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Induction of prophage λ does not require full induction of RecA protein synthesis
BIOCHIMIE, 1~80, 62, 687-694.
Induction of prophage ), does not require full induction of RecA protein synthesis ('). Patrice L. MOREAU, Mich61e FANICA and R a y m o n d DEVORET <>. (Re~a le 25-2-1980. Accept~ apr~s r e m a n i e m e n t le 28-~-1980).
Radiobioloffie Cellulaire, Laboratoire d'Enzymologie, C.N.R.S., 91190 Gi[-sur-Yvette, France.
Mots cl~s : prot6ine ReeA ; induction lysog~nique.
Key words : RecA protein ; ), repressor cleavage ; rifampiein ; mitomycin C.
R~sum6.
Summary.
Les bact6ries 1ysogbnes Escherichia coli K12 (X) sont induites par la mitomycine C alors que la vitesse de synthbse de la prot6ine RecA d'E. coli est s p ~ i f i q u e m e n t r~duite par une faible concentration de rifampicine (4 ~tg/ml). L'induction du prophage X a pu ~tre raise en 6vidence par la Iyse des bact6ries lysog~nes, par la production de phages ]ibres et par la destruction du r6presseur du prophage ~. La vitesse avec laquelle se produit le clivage du r6presseur et l'efficacit6 de ce clivaqe sont pratiquement inchang6es bien que la d6r6pression de la synth~se de la prot6ine RecA soit fortement inhib6e. Toutefois, la production de phages libres est diminu6e en pr6sence de rifampicine et la lyse des cellu|es un peu retard6e. On peut donc conclure de ces r6sultats que ches des bact~ries de type s a u v a g e (RecA÷), l'induction du prophage ~ n'exige pas pour se produire la d6r6pression complete de la synth~se de la prot6ine RecA.
In mitomycin C-treated ~ 1ysogens. even though the rate of synthesis of Reck protein w a s greatly reduced b y a l o w c o n c e n t r a t i o n of
Introduction. The R e c A gene in E s c h e r i c h i a colt controls genetic recombination [1] and various cellular responses to DNA damage, the so-called (( SOS phenomena >> ~2]. Not only do recA mutations affect recombination proficiency but lhey also prevent
* This paper is dedicated to the memory of Jacqueline George. © To whom all correspondence should be addressed.
rifcunpicin (4 ~tg/ml). i n d u c t i o n of p r o p h a q e
occurred readily as assessed b y (i) ceil lysis of the l y s o g e n s , (ii) p r o d u c t i o n of p r o g e n y p h a g e , a n d (iii) e x t e n s i v e c l e a v a g e of ~ r e p r e s s e r . T h e extent a n d the rate of c l e a v a g e of ~ repressor were not s i g n i f i c a n t l y a f f e c t e d b y the l o w rate of s y n t h e s i s of R e c A p r o t e i n r e s u l t i n g f r o m ri. f a m p i c i n action. H o w e v e r , the y i e l d of p h a g e p r o g e n y w a s r e d u c e d a n d Iysis of the cells w a s sliqhtly d e l a y e d . W e c o n c l u d e that in RecA + b a c t e r i a , i n d u c t i o n of p r o p h a g e ~ d o e s not r e q u i r e full i n d u c t i o n of R e c A p r o t e i n syn-
thesis.
inducible error-prone repair (SOS repair), cell filamentation and p r o p h a g e ), induction from o c c u r r i n g [2]. The p r o d u c t of the recA gene [3] was first identified as protein X [4-8], whose rate of synthesis increases sharply when SOS functions are provoked [4, 5, 9~. Induction of prophage ), is one of the most dramatic cellular responses to DNA d a m a g e ; its m e c h a n i s m is presently the most amenable to molecular investigation. In a )~ lysogen, p r o p h a g e )~ is maintained in a dormant state by a repressor protein, the p r o d u c t of the p r o p h a g e cl gene [101.
688
P. L. Moreau and coll.
I n a m o n o l y s o g e n , w h e n all t h e )~ r e p r e s s o r m o l e c u l e s , p r e s e n t at a c o n c e n t r a t i o n of a b o u t 140 c o p i e s [11], a r e i n a c t i v a t e d [12, 13], a n d c l e a v e d i n t o t w o a l m o s t e q u a l f r a g m e n t s [14], p r o p h a g e ), is i n d u c e d t o d e v e l o p v e g e t a t i v e l y . P r o t e o l y t i c c l e a v a g e of ), r e p r e s s o r o c c u r s a l s o in vitro w i t h p u r i f i e d R e c A p r o t e i n , it also r e q u i r e s A T P a n d s i n g l e - s t r a n d e d p o l y n u c l e o t i d e m o l e c u l e s [15-17]. G e n e t i c d a t a [13, 18, 19] s u p p o r t t h e i d e a t h a t , in oivo, t h e i n a c t i v a t i o n of ), r e p r e s s o r r e s u l t s f r o m t h e e x p r e s s i o n of a RecA g e n e f u n c t i o n . It h a s b e e n w i d e l y
s u r m i s e d t h a t i n d u c t i o n of induction, as it m a y b e f o r i n d u c t i o n of o t h e r c e l l u l a r r e s p o n s e s t r i g g e r e d b y D N A d a m a g e , s u c h as cell f i l a m e n t a t i o n , i n h i b i t i o n of D N A d e g r a d a t i o n , o r i n d u c i b l e e r r o r - p r o n e r e p a i r [6, 7, 16, 20, 21]. W e report here evidence that full induction of ReeA p r o t e i n is n o t r e q u i r e d f o r i n d u c t i o n of p r o p h a g e ), i n w i l d - t y p e l y s o g e n s .
