Increased UV-inducibility of SOS functions in a dam-3 mutant of Escherichia coli K12 uvrA

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Mutation Research, 52 (1978) 323--331 © Elsevier/North-Holland Biomedical Press

INCREASED UV-INDUCIBILITY OF SOS FUNCTIONS IN A dam-3 MUTANT O F Escherichia coli K12 uvrA

ALBERT GOZE * and STEVEN G. SEDGWICK ** Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, 91190 Gif-sur- Yvette (France) (Received 12 January 1978) (Revision received 14 July 1978) (Accepted 26 July 1978)

Summary The dam-3 mutation caused a 2--4 fold increase in the susceptibility of E. coli K-12 uvrA to UV induction of prophage k, induced reactivation and mutagenesis of k, and mutation to histidine prototrophy. The increased inducibility exceeded the level expected by UV and dam-3 acting additively and independently, and suggests that the effects of UV and dam-3 interact in some way to potentiate induction of SOS functions.

Introduction dam-3 mutants of Escherichia coli [17] have less than 15% of wild-type levels of 6-methyladenine (6MeA) in their DNA compared with wild-type bacteria [19]. Methylation of adenine in wild-type bacteria normally occurs in situ so that for a short period after replication old and new DNA are distinguishable by their different contents of 6MeA [16]. It has been postulated that the difference in methylation permits excision of mismatched bases to be directed so that the old, methylated, strand is conserved, and the new, undermethylated DNA is repaired [10,31]. The phenotypic effects of 6MeA deficiency in dam-3 mutants are very varied, dam-3 bacteria are sensitive to the lethal and mutagenic effects of base analogues such as 2-aminopurine and 5-bromouracil, presumably because there is no discrimination against mispairing bases in new DNA [10]. There is also an increased incidence of genetic recombination with * Present address: Institut de Biologie Moldculai~e, Facultd des Sciences, Quai St. Bernard, Paris (France). ** Present address: Genetics Division, National Institute for Medical Research, The Ridgeway, Mill Hill, L o n d o n N W 7 1 A A (Great Britain).

324 F factor DNA in dam-3 mutants [20]. Furthermore, the p h e n o t y p e of dam-3 mutants features higher incidences of naturally occurring mutagenesis [18], lysogenic induction [19] and cell filamentation [18]. These latter processes have been called "SOS functions" as they normally arise in wild-type bacteria in response to DNA damage [24,33]. In population terms the effect of dam-3 on the induction of SOS functions would at first appear to be quite small. Although dam-3 causes a 10-fold increase in ~ lysogenic induction, this only represents a change in approximately 1% of the population. 99% of the population remains uninduced for ~ and apparently unaffected b y the dam-3 mutation. The purpose of this communication is to show that the dam-3 mutation does, in fact, affect SOS functions on a much larger scale. Its effect was to reduce the UV doses required to cause mass induction of several SOS functions. Materials and methods Bacterial and phage strains The properties and sources of the bacterial strains used are listed in Table 1. GY 515 was used as indicator for the induced reactivation experiments. Its uvrA16 mutation prevents host cell reactivation [26]. GY 4015 was used as indicator bacteria for the X lysogenic induction experiments [22]. The phage strain used was k papa. Media Cultures for mutagenesis experiments were grown in Bacto Davis minimal medium supplemented with 0.5 #g/ml thiamine hydrochloride, 50 pg/ml threonine, leucine, proline, arginine, histidine and thymine. Semi-enriched (SEM) plates contained supplemented Bacto Davis minimal medium without histidine, 5% (v/v) Oxoid nutrient broth and was solidified with 1.2% (w/v) agar. Minimal (MIN) plates lacked the supplement of histidine. All other cultures were grown in Luria broth (LB) which contains 5 g/1 Difco yeast extract, 10 g/1 Difco tryptone and 10 g/1 NaC1. Luria agar (LA) was LB solidified with 1.5% (w/v) Biomar agar. GT agar, used for scoring k plaques, contained 8 g/1 peptone, 5 g/1 Difco tryptone, 5 g/1 NaC1 and 12 g/1 Biomar agar. GTamp is GT containing 20 #g/ml of D-ampicillin [22]. Soft agar containing 7.5 g Difco agar per litre of demineralised water was used to plate phage and bacteria.

