetics already reported by Witkin [21], Defais et al. [8] and Monk and Kinross [13], regarding bacterial UV mutagenesis, W reactivation of UV-irradiated phage )~ and prophage induction, resp. The synthetic antibiotic, nalidixic acid (NAL), is believed to act on the DNA gyrase resulting in underwound DNA and causing preferential, rapid and reversible inhibition of DNA synthesis [5]. One of the consequences of inhibition of DNA synthesis by NAL is induction of synthesis of protein X, since identified as the product of the bacterial recA gene [10,11]. The induction pattern of the recA protein showed a gradual increase up to 30 rain and then remained more or less constant for about another 30 rain after inhibition of DNA synthesis by NAL. Therefore, it seems reasonable to conclude that the mutagenic reactivation of UV-irradiated ~X174 shares with the other error-prone DNA repair modes a c o m m o n pathway involving the reck4 protein. Acknowledgement We are indebted to the Department of Atomic Energy, Government of India, and the University Grants Commission for financial assistance. We also sincerely thank Dr. R. Devoret for critically going through the manuscript, Drs. C.K. Dasgupta, A. Ghosh, U. Chaudhuri and A.R. Thakur for cooperation, and Dr. R.L. Sinsheimer for generously supplying the phage and bacterial strains. References 1 Adams, M.H., Bacteriopha~es, Wfley/lntersclenee, New York, 1959. 2 Bleichrodt0 J.F., and W~.D. VerheJj, Mute~enesis by ultraviolet radiation in b a e t e r i o p h ~ e ~X174; o n the m u t a t i o n stimulation processes induced by ultraviolet radiation in the h o s t bacterium, Mol. Gen. Oenet., l S 5 (1974) 19--27. 3 Boyle, J.V., W.A. Gross and T.M. C o o k , I n d u c t i o n of excessive deoxyribonucleic acid synthesis in Escher~ehla t o l l by n~d/dixic acid, J. Bactez~ol., 94 (1967) 1664--1671. 4 Bridges, B.A., Evidence for a further dark repclr process in bacteria, Nature ( L o n d o n ) , N e w Biol., 240 (1972) 52--53.

62 5 Cozzaralli, N.R., The m e c h a n i s m of a c t i o n of i n h i b i t o r s of DNA synthesis, Annu. Rev. Biochem., 46 (1977) 6 41--668. 6 Dasgupta, C.K., and R.K. Poddar, Ultraviolet reactivation of the single stranded DNA phage ~ X 1 7 4 , Mol. Gen. Genet.0 139 (1975) 77--91. 7 Dasgupta, C.K., and R.K. Poddar, Influence of host m e t a b o l i c state on the UV reactivation (UVR) of ~ X 1 7 4 , Proc. Syrup. Structtu-al and F u n c t i o n a l Aspects of Chromosomes, B.A.R.C., Bombay, 1975, p. 130. 8 Defais, M., P. Calllet-Fauquet, S.M. Fox and M. R a d m a n , I n d u c t i o n ki ne t i c s of mut a ge ni c DNA repair activity in E. coli following ultraviolet irradiation, MoL Gen. Genet., 148 (1976) 125--130. 9 Devoret, R., M. Blanco, J. George and M. R a d m a n , Re c ove ry of phage k from ul t ra vi ol e t damage, in: P. Hanawalt and R.B. Setlow (Eds.), Molecular Mechanisms for Repair of DNA, Part A, Plenum, New York, 1975, pp. 155--171. 10 Gudas, L.J., and A.B. Pardee, DNA synthesis i n h i b i t i o n and the i n d u c t i o n of prot e i n X in E s c h e r i c h i a coil, J. Mol. Biol., 101 (1976) 459--477. 11 Gudas, L.J., and D.W. Mount, I d e n t i f i c a t i o n of the r e c A (tif) gene p r o d u c t of E s c h e r i c h i a coli, Proc. Natl. Acad. Sci. (U.S.A.), 74 (1977) 5280--5284. 12 Hewitt, R., D. BiLien and G. Jorgensen, R a d i a t i o n - i n d u c e d r e o r i e n t a t i o n of c h r o m o s o m e replication sequence generality in E s e h e r t c h i a coli, I n d e p e n d e n c e of prophage or 5-bromouracfl t o x i c i t y , Radiat. Res., 32 (1967) 214--226. 13 Monk, M., and J. Kinross, The k i n e t i c s of darepression of prophage following ultraviolet irradiation of lysogenic cells, Mol. Gen. Genet., 137 (1975) 263--268. 14 Poddar, R.K., and R.L. Sinaheimer, Nature of the c o m p l e m e n t a r y strands s ynt he s i z e d in vitro u p o n the single stranded circular DNA of bacteriophage ~ X 1 7 4 after ultraviolet irradiation, Biophys. J., 11 (1971) 355--369. 15 Pritehard, R.H., and K.G. Lark, I n d u c t i o n of replication by t h y m i n e starvation at the c h r o m o s o m e origin i n E s c h e r i c h i a coil, J. MoL Biol., 9 (1964) 288--307. 16 R a d m a n , M., SOS repai~ hypothesis, P h e n o m e n o l o g y of an inducible DNA repair which is accompanied by mutagenesis, in: P. Hanawalt and R.B. Setiow (Eds.), Molecular Mechanism for R e pa i r of DNA, Part A, Plenum, New Y o r k , 1975, pp. 355--367. 17 Sedgwick, S.G., Mis~epair of overlapping d a u g h t e r strand gaps as a possible m e c h a n i s m for UVinduced mutagenesis in u v r strains of E s c h e r i c h i a coli, A general mode l for induced mutagenesis by m i s ~ p a i ~ (SOS repair) of closely spaced DNA lesions, M ut a t i on Res., 44 (1976) 185--206. 18 Tessman, E.S., and T. Ozaki, The i n t e r a c t i o n of phage $13 w i t h ultraviolet irradiated hos t cells and p ro perties of the ultraviolet in'adiated phage, Virology, 12 (1960) 431--449. 19 Thakur, A.R., and R.K. Poddar, G r o w t h and reactiva t i on of single stranded DNA phage ~PX174 in E. co|i tmdergoing "thymine-less death ", Mol. Gen. Genet., 151 (1977) 313. 20 Weigle, J.J., I n d u c t i o n of m u t a t i o n in a bacterial virus, Proc. Natl. Acad. Sci. (U.S.A.), 39 (1953) 628--636. 21 Witkin, E.M., Persistence and decay of t h e r m o i n d u c i b l e error-prone repair activity in n o n f i l a m e n t o u s derivatives of tif-1 E s c h e r i c h i a c o | i B/r: the timing of some critical events in ultraviolet mutagenesis, Mol. Gen. Genet., 142 (1975) 87--103. 22 Yarus, M., and R.L. Sinsheimer, The UV resistance of double stranded ~ X 1 7 4 DNA, J. Mol. Biol., 8 ( ! 9 6 4 ) 614--615.