RecA p r o t e i n is r e q u i r e d f o r p r o p h a g e
excess) was not p r e c i p i t a t e d by a d d i t i o n of a second a n t i s e r u m b u t e h r o m a t o g r a p h e d on columns of protein A-Sepharose CL-4B ( P h a r m a e i a ) [22]. Samples were dispersed in ¢ final sample buffer [23], h e a t e d for 2 rain at 100°C a n d subjected to sodium dodeeyl sulfate p o l y a e r y l a m i d e gel eleetrophoresis (SDS-PAGE) [23] in o r d e r to separate labeled proteins. The r a d i o a c t i v i t y in 35S per slot was a d j u s t e d to be the same in each e x p e r i m e n t . After eleetrophoresis, gels were dried a n d placed in contact w i t h Kodirex X-ray films. On the a u t o r a d i o g r a m s , the optical density of the b a n d of interest was m e a s u r e d w i t h a double b e a m a u t o m a t i c recording m i e r o d e n s i t o m e t e r (Joyee-Loebl a n d Co.). U n d e r o u r e x p e r i m e n t a l conditions a l i n e a r r e l a t i o n s h i p w a s observed between t h e n u m b e r of 85S counts in RecA p r o t e i n (expressed b y the t r a n s d u c i n g phage )~precA÷) a n d the optical density of corresponding b a n d on a u t o r a d i o g r a m s . Thus, w h e n t h e cellular p r o t e i n s were pulse-labeled w i t h [35S] m e t h i o n i n e , the optieal density of the ReeA b a n d on a u t o r a d i o g r a m s was a m e a s u r e of the rate of synthesis of ReeA p r o t e i n at the t i m e of the pulse. As for the optical d e n s i t y of ). r e p r e s s o r b a n d , it eorrespond.ed to t h e a m o u n t of )~ repressor r e m a i n i n g in the lysogens at a given time of i n c u b a t i o n .
Materials and Methods. Results. Strains and growth conditions. Escheriehia colt K12 C600 0,) and C600 (Xind-) lysogens were grown in, unless otherwise specified, e n r i c h e d YM9 m e d i u m eont a i n i n g per l i t e r of w a t e r : Na2I-IPOo 7.0 g ; KI-I~P04, 3.0 g ; NH4C1, 1.0 g ; NaC], 5.0 g ; MgCl.o 0.5 m m o l ; CaCti, 0.1 mmol ; MgSO4, 1 m M ; glucose, 2.0 g ; t h i a mine, 1 m g ; 14 essential L-amino acids, 50 m g each, t h e s u l f u r e o n t a i n i n g a m i n o acids b e i n g excluded. Low s u l f u r m e d i u m w a s b a s i c a l l y enriched YM9 m e d i u m w i t h o n l y 50 ~tM MgSO~. All cultures were grown or i n c u b a t e d w i t h drugs, at 37~C. The t u r b i d i t y of the c u l t u r e s was m o n i t o r e d w i t h a Lange p h o t o c o l o r i m e t e r ; a t u r b i d i t y v a l u e of 0.15 corresponded to a cell c o n c e n t r a t i o n of 2 × 108 b a c t e r i a per ml.
Chemicals. R i f a m p i e i n fRIF) (Boehringer) and chlora m p h e n i e o l (CAP) (Sigma) were dissolved in 0.4 per cent of d i m e t h y l sulfoxide (DMSO). Mitomycin C (MMC) (Sigma) was dlssolved in distilled water. L-[85S] m e t h i o n i n e (specific activity 5,0.0 C i / m m o l ) a n d carrierfree H235SO4 (specific activity 25 m C i / m l ) were o b t a i n e d f r o m New E n g l a n d Nuclear. Titration of RecA protein and of ~ reprcssor. RecA p r o t e i n was t i t r a t e d in whole cell e x t r a c t s as described b y Gndas ,~ Pardee [5]. RecA p r o t e i n was identified on a u t o r a d i o g r a m s b y c o m p a r i n g its m o b i l i t y to t h a t of ReeA protein m a d e b y t h e t r a n s d u c i n g phage ~precA ÷ [3] ; following infection of h e a v i l y UV-irradiated C600 (~inds) lysogens b y ~precA ÷, ReeA protein expressed b y the t r a n s d u c i n g phage was the only protein labeled d u r i n g i n c u b a t i o n w i t h [snS] m e t h i o n i n e . Cleavage of )~ repressor was m e a s u r e d b y i m m u n o precipitation e s s e n t i a l l y as described b y Roberts & Roberts [14] except t h a t the soluble complex ~ repress o r - a n t i b o d y (anti-)~ repressor s e r u m was a l w a y s in
BIOCHIMIE, 1980, 62, n ° 10.