TABLE 1 BACTERIAL STRAINS Strain

Relevant genotype

Source

AB GM GM GY GY GY GY

his-4 u v r A 6 u v r A 6 dam-3 hi8-4 u v r A 6 d a m - 3 uvrA6 ampA601 GM 96 (k) AB 2 5 0 0 ( k )

P. H o w a r d - F l a n d e r s M. M a r i n u s M. M a r l n u s a A.M. D e l v a u x a n d R . D e v o r e t M o r e a u et al. [ 2 2 ] This work This work

2500 55 96 515 4015 5802 5803

a S t r a i n G M 9 6 w a s g e n e r o u s l y c o n s t r u c t e d b y D r . M. M a r i n u s for the p u r p o s e o f t h e s e e x p e r i m e n t s .

325 UV irradiation UV-radiation came from a General Electric germicidal lamp having a maximum o u t p u t at 253.7 nm. Fluence was measured with a Latarjet dosimeter. Phage and exponentially growing bacteria (1--3 × 10 s cells per ml) were irradiated in 0.01 M MgSO4 in glass petri dishes. The thickness of the suspension was approx. 1 mm. Prophage X induction )~ infective centers were detected on GTamp plates using GY 4015 ampR as indicator bacteria [23]. Bacterial survival was measured on LA plates. Induced reactivation o f phage X Bacteria were UV-irradiated, diluted 2-fold in double strength LB and incubated with aeration for 30 min. Phage X, UV-irradiated with 50 J/m 2, was diluted and incubated with aliquots of UV-treated cells for 20 min to permit absorption. The mixtures were then poured with soft agar containing GY 515 indicator bacteria onto GT plates. To measure induced reactivation of the phage, the top agar contained 100-1000 p.f.u. (m.o.i. about 10 -4) per plate, whereas there were 5000--20 000 p.f.u. (m.o.i. a b o u t 10 -2) per plate in measurements of phage mutagenesis. Phage survival was estimated after overnight incubation. Clear plaque mutants were more easily scored after a full day's growth and were probably caused by mutagenesis at cI, cII and cY loci. Repair efficiency was calculated according to Blanco and Devoret [ 1 ]. UV-induced and spontaneous mutagenesis to histidine prototrophy Reversion to histidine p r o t o t r o p h y was measured using the semi-enriched plate technique described in detail by Green et al. [ 11 ]. Mutation frequencies were calculated according to the method of Bridges [3]. SDS--polyacrylamide slab gel electrophoresis The proteins of bacteria grown in supplemented Bacto Davis minimal medium were radioactively labelled for 20 min by the addition of 2 pCi of [3sS] methionine (149 Ci mmo1-1) (New England Nuclear Corp.) and 0.02 pg of methionine to 0.1-ml aliquots of culture. Samples were prepared for electrophoresis exactly as previously described [30]. Electrophoresis was through 10% SDS--polyacrylamide gels for 75 min at 200 V using the buffer system of Laemmli [14]. Results

L ysogenic induction Spontaneous phage k production was 10--20 times greater in dam-3 lysogens compared to the isogenic dam ÷whether assayed by measurement of free phages or infective centers in the presence or absence of ampicillin [19] (Fig. 1). After UV-irradiation maximal induction of X was achieved with approx. 2 J / m : of UV in uvrA6 dam-3 bacteria compared with approx. 4 J/m 2 in vvrA6 dam ÷ cells. This reduction in UV dose for )~ induction was accompanied by an

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F i g . 1. P r o p h a g e ~. i n d u c t i o n after UV-irradiation. G Y 5 8 0 3 uvrA dam-3 ( k ) ( ~ 4 ) and G Y 5 8 0 2 uvrA d a m + (k) ( o e ) w e r e ~ o w n o n c o m p l e t e m e d i u m t o m i d - l o g phase, c e n t r i f u g e d , r e s u s p e n d e d in 0 . 0 1 M M g S O 4 , U V - i r r a d i a t e d , and plated on L A for m e a s u r i n g cell survival ( c l o s e d s y m b o l s ) or o n G T a m p s e e d e d w i t h G Y 4 0 1 5 ampA601 for m e a s u r i n g i n f e c t i v e c e n t e r s ( o p e n s y m b o l s ) . F i g . 2. U V - i n d u c e d r e a c t i v a t i o n o f UV-irradiated phage k. A B 2 5 0 0 u v r A 6 d a m + (oe) and G M 9 6 u v r A 6 d a m - 3 (AA) g r o w n to m i d - l o g phase w e r e UV-irradiated in 0 . 0 1 M M g S O 4. A f t e r 3 0 m i n i n c u b a t i o n w i t h

