Participation of rec genes of Escherichia coli K12 in W-reactivation of UV-irradiated phage λ

Mutation Research, 243 (1990) 159-164

159

Elsevier MUTLET 0308

Participation of rec genes of Escherichia coli K 12 in W-reactivation of UV-irradiated phage 2 Draga Simi6, Branka Vukovi6-Ga6i6 and Jelena K~ne2evib-Vuk6evi6 ¢~

Botanical Institute and Garden, University of Belgrade, Takovska43, 11000Belgrade (Yugoslavia)

(Accepted 28 July 1989) Keywords."Recombinationdeficiency;W-reactivation;Bleomycin;UV-irradiation

Summary The effect of the recombinational deficiency on W-reactivation of UV-damaged phage k was explored. In this paper we show that W-reactivation is reduced by the recB21 and recF143 mutations after bleomycin (BM) and UV treatment. Combination of these mutations in the recB21recF143 double mutant blocks Wreactivation completely after BM induction, but leaves residual W-reactivation ability after UVoirradiation, which is abolished by the introduction of uvrB deficiency (za(uvrB-chlA)). W-reactivation has been rendered constitutive in recB21C22sbcB15, but the efficiency of reactivation remained virtually constant over the range of BM and UV doses, indicating the role o f the RecBC(D) enzyme in W-reactivation.

In our previous work we attempted to determine the role(s) of alternative pathways of recombinational repair of damaged DNA in RecA protein activation (Simi6 et al., 1985; Kne2evi6-Vuk~evi6 et al., 1987), which is known to be of key importance for SOS induction (Burckhardt et al., 1988; Little, 1984; Roberts and Devoret, 1983; Walker, 1984, 1985). The obtained data indicated that the proficient RecBC(D) pathway as well as the fully expressed RecF pathway (switched on by the sbcB mutation in a recBC background) are required for bleomycin (BM)-induced damage processing and SOS signal generation. Moreover, our data confirmed that a functional recF gene is required for Correspondence:Dr. D. Simid, BotanicalInstitute and Garden, University of Belgrade, Takovska 43, 11000 Belgrade (Yugoslavia).

efficient SOS induction by UV-irradiation (Armengod and Blanco, 1978; Thorns and Wackernagel, 1987; Volkert and Hartke, 1984). In this study we examined W-reactivation (Weigle, 1953), the induced repair of damaged phages, mediated by pathways of recombinational repair. W-reactivation belongs to the class of SOS functions that are umuDC- and uvrABCdependent (Walker, 1984) and in UV-treated hosts requires the functional recF gene product (Rothman et al., 1979; Volkert and Hartke, 1987).

Material and methods Bacterial and phage strains Bacterial strains, all E. coli K12 derivatives, are

listed in Table 1. The k virulent phage mutant 0, vir) was used in this work.

0165-7992/90/$ 03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)

160

0.0-12 /~g/ml), was applied to bacterial cultures, which were then incubated for l0 min in the dark.

TABLE 1 BACTERIAL STRAINS Strain a

Relevant genotype

Source

AB1157 AB2470 DL51 D L 131 DL132 DL 148 SP254 SR1352 SR 1374 SR1350 SR 1372

rec* recB21

K.B. Low ibid. A. Taylor ibid. ibid. ~ ibid. M. Radman K. Smith ibid. ibid. ibid.

recB21C22sbcB 15 recF 143 recB21recF143 recB21 C22sbcB 15recF 143 recN262 rec ÷uvrB recB21 uvrB recF143uvrB recB21 recF 143 uvrB

UV-irradiation This was carried out as described earlier (Simi6 et al., 1985). W-reactivation The phage lysate was diluted to about 10 7 plaque-forming units (pfu)/ml in 0.01 M MgSO4 and irradiated with a C a m a g germicidal lamp (254 rim). Unirradiated and irradiated phages were adsorbed for 20 min on BM- or UV-treated hosts (multiplicity of infection (MOI) about 0.01). After incubation at 37°C, samples were diluted and plated on the indicator strain.

auvr + strains with initials DL and SP are as ABl157; uvrB strains with initial SR have a KH21(A(uvrB-chlA)) background

(Wang and Smith, 1986a).

Results and discussion

Media and growth conditions Experiments were performed on bacteria grown in LB medium at 37°C, with aeration, to the midlog phase (OD61o-0.2).