1. R i f a m p i c i n decreases RecA protein synthesis induced by m i t o m y c i n C. M i t o m y c i n (MMC), a D!NA a l k y l a t i n g a n d c r o s s l i n k i n g a g e n t [241, i n h i b i t s DNA r e p l i c a t i o n ; it is a p o t e n t i n d u c e r of p r o p h a g e k d e v e l o p m e n t as w e l l as of R e c A p r o t e i n s y n t h e s i s . MMC t r e a t m e n t of E. colt is k n o w n to c a u s e i n d u c i n g e f f e c t s s i m i l a r to U V - i r r a d i a t i o n [9, 12, 14, 25]. R i f a m p i c i n ( R / F ) , at a h i g h c o n c e n t r a t i o n (80 ~ g / m l ) , i n h i b i t s R N A p o l y m e r a s e r a t h e r s p e c i f i c a l l y . Most n o t a b l y , R I F , at a l o w c o n c e n t r a t i o n (4 ~ g / m l ) , h a s b e e n r e p o r t e d to p r e v e n t s e l e c t i v e l y t h e s y n t h e s i s of RecA protein induced by nalidixic acid treatment
[20, 21]. F i r s t , w e c h e c k e d to see w h e t h e r o r n o t R I F at a c o n c e n t r a t i o n of 4 ~ g / m l p r e v e n t e d R e e A protein synthesis from being fully induced by MMC t r e a t m e n t . C600 ('~ind-) l y s o g e n s w e r e t h e r e fore incubated with RIF ~rst and, 5 min later, with MMC or, as a c o n t r o l , w i t h MMC a l o n e . At v a r i o u s t i m e s of i n c u b a t i o n , t h e c e l l u l a r p r o t e i n s w e r e p u l s e l a b e l e d f o r 3 m i n w i t h [35S l m e t h i o n i n e . T h e cellular proteins were separated by SDS-PAGE (fig. 1), a n d a u t o r a d i o g r a m s w e r e s u b j e c t e d to s c a n n i n g d e n s i t o m e t r y t o q u a n t i t a t e t h e r a t e of s y n t h e s i s of R e c A p r o t e i n (fig. 2). It is c l e a r t h a t i n d u c t i o n of R e c A p r o t e i n b y MMC t r e a t m e n t w a s g r e a t l y r e d u c e d b y t h e p r e s e n c e of R I F i n t h e
r e p r e s s o r cleavage.
PrecA synthesis and
689
b y R I F i n d e p e n d e n t l y of t h e p r e s e n c e of MMC (fig. 1) b u t w a s not a f f e c t e d b y C ~ P ( d a t a n o t shown).
i n c u b a t i o n m e d i u m (fig. 1). T h e r e l a t i v e r a t e of s y n t h e s i s of R e c A p r o t e i n i n c r e a s e d r o u g h l y l i n e a r l y f o r 3,0 m i n f r o m I to 4.'5 w i t h o u t R I F , a n d f r o m 1 to 1.5 w i t h R I F (fig. 2). At 30 m i n of i n c u bation, t h e r a t e of s y n t h e s i s of R e c A p r o t e i n i n d u c e d b y MMC w a s t h e r e f o r e r e d u c e d b y R I F by about 77 p e r cent. In c o n t r a s t , the o v e r a l l r a t e of s y n t h e s i s of c e l l u l a r p r o t e i n s w a s r e d u c e d o n l y by 10 p e r c e n t at t h e s a m e c o n c e n t r a t i o n of R I F (data not s h o w n ) .
2. I n d u c e d ~ lysogens lyse in the presence of RIF. Satta et el. [213 h a v e r e p o r t e d t h a t R I F at a c o n c e n t r a t i o n of 4 ~ g / m l r e d u c e d b a c t e r i o p h a g e P1 <> in E. colt B / r by l o w e r i n g t h e i n d u c e d s y n t h e s i s of R e c A p r o t e i n . In c o n t r a s t ,
m recA
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FxG. 1. - - Induction of ReeA protein synthesis. C600 O~ind-) lysogens grown in enriched YM9 medium up to 0.15 Lange units were incubated, or not, with 4 ~ g / m l RIF for 5 min before addition of 5 ~g/ml MMC. As a function of the time of incubation after the addition of MMC, 2@0-~1 samples were withdrawn and incubated further with 10 ~Ci/ml [35S] methionine for 3 rain and quickly frozen after the addition of cold YM9. Labeled cellular proteins were then analysed by SDS-PAGE (on a 12 per cent acrylamide gel) and autoradiography. The radioactivity in 35S per slot was adjusted to 10~ epm. Below each lane on the autoradiogram are indicated the times of incubation at which 3-min pulses were started. In the first slot at the left was an extract of heavily UV-irradiated C600, (kind') lysogens infected with phage )~precA+ and incubated with 10 ~Ci/ml [sss] methioninc for 30 min ; the sample contained 7 X 103 epm. The molecular weight of the ReeA protein was about 4~),000.