a e r a t i o n in rich m e d i u m , t h e y w e r e i n f e ~ t e d w i t h U V - i r r a d i a t e d phage k and p l a t e d o n G T s e e d e d w i t h G Y 5 1 5 u v r A 6 bacteria. S y m b o l s : e f f i c i e n c y o f repair ( c l o s e d s y m b o l s ) ; clear plaque m u t a t i o n f r e q u e n c y ( o p e n s y m b o l s , d o t t e d lines).

increase in the percentage of cells induced and was seen in 6 out of 6 experiments performed. The greater UV inducibility of ~ in dam-3 bacteria is still evident after subtracting the high numbers of spontaneously produced infective centers from the total induced. Thus the dam-3 mutation potentiates the effect of UV in k lysogenic induction. A similar increase in UV inducibility was found with uvr+dam-3 bacteria compared with wild-type {data not shown). UV-induced reactivation and mutagenesis o f UV-irradiated phage The increases in survival and mutagenesis of UV irradiated phage k plated on host bacteria which have also been irradiated [13] have been attributed to the activity of a bacterial, inducible error-prone repair system [1,8]. This system is apparently more readily induced in dam-3 bacteria because (a) maximal UVinduced reactivation of phage k occurred after irradiation of uvrA6 dam-3 with 5 J/m 2 of UV, whereas 10 J/m 2 of UV were required to maximally induce the uvrA6 dam+ strain (Fig. 2), (b) the yield of clear mutations at any UV fluence was always greater from uvrA6 dam-3 infective centers. No differences were detectable in the survival and mutagenesis of UVdamaged phage k plated on unirradiated uvrA6 dam-3 or uvrA6 dam + bacteria. This observation is in apparent contrast to the increase from 0.1% to 1% in the frequency of spontaneously induced k prophage in dam-3 mutants. It must be remembered, however, that induced reactivation measures the capability of a whole-cell population to reactivate damaged phage. A 10-fold change from 0.1

327

to 1% in the number of cells spontaneously induced for SOS repair would only represent an increase, at the most, of 1% in phage survival and would not be detectable. M u t a g e n i c reversion to histidine p r o t o t r o p h y

Spontaneous His + revertants were detected with frequencies of 1--5 × 10 -7 in AB 2500 u v r A 6 dam + and 5 × 10 -6 in GM 96 u v r A 6 dam-3. This confirms that the d a m - 3 mutation causes an increase in spontaneous mutation frequency [18, 19]. Mutagenesis to histidine prototrophy was also 2--4 times more frequent after UV irradiation of u v r A 6 d a m - 3 bacteria than of u v r A 6 d a m + cells (Fig. 3).

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328 In the calculation of induced mutation frequencies the numbers of mutants in unirradiated cultures are subtracted. Thus the additional mutants detected in irradiated GM 96 u v r A 6 darn-3 are not explicable as the sum of the separate effects of the UV radiation and of the high spontaneous mutability caused by the dam-3 mutation, but rather by interaction between the radiation treatment and the presence of the dam-3 allele. As previously reported, the dam-3 mutation slightly increased UV sensitivity [ 18]. I n d u c t i o n o f n e w p r o t e i n s in U V irradiated and unirradiated d a m ÷ and dam-3 bacteria SDS--polyacrylamide electrophoresis of protein extracts from E. coli