Quantitative survival of rec mutants was compared over the range of different BM and UV doses. All strains defective in recombinational repair ~xhibited an increased sensitivity to both agents, but with different patterns (Fig. 1). It should be noted that recB-deficient strains are ex-

B M treatment BM, purchased from Krka (final concentration

rec o I 0 -~

•--~

•

ld

[ ~ A

6~ ~

re~"

rec B2% recC22 5boB15

I

~o-~

o

;

;

~ B~

FiB. ]. Compa[ison of survival of dependen[ experimen[s.

rec

~,

(~g/ml)

s[rains af[er B ~

~ ~.~ ~ / , ~ U a 2 o

~

~o

~ ~"~

~o

UV ~o~

, ,~ ~o go ( J / m 2)

[rea[men[ and UV-irradiafion. Each value represen[s [he m e a n o[ a[ leas[ 3 in-

161

tremely sensitive to BM, while the recBCsbcB mu° tant (recombination-proficient) is least sensitive with survival close to that of the wild type. Introduction of the uvrB deficiency (A(uvrBchlA)) into recB and recF mutants does not significantly increase the sensitivity to BM treatment (data not shown), indicating that BMinduced lesions, single-strand and double-strand DNA breaks (Mfiller et al., 1972; Takeshita et al., 1978) are repaired mostly by recombinational repair. Thus, sensitivity to BM would be consistent with a reduction in recombinational repair, but it could be enhanced if the RecBC(D) enzyme (exoV) was also required to generate the SOS-inducing signal (Chaudhury and Smith, 1985). The indication that the RecBC(D) enzyme is required for DNA repair is confirmed by following incision and reformation of high-molecular-weight DNA after BM treatment (Kne~evi6-Vuk~evi~, 1989). Moreover, our previous results indicate that in recB the SOS response is delayed and reduced, and completely abolished in the recBrecF double mutant after the same treatment (Simid et al., 1985; Kne~evi~-Vuk~evi~ et al., 1987). We continue to explore the inducible phenomena in different rec mutants by studying W-reactivation o f UV-irradiated k virulent phage in BM- and UVtreated hosts. As can be seen in Table 2, differences in induced reactivation depending on BM concentration a n d / o r genetic background of the bacterial strains were observed. The effect of recB and recF mutations on W-reactivation of the UVoirradiated phage was greatly reduced, demonstrating the requirement for both gene products. Moreover, Wreactivation ability was virtually abolished in the recBrecF mutant. The maximum amount of Wreactivation in recN occurred at a lower BM dose (3 /~g/ml) than observed with the wild type (6/~g/ml) and was slightly reduced, probably due to its repair defect. Above 3 ~g/ml of BM similar patterns of Wreactivation induction were obtained for the recB and recBCsbcB strains (Table 2). The same phenomenon was observed in these strains after UV-irradiation (data not shown). However, W-

reactivation has been rendered constitutive by the sbcB mutation in the recBC cells as shown by the

high efficiency of reactivation (E) in this strain (Table 2), and supported by a survival of the UVo irradiated phage in untreated recBCsbcB higher than seen in the wild type or other rec host strains used in this study (Table 3). In BM-induced wild-type cells, as compared to UV-induced cells, there is an intermediate state of SOS induction (Table 3), probably not allowing full expression of the specific genes required for Wreactivation (Walker, 1984). The situation could be similar to that reported by Barb6 et al. (1985) that nalidixic acid, in the wild type, induced recA protein amplification, cell filamentation and prophage induction, without triggering the expression of the u m u C gene. Surprisingly, recB and recF show essentially the same efficiency in the repair of UV-irradiated k regardless of the inducing treatment. This is in contrast to our previous results indicating a different relative importance of these genes in the SOS response depending on BM or UV treatment. No residual component of W-reactivation is found in BM-treated recBrecF and recBCsbcBrecF hosts. The lack of SOS induction by BM in these strains, but not after UVoirradiation, has already been shown in our published data. By comparing genetic backgrounds of strains with complete loss of W-reactivation ability (Table 3), it can be stated that the recF gene product determines one component of W-reactivation, the other component(s) being dependent on recB and uvr genes. The complete block of W-reactivation in the BM-treated recBrecF double mutant confirms that recombinational repair is the only component required for SOS signal generation by BM. On the contrary, residual UV-induced W-reactivation is observed in this strain, while introduction of the uvrB deficiency in recF and recBrecF blocks Wreactivation completely. This is in agreement with data already reported on the uvr-mediated pathway of UV-induced W-reactivation (Volkert and Hartke, 1987; Walker, 1984). The requirement for the RecBC(D) enzyme is indicated by the severe reduction of W-reactivation

162

TABL E 2 W - 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 ON B M-TR EA TED HOST a Host genotype

Strain

BM concentration (~g/ml) 0

rec + recB21 recB21C22sbcB15 recF143

recN262

3

6

12

Rb

E

R

E

R

E

R

E

ABl157 AB2470 DL51

1.0 1.0 3.0

0.0 0.0 25.0

4.9 1.3 3.6

23.0 5.5 29.0

6.1 1.4 3.4

27.0 8.8 31.0

2.2 1.3 3.3

12.0 5.5 27.0

DLI31 SP254

1.0 1.0

0.0 0.0

1.7 4.4

11.5 22.0

1.7 3.1

12.3 17.0

1.0 1.1

0.6 1.3

aln r e c B 2 1 r e c F 1 4 3 (DL132) W-reactivation was not detected. bR and E values were calculated as described (Devoret et al., 1975; R ot hma n et al., 1979). Each value represents the mean of at least 3 independent experiments.