When chloramphenicol (CAP) ~vas u s e d at 1.4 ~ g / m l , a c o n c e n t r a t i o n t h a t m i m i c k e d R I F effects on c e l l g r o w t h a n d p r o t e i n s y n t h e s e s ( d a t a n o t s h o w n ) , it d i d n o t affect t h e i n d u c t i o n of R e c A p r o t e i n s y n t h e s i s b y MMC (fig. 2). T h e s y n t h e s i s of a n o t h e r p r o t e i n , n o t yet i d e n tified, of a p p r o x i m a t e l y 50 Kd, w a s also i n h i b i t e d
BIOCHIMIE,
1980,
62, n °
10.
w e o b s e r v e d full cell l y s i s of k l y s o g e n s i n c u b a t e d w i t h R,IF a n d MMC, C ~ P a n d MMC, o r nvith MMC a l o n e (fig. 3). A l t h o u g h in t h e first c u l t u r e ( R I F + MMC) cell lysis w a s s l i g h t l y d e l a y e d a n d t h e final p h a g e t i t e r w a s 2.5-fold l o w e r t h a n in t h e c o n t r o l c u l t u r e , t h e r e is n o d o u b t t h a t p r o p h a g e k w a s fully i n d u c e d in all t h e t r e a t e d lysogens.
P. L. Moreau a n d coll.
690
T o a c c o u n t f o r t h e effect of R I F o n p h a g e k production, we must consider two alternative m e c h a n i s m s . E i t h e r t h e i n a c t i v a t i o n of k r e p r e s s o r w a s d e l a y e d a n d , c o n s e q u e n t l y , t h e o n s e t of p h a g e
-
incubated
f o r d i f f e r e n t t i m e s w i t h MMC.
Samples were t h e n lysed a n d labeled k repressor was titrated i n the crude cell extracts (figs 4 a n d 5). F i g u r e 5 s h o w s t h a t t h e d e c a y of k r e p r e s s o r was deIayed by approximately 3 rain when the l y s o g e n s w e r e i n c u b a t e d w i t h R I F a n d MMC, as c o m p a r e d t o l y s o g e n s i n c u b a t e d w i t h MMC a l o n e . When the lysogens were incubated with CAP and MMC, t h e d e c a y of k r e p r e s s o r w a s a l i t t l e m o r e rapid than in the control culture. In any case, a ,5 t o 10 r a i n l a g p e r i o d p r e c e d e d t h e d e c a y of k r e p r e s s o r a n d m o r e t h a n 90 p e r c e n t of k r e p r e s s o r ~vas d e s t r o y e d b y 40 m i n of i n c u b a t i o n .
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0
10 20 3 0 . 4 0 50 60 minutes Fla. 2. - - Time course of induction of RecA protein
synthesis. C600 (kind-) lysogens were pulse-labeled w i t h lasS] m e t h i o n i n e (as described in fig. 1) as a f u n c t i o n of the time of i n c u b a t i o n w i t h RIF a n d M'~C (A)., CAP a n d MM~C (V), D~SO a n d MMC (O), or MMC alone (O). The relative rate of s y n t h e s i s of RecA p r o t e i n w a s calculated as the r a t i o of the optical density (on a u t o r a d i o g r a m s ) of the b a n d at a given t i m e of i n c u b a t i o n with MM,C over t h a t observed before MMC addition. The b a s a l rate of RecA p r o t e i n s y n t h e s i s was not significantly different w h e t h e r the b a c t e r i a were prei n c u b a t e d or not w i t h RIF or CAP.
d e v e l o p m e n t , o r t h e i n a c t i v a t i o n of k r e p r e s s o r was complete but not the repair of the MMC-damaged prophage whose replication, and therefore maturation into progeny phage might have been hampered.
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0.05-
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minutes FIO. 3. - - Cell lysis produced by phage "h deoelopment. G600 0,) lysogens were g r o w n in enriched YM9 med i u m up to 0.15 Lunge units. The culture was split into 4 samples : (a) was t r e a t e d w i t h 4 ~ g / m l RIF, (b) w i t h 1.4 I~g/ml CAP, (c) a n d (d) were left u n t r e a t e d . The f o u r samples were i n c u b a t e d for 5 m i n a n d t h e n 5 ~ g / m l MMC were added (arrow) to (a), (b) a n d (e), b u t not to (d). At v a r i o u s t i m e s a f t e r MMC a d d i t i o n (zero time), the t u r b i d i t y of the cultures was m e a s u r e d (values h i g h e r t h a n 0.3 Lange u n i t s are u n d e r e s t i mated). After i n c u b a t i o n for 180 min, the t i t e r of phage released is indicated in p a r e n t h e s e s on the graph, a : RIF + MM,C ( A ) ; b : CAP + M M C ( V ) ; c : MMC (0) ; d : u n t r e a t e d (X).
3. R I F does n o t a f f e c t the rate of cleavage
of ~ repressor. To differentiate b e t w e e n the two m e c h a n i s m s e n u n c i a t e d above, w e m e a s u r e d the time-course of i n a c t i v a t i o n b y cleavage of k r e p r e s s o r [14]. C600 0,) lysogens w e r e first i n c u b a t e d for 70 rain (time for 1.5 cellular divisions) w i t h H 2 35504 ; after a 5-min chase b y cold MgSO 4, in the p r e s e n c e or the absence of R I F or CAP, the lysogens were BIOCHIMIE, 1980, 62, n ° 10.
U p o n i n c u b a t i o n of l y s o g e n s w i t h MMC, i n t h e p r e s e n c e o r t h e a b s e n c e of R I F (fig. 4) o r C A P ( d a t a n o t s h o w n ) , t h e k r e p r e s s o r m o n o m e r (R) was transformed into characteristic cleavage fragments (R'), which are approximately one-half the size of k r e p r e s s o r [14, 26]. T h e a m o u n t of R' f r a g m e n t s i n c r e a s e d f r o m 10 to 30 r a i n i n c u b a t i o n time and declined rapidly thereafter. Note that
PrecA synthesis and ~ repressor cleavage. t h e a m o u n t of g r a m s (fig. 4) sence of RIF. lysogens with
c a n r e a c h 3 p e r c e n t of t h e t o t a l o f t h e c e l l u l a r p r o t e i n s a f t e r 5,0 r a i n o f i n c u b a t i o n [5]. S u c h t r e a t m e n t s i n d u c e also t h e s o - c a l l e d SOS f u n c t i o n s , s u c h as c e l l f i l a m e n t a t i o n , i n h i b i t i t o n o f DNA
R ' f r a g m e n t s s e e n on t h e a u t o r a d i o was slightly increased by the preA f t e r 10 m i n of i n c u b a t i o n o f t h e R I F a n d MMC, R' f r a g m e n t s w e r e
mitt M M C
0
10
--
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20
30
40
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+
+
691
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0 --
10 2 0 3 0 4 0 4 0 +
+
+
+
-
RIF
Fro. 4. - - Cleavage of ~ repressor. C600 00 lysogens were grown in low sulfur m e d i u m containing 0.4 m C i / m l HeaSSO~ f r o m 0.05 to 0.15 Lange units. The culture was then split into two samples : one h a l f was treated with RIF and 10 mM cold M'gSO4, to the other h a l f cold MgS04 was added. The two cultures were incubated for a f u r t h e r 5 min, centrifuged and t h e n resuspended in w a r m enriched YM9 m e d i u m containing RIF and MMC, or MMC alone, as indicated on the autoradiogram. As a function of the time of incubation w i t h MMC, 1-ml samples were removed and processed for )~ repressor precipitation by an a n t i - r e p r e s s o r serum. Purified labeled p r o t e i n s were analysed by SDS-PAGE (on a 15 per cent acrylamide gel) and autoradiography. The radioactivity in 85S per slot was a d j u s t e d to 6 × 103 cpm. Below each lane on the autoradiogram arc indicated the t i m e s at which the samples were removed and quickly frozen. The molecular weight of the ~. rcpressor m o n o m e r (R) and of its f r a g m e n t (R') were ahout 27,000 and 14,000 respectively.
m o r e a b u n d a n t t h a n w i t h o u t R~F, a n d t h e i r d i s a p p e a r a n c e w a s also d e l a y e d ; n o s i m i l a r a f f e c t w a s o b s e r v e d i n t h e p r e s e n c e of CAP ( d a t a n o t s h o w n ) .
Discussion. U p o n t r e a t m e n t of E. colt w i t h D N A d a m a g i n g a g e n t s , t h e s y n t h e s i s of R e c A p r o t e i n is i n d u c e d [4, 5, 9, 27] ; t h e c o n e e n t r a t i o n of R e c A p r o t e i n
BIOCHIMIE, 1980, 62, n ~ 10.
degradation and prophage s i o n of w h i c h a p p e a r s to t r o l l e d [21. D o e s t h i s m e a n R e c A p r o t e i n is r e q u i r e d to t i o n s as h a s b e e n p r o p o s e d 16, 20, 211 ?
induction, the expresbe coordinately cont h a t full i n d u c t i o n o f p r o m o t e all SOS f u n c b y m a n y a u t h o r s [6, 7,
W e s h o w in t h i s p u b l i c a t i o n t h a t t h i s is n o t so f o r i n d u c t i o n of p r o p h a g e k. I n d e e d , i n MMCt r e a t e d l y s o g e n s , e v e n t h o u g h t h e r a t e of s y n t h e s i s of RecA p r o t e i n was quite specifically r e d u c e d by a l o w c o n c e n t r a t i o n of R I F , i n d u c t i o n of p r o p h a g e ), o c c u r r e d r e a d i l y as a s s e s s e d b y : (i) cell
P. L. M o r e a u a n d coll.
692
lysis of the lysogens, (it) p r o d u c t i o n of p r o g e n y phage, and (iii) e x t e n s i v e c l e a v a g e of ~ repressor. In p a r t i c u l a r , the rate and the extent of cleavage of X r e p r e s s o r w e r e not significantly affected by the d e c r e a s e d rate of synthesis of RecA p r o t e i n
I 0 0 '_-~ .-a
7"V~X
N o t w i t h s t a n d i n g , it is c l e a r that c l e a v a g e of r e p r e s s o r , w h i c h is t h o u g h t to be t h e t e r m i n a l c e l l u l a r event l e a d i n g to p r o p h a g e )~ i n d u c t i o n , did not r e q u i r e full i n d u c t i o n of ReeA p r o t e i n .
0'3
[ a.) L.
~
10
e-.-
._~ --
A k..)
I
i
i
I
I
0 10 20 30 40 minutes FIG. 5. Time coarse of cleavage of )~ repressor. C600 00 bacteria were incubated as described in figure 4 with R~F and MMIC (A), CAP and M.MG , V', or with I~M!Calone (0). The amount of )~ repressor was measured on autoradiograms by densitometry of the repressor (R) band. The plotted values on the ordinate are expressed in percentage of )~ repressor in the control culture (RIF and GAP free) just before MMC addit i o n ; they represent the mean of two experiments. On the abscissa is indicated the time of incubation with MMC. -
-
resulting from R I F action. T h e lag p e r i o d before the d e c a y of )~ r e p r e s s o r was, h o w e v e r , slightly e x t e n d e d in the p r e s e n c e of R I F (fig. 5) and the d i s a p p e a r a n c e of ), r e p r e s s o r f r a g m e n t s (R') was slow (fig. 4) ; in contrast, the t i m e course of prod u c t i o n of R' r e p r e s s o r f r a g m e n t s w a s r a t h e r accel e r a t e d by R I F (fig. 4). T h e a p p a r e n t d i s c r e p a n c y b e t w e e n the t w o results m i g h t reflect the c o m p l e x c e l l u l a r effects of R I F at low c o n c e n t r a t i o n . It has been s h o w n that R I F at low c o n c e n t r a t i o n can stim u l a t e the t r a n s c r i p t i o n of some o p e r o n s and r e d u c e that of others s e l e c t i v e l y [28, 291. In our e x p e r i m e n t s , w e m i g h t h a v e o b s e r v e d the net result of v a r i o u s effects of RIF. R I F c l e a r l y reduced the i n d u c e d s y n t h e s i s of RecA p r o t e i n but might have also d e l a y e d the d e g r a d a t i o n of ),
BIOCHIMIE, 1980, 62, n ° 10.
r e p r e s s o r f r a g m e n t s , w h i c h is n o r m a l l y o c c u r r i n g r a p i d l y in g r o w i n g cells [30]. A l t e r n a t i v e l y , R I F at l o w c o n c e n t r a t i o n m i g h t have a l l o w e d a h i g h e r synthesis of X r e p r e s s o r d u r i n g t h e 5-min chase p e r i o d before t h e a d d i t i o n of MMC, w h e r e a s CAP at l o w c o n c e n t r a t i o n m i g h t h a v e i n h i b i t e d the synthesis of k r e p r e s s o r , a n d t h e r e f o r e a c c e l e r a t e d the cleavage of k r e p r e s s o r (fig. 5) and thus i n d u c tion of p r o p h a g e )~ (fig. 3).
W e do not c l a i m that the basal level of RecA p r o t e i n is sufficient to c l e a v e )~ r e p r e s s o r s i n c e R I F did not c o m p l e t e l y abolish the i n d u c t i o n of RecA p r o t e i n synthesis. Moreover, our data do not e x c l u d e the p o s s i b i l i t y that a b a c k g r o u n d of cellular p r o t e i n s o t h e r t h a n RecA (of s i m i l a r m o l e c u l a r w e i g h t ) m i g h t h a v e led to u n d e r e s t i m a t e the i n c r e a s e in t h e r a t e of synthesis of RecA p r o t e i n . This is all the m o r e possible as the t i t r a t i o n of RecA p r o t e i n ~vas not p e r f o r m e d w i t h a techn i q u e as specific as that used for X r e p r e s s o r titration. Since RecA m o l e c u l e s are stable for at least I h [4, 33], the a c c u m u l a t i o n of RecA p r o t e i n induced by MMC m i g h t h a v e been u n d e r e s t i m a t e d in the p r e s e n c e of RIF. F u r t h e r m o r e , it is k n o w n that (i) de novo protein synthesis is r e q u i r e d for cleavage of )~ r e p r e s sor in MMC-induced lysogens [31], (it) lexA mutants, in w h i c h RecA p r o t e i n synthesis is p o o r l y i n d u c e d after DNA damage [32!, do not express SOS f u n c t i o n s [2], and (iii) the b e g i n n i n g of ~ r e p r e s s o r b r e a k d o w n is p r e c e d e d by a lag p e r i o d [12, see also fig. 5]. These facts and our results taken t o g e t h e r m a y simply m e a n that a small a c c u m u l a t i o n of RecA p r o t e i n is n e c e s s a r y to obtain full cleavage of k repressor. It m a y also be that the cellular events p r e c e d i n g the d e c a y of ~ r e p r e s s o r ore related to the occurr e n c e of DNA r e p a i r processes, w h i c h m a y lead to the a c t i v a t i o n of RecA p r o t e i n to cleave )~ r e p r e s sor [17]. T h a t RecA p r o t e i n must be a c t i v a t e d for the c l e a v a g e r e a c t i o n to o c c u r is s u p p o r t e d by v a r i o u s g e n e t i c and b i o c h e m i c a l data : (i) Although RecA p r o t e i n is p r o d u c e d at a high cellular c o n c e n t r a t i o n as a result of m u l t i p l e lexA m u t a t i o n s (lexA3 spr-51 or lexA3 tsl-1) or of amplification due to a plasmid, i n d u c t i o n of prop h a g e ), does not o c c u r constitutively, lhat is, ill
PrecA
synthesis
and
the a b s e n c e of DNA d a m a g e [6, 15, 34, 35]. Moreover, in some b a c t e r i a l mutants, such as i n f A , DNA damage i n d u c e s RecA p r o t e i n synthesis, w h e r e a s there is no p r o p h a g e ), i n d u c t i o n [9, 36, L. J., Gudas, pers. c o m m . ] . (it) D e n o v o RNA a n d p r o t e i n syntheses are r e q u i r e d in o r d e r for )~ r e p r e s s o r to be i n a c t i v a t e d in a ti[-1 m u t a n t at 42°C w h e r e a s the a m p l i f i c a t i o n of RecA p r o t e i n m o d i f i e d by the t i f m u t a t i o n [68] is not n e e d e d [~7]. Thus, in the case of the ti[ mutant, the a c t i v a t i o n only of the (RecA) Tif protein seems sufficient for p r o p h a g e k i n d u c t i o n . H o w e v e r , note that : (1) the ti[ mutant synthesizes RecA p r o t e i n at a faster rate than t h e w i l d type strain even at 30°C [3~], (2) purified T i f p r o t e i n is c o n s i d e r a b l y m o r e active t h a n the RecA + p r o t e i n as r e g a r d s cleavage of ), r e p r e s s o r [16]. W h a t applies to the ti[-1 m u t a n d does not a p p l y necessarily to w i l d t y p e b a c t e r i a c o n s i d e r e d in our experiments. (iii) Cleavage of ), r e p r e s s o r r e q u i r e s , a p a r t f r o m the p u r i f i e d ( R e e A ) T i f p r o t e i n , s i n g l e - s t r a n d e d DNA a n d also A T P [17]. If a c t i v a t i o n of R e c A p r o t e i n is r e q u i r e d for cleavage of p h a g e k r e p r e s s o r , our results i n d i c a t e that only a small (if any) i n c r e a s e o v e r the b a c k g r o u n d l e v e l of RecA p r o t e i n is n e c e s s a r y to p r o m o t e total ), r e p r e s s o r cleavage. T h i s m a y be i n t e r p r e t e d as i n d i c a t i n g that the m o l e c u l a r signals that cleaves ~ r e p r e s s o r , the m o l e c u l a r signals activating ReeA protein, whatever their nature [17], are p r o d u c e d in l i m i t e d a m o u n t s after a DNA d a m a g i n g treatment. This h y p o t h e s i s can a c c o u n t in a s i m p l e w a y for the a p p a r e n t paradox that t h o u s a n d s of ReeA m o l e c u l e s must take 40 rain (about a g e n e r a t i o n time) to cleave a hund r e d or so m o l e c u l e s of k r e p r e s s o r in an i n d u c e d k lysogen. A f r a c t i o n only of i n d u c e d RecA molecules m a y act as a p r o t e a s e [13, 34], in s u c h a w a y that a r e d u c t i o n in the total level of ReeA p r o t e i n w i l l not affect p r o p h a g e ), i n d u c t i o n , even t h o u g h o t h e r c e l l u l a r p r o c e s s e s , s u c h as DN,A r e p a i r , m a y then be i n h i b i t e d . A l t e r n a t i v e l y , in an i n d u c e d lysogen, t h e r e m a y be an a b u n d a n c e of active RecA p r o t e i n but a l i m i t e d a m o u n t of ), r e p r e s s o r susceptible to cleavage at a given time. E x p e r i m e n t s p e r f o r m e d in o u r l a b o r a t o r y h a v e s h o w n that u p o n UV-irradial i o n of k lysogens, the rate of i n a c t i v a t i o n of r e p r e s s o r did not c h a n g e in spite of a 5-fold i n c r e a s e due to a p l a s m i d in the c e l l u l a r level of k r e p r e s s o r ; t h e i n a c t i v a t i o n system, h o w e v e r , d i d not a p p e a r to be s a t u r a t e d [13]. One i n t e r p r e t a t i o n of this result is that t h e r e are t w o f o r m s of )~
BIOCHIMIE, 1980, 62, n o 10.
)~ r e p r e s s o r
cleavage.
693
repressor, cleavable and non cleavable, and that t h e r e is a d y n a m i c e q u i l i b r i u m b e t w e e n these t w o forms !113, J. W. Roberts, pers. c o m m . ] . T h e speed of g e n e r a t i o n of c l e a v a b l e (e.g., m o n o m e r i c ) f o r m s d r a w n f r o m a pool of non c l e a v a b l e (e.g., o l i g o m e r i c [26]) forms of k r e p r e s s o r m a y be t h e l i m i t i n g factor of the cleavage r e a c t i o n a n d m a y r e n d e r it i n d e p e n d e n t of the cellular level of RecA p r o t e i n w i t h i n a r e l a t i v e l y b r o a d range. Finally, we cannot rule out the p o s s i b i l i t y that RecA p r o t e i n might not be itself the r e p r e s s o r c l e a v i n g e n z y m e but m i g h t be the r e q u i r e m e n t for a n o t h e r u n k n o w n i n d u c i b l e p r o t e a s e to cleave k r e p r e s s o r . The p r o t e a s e m i g h t be p r e s e n t in t r a c e a m o u n t s associated w i t h purified RecA p r o t e i n I17]. W h a t e v e r the exact role or f u n c t i o n of RecA p r o t e i n in p r o p h a g e )~ i n d u c t i o n is, w h e n t h e cleavage of ), r e p r e s s o r starts, t h e r e is no a p p a r e n t c o r r e l a t i o n b e t w e e n the rate of c l e a v a g e of k r e p r e s s o r and the rate of synthesis of RecA p r o tein. Recently, a lack of c o r r e l a t i o n has also been o b s e r v e d b e t w e e n the synthesis of RecA p r o t e i n and a n o t h e r SOS function, n a m e l y t h e i n h i b i t i o n of cell d i v i s i o n [33]. Note added in p r o o f : While this paper was in press, a paper by Balueh et al. (198ff) was published whose title is : Induction of prophage )~without amplification of ReeA protein, Mol. Gen. Genet., 178, 317-323.
Acknowledgements.
W e are grateful to M. K a m i n s k i and P. Liacopoulos f o r their help in the preparation of the anli-repressor sera. W e t h a n k D. P a n t a l o n i f o r advice, M. O. Mossd for assistance w i t h the d e n s i t o m e t e r and J. Nappd f o r typing the manuscript. This work was aided by grants f r o m E u r a t o m and Fondation pour la Recherche M~dicale.
REFERENCES. 1. Clark, A. J. ,~ Margulies, A. D. (1965) Proc. Natl. Acad. Sci. U.S.A., 53, 451-459. 2. Witkin, E. M. (19,76) Bacteriol. Bey., 40, 869-907. 3. McEntee, K., Hesse, J. E. ~ Epstein, W. (1976) Proc. Natl. Acad. Sci. U.S.A., 73, 3979-3983. 4. Inouye, M. ,~ Pardee, A. B. (1970) J. Biol. Chem., 245, 5813-58:19. 5. Gudas, L. J. ~ Pardee, A. B. (1976) J. Mol. Biol., 1Ol, 459-477. 6. MeEntee, K. (1977) Proc. Natl. Acad. Sci. U.S.A., 74, 52,75-5279. 7. Gudas, L. J. • Mount, D. W. (1977) Proc. Natl. Acad. Sci. U.S.A., 74, 52~,0~52~84. 8. Emmerson, P. T. ~ West, S. C. (1977) Mol. Gen. Genet., 155, 77-85. 9. West, S. C. ,~ Emmerson, P. T. (197'7) Mol. Gen. Genet., 151, 57-67.
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10. Ptashne, M., Backman, K., H u m a y u n , M. Z., Jeffrey, A., Maurer, R., Meyer, B. & Sauer, R. T. (1976) Science, 194, 156-161. 11. Levine, A., Bailone, A. & Devoret, R. (1979) J. Mol. Biol., 131, 655-661. I2. Shinagawa, It. & Itoh, T. (1973) Mol. Gen. Genet., 126, 103-110. 13. Bailone, A., Levine