induced for SOS functions by treatment with radiations or chemicals shows the characteristic induction of Protein X [11] which is now known to be the recA ÷ protein [9, McEntee, cited in ref.9]. We have tested whether recA ÷ protein synthesis occurs constitutively in unirradiated dam-3 bacteria and whether or not there is more of this protein in UV irradiated dam-3 mutants compared with wild-type cells. After UV irradiation of AB 2500 d a m ÷ two new protein species were detectable by SDS polyacrylamide gel electrophoresis (Fig. 4, column 2). One, having a molecular weight of 40 000 Daltons, is Protein X [12]. The other protein with a molecular weight of 45 000 Daltons has n o t been previously described and its properties have not y e t been investigated. The proteins were not detected in extracts of unirradiatetl cultures of two d a m - 3 strains (Fig. 4, tracks 3 and 5). They were nevertheless detectable after UV-irradiation (Fig. 4, tracks 4 and 6). It is concluded that a population as a whole of dam-3 bacteria is not spontaneously induced for r e c A ÷ protein synthesis. A second type of band change was also detected which seemed to be associated specifically with the dam-3 mutation. A band with a molecular weight of approximately 36 000 in wild-type bacteria was found at a lower molecular weight position of 33 000 Daltons in dam-3 bacteria (Fig. 4, arrow d a m ) . Discussion In confirmation of the work of Marinus and Morris [18,19] we found that the phenotype of d a m - 3 bacteria resembles that of wild-type cells which have been slightly UV-irradiated. The frequency of spontaneous lysogenic induction increased from approximately 0.1 to 1%. Similarly the incidence of spontaneous reversion to histidine p r o t o t r o p h y increased 10--40 fold. However these increased fractions of apparently induced cells are still small in comparison with the large part of the population which remained uninduced. When whole populations of dam-3 bacteria were examined for increased spontaneous levels of other SOS functions such as recA ÷ protein (Fig. 4) and induced reactivation of phage ?~, no changes were detectable. Nevertheless the d a m - 3 mutation did exert an effect on whole populations of bacteria in making them more readily UV inducible for reversion to histidine p r o t o t r o p h y (Fig. 3) or clear plaque mutagenesis of ~ (Fig. 2). Similarly there was an approximate halving of the UV dose required for maximal induction of prophage ?t (Fig. 1) or for induced reactivation of damaged phage (Fig. 2).

329 Increased inducibility was more than the sum totals of the separate effects of UV and the dam-3 mutation indicating that dam-3 potentiates the effect of UV in inducing SOS functions. The means of SOS induction by UV alone is n o t clear. The photoreversibility of many SOS functions induced by UV implicates the pyrimidine dimer as the lesion causing SOS induction. However to cause induction many hundreds of dimers must be present in the DNA of the treated cells; and although dimers are introduced linearly with dose [29], induction occurs with 2-hit kinetics [15]. This has been taken to indicate that two radiation events per cell must occur for induction [15] and that at least one must be a pyrimidine dimer. Indeed the level of induction after UV irradiation can be related to the frequency of pairs of pyrimidine dimers close enough in opposite DNA strands so that they interact and inhibit each other's constitutive repair [28]. This would occur when one pyrimidine dimer was in the tract of DNA required for the repair replication of the other. It can also be envisaged that the inducing potential of these, or any other lesions, is also related to the length of time they persist in DNA before being repaired. Thus the effect of dam-3 in increasing UV inducibility of SOS functions could be due to increasing the probability of lesion interaction and/or to increasing the time lesions remain in DNA before bein~ repaired. For example dam-3 could potentiate the SOS inducing effect of UV b y generating " h y b r i d " inducing lesions comprising of a dam-3 associated DNA strand break [18] in one strand, opposite a pyrimidine dimer in the other. A second possibility is that dam-3 increases the chances of pyrimidine dimer interaction by increasing the length of tracts of DNA involved in repair. This effect is seen in p o l A mutants where an increase in inducibility of SOS functions [21,32] can be related to an increase in excision-repair gap size [6] and thus an increase in the probability of lesion interaction [4,5]. This could occur if dam-3 lesions and pyrimidine dimers competed for some limiting quantity of repair enzyme. Thirdly DNA methylation may be essential for the efficient repair of inducing lesions, which, if unrepaired, would continue to exert their inducing influence. This suggestion stems from proposals that dam-3 methylation is used by a mismatch repair system to discriminate between old, methylated, and new, unmethylated DNA [10,31]. Such strand discrimination would permit conservation of the old template strand and excision of the mismatched base from the new DNA [10]. Mismatched base incorporation also occurs during SOS repair where bases are randomly inserted opposite non-coding lesions in the template strand [25]. In contrast to mismatch-base repair occurring near the replication fork, succesful SOS-repair requires conservation of the new DNA and elimination of the non-coding base in the old DNA. It is suggested that the distinction between old and new DNA needed for SOS repair could again be due to dam ÷ methylation. However, the direction of basepairing correction would be opposite to that occurring at the replication fork and would conserve the new DNA strand. Thus a reduction in DNA methylation in dam-3 mutants would prevent strand discrimination from occurring so that there would be a tendency for the mismatched bases inserted by SOS repair to be removed. The original non-coding lesion in the template strand would be revealed again and so would continue to exert its SOS inducing signal. UV and the dam-3 mutation continued to interact in mutagenesis after

330

irradiation with doses larger than the 2 J/m 2 required to fully induce repair in the uvr backgroun [27]. It is concluded that UV and d a m - 3 also interact to provide a substrate for error-prone repair. Similar interactions have been found for combinations of X- and UV-irradiations which induced more mutations than the sum totals of both treatments administered separately [2,7]. These results are consistent with the proposal that interacting lesions are both the inducing signal for, and the substrate of, inducible error-prone repair [28]. Acknowledgements S.G.S. is grateful to Dr. R.B. Setlow in whose laboratory part of this work was performed. We also thank Dr. R. Devoret for hospitality and laboratory facilities. The authors are very grateful to Dr. M.G. Marinus for advice and strains. This work was supported by a Euratom grant No. 147-75-I.BIO F. to Dr. R. Devoter and a Euratom fellowship to S.G.S. References 1 Blanco, M., a n d R. D e v o r e t , R e p a i r m e c h a n i s m s i n v o l v e d in p r o p h a g e r e a c t i v a t i o n a n d UV r e a c t i v a t i o n of U V - i r r a d i a t e d p h a g e k, M u t a t i o n Res., 17 ( 1 9 7 3 ) 2 9 3 - - 3 0 5 . 2 Bridges, B.A., R.J. M u n s o n , C.F. A r l e t t a n d D.R. Davies, I n t e r a c t i o n b e t w e e n u l t r a v i o l e t light a n d ~,r a d i a t i o n d a m a g e in t h e i n d u c t i o n of m u t a n t s o f Escherichia coli: t h e e f f e c t of s o m e m o d i f y i n g t r e a t m e n t s , J. G e n . Microbiol., 46 ( 1 9 6 7 ) 3 3 9 - - 3 4 6 . 3 Bridges, B.A., S i m p l e b a c t e r i a l s y s t e m f o r d e t e c t i n g m u t a g e n i c agents, Lab. Pract., 21 ( 1 9 7 2 ) 4 1 3 - 419. 4 B o n u r a , T., a n d K.C. S m i t h , E n z y m a t i c p r o d u c t i o n of d e o x y r i b o n u c l e i c acid d o u b l e s t r a n d b r e a k s a f t e r u l t r a v i o l e t i r r a d i a t i o n of Escherichia coli K 1 2 , J. 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Gen. Microbiol., 46 ( 1 9 6 7 ) 3 2 9 - - 3 3 8 . 8 Defais, M., P. F a u q u e t , M. R a d m a n a n d M. E r r e r a , U l t r a v i o l e t r e a c t i v a t i o n a n d u l t r a v i o l e t m u t a g e n e s i s of k in d i f f e r e n t g e n e t i c s y s t e m s , V i r o l o g y , 43 ( 1 9 7 1 ) 4 9 5 - - 5 0 3 . 9 E m m e r s o n , P.T., a n d S.C. West, I d e n t i f i c a t i o n of P r o t e i n X of Escherichia coli as the recA+/tif + gene p r o d u c t , Mol. Gen. G e n e t . , 1 5 5 ( 1 9 7 7 ) 7 7 - - 8 6 . 10 G l i c k m a n , B.W., P. v a n d e n Elsen a n d M. R a d m a n , I n d u c e d m u t a g e n e s i s in d a m - m u t a n t s o f Escherichia coli: a role f o r 6 - m e t h y l a d e n i n e r e s i d u e s in m u t a t i o n a v o i d a n c e , Mol. G e n . G e n e t . , 1 6 3 ( 1 9 7 8 ) 307--312 11 G r e e n , M . H . 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