in the recB mutant (Table 3). Moreover, in spite of the high efficiency of reactivation in recBCsbcB, most probably due to the high basal level of activated RecA (Tables 2 and 3; Karu and Belk, 1982), as well as fully expressed repair genes, the effect of the inducing dose is insignificant. While the net W-reactivation maximum is found in the wild type, the efficiency of reactivation remains virtually constant in recBCsbcB over the range of BM doses, and similar to recB (Table 2). The same results are obtained after UV-irradiation. In our opinion, these data also indicate the direct a n d / o r indirect role of the RecBC(D) enzyme in W-

reactivation. The striking similarity of W-reactivation efficiency in recB and recF mutants following BM and UV treatment is intriguing. Both recB and recF gene products participate in SOS induction (Armengod and Blanco, 1978; Karu and Belk, 1982; Volkert et al., 1984), as well as in the repair of BM- and UV-induced lesions (Fig. 1; Wang and Smith, 1983, 1986a, b). The higher level of activated RecA protein resulting from SOS induction enhances the capability for DNA repair (Peterson et al., 1988). Besides, activation of RecA is modulated by its interaction with the RecF protein

TABL E 3 E F F I C I E N C Y OF 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 A F T E R BM OR UV A P P L I E D TO T H E HCST a Host genotype

rec ÷ recB21 recF143 recB21recF143 r e c B 2 1 C 2 2 s b c B 15

Survival of UV-irradiated phage on untreated host b

Emax of UV-irradiated phage on treated host

UVF+

/~vr -

$/yr +

//vr -

//vr +

1.0 0.57 0,71 0.78 2.31

1.0 0.39 0.52 0.53

100.0 15.3 26.5 17.7 62.1 25.8 91.0

40.7 4.9 0.0 0.0

46,5 15.5 21.5 0.0 53.4 0.0 37.9

r e c B 2 1 C 2 2 s b c B 15recF143 recN262

0.72

UV

BM

aEfficiency of reactivation (Emax %) was calculated as in Table 2; the value in the UV-irradiated wild type is taken as 100.0%. bRelative phage survival was calculated: surviving fraction on r e c - divided by surviving fraction on rec ÷ . For u v r ÷ strains the phage was irradiated with 250 J / m 2 (survival on AB1157 was 1.6 × 10 - 2) and for u v r - strains the phage was irradiated with 50 J / m z (survival on SR1352 was 1.2× 10-2). Each value represents the mean of at least 3 independent experiments.

163 ( M o r e a u , 1988; T h o m s a n d W a c k e r n a g e l , 1987). T a k i n g into c o n s i d e r a t i o n the above, the results o b t a i n e d in o u r study o f W - r e a c t i v a t i o n are n o t easily interpretable. Nevertheless, we p r e s u m e that processing by RecBC(D) of D N A lesions i n d u c e d by BM, most p r o b a b l y the D N A - u n w i n d i n g activity o f exoV ( C h a u d h u r y a n d Smith, 1985; O s s a n n a a n d M o u n t , 1989), is needed for efficient SOS ind u c t i o n . R e d u c t i o n o f W - r e a c t i v a t i o n in BMtreated r e c B m u t a n t s is most likely due to the low level o f SOS i n d u c t i o n . In this situation the stabilizing role of the RecF p r o t e i n w o u l d be critical for the SOS response. T h e reason for the severe r e d u c t i o n o f W - r e a c t i v a t i o n in U V - i n d u c e d r e c B is n o t clear since the experimental evidence o n the effect o f the r e c B m u t a t i o n o n the U V - i n d u c e d SOS response is c o n t r a d i c t o r y (Barb6 et al., 1985; Simi6 et al., 1985; T h o m s a n d W a c k e r n a g e l , 1987). T h e reduced W - r e a c t i v a t i o n in B M - i n d u c e d recF m u t a n t s (Tables 2 a n d 3) together with data from the literature ( R o t h m a n et al., 1979; Volkert a n d H a r t k e , 1987) suggest the a d d i t i o n a l role o f RecF p r o t e i n in the alleviation o f U V d a m a g e to the infecting phage g e n o m e . M u t a t i o n in r e c N produces a deficiency in the induced capacity o f d o u b l e - s t r a n d break repair (Fig. 1), b u t has n o m a r k e d effect o n SOS signal generat i o n (Table 3). E x p e r i m e n t s different to those yet described are needed to d e t e r m i n e the relative c o n t r i b u t i o n o f D N A repair in W - r e a c t i v a t i o n .

Acknowledgements W e are grateful to Miroslav R a d m a n , Kendric S m i t h a n d Neil Sargentini for the E . coli strains. This study was s u p p o r t e d by Yu P r o g r a m U1-BL-B 83/88 ( P 2 2 / B B 3 ) a n d N a t i o n a l Scientific G r a n t 2901/1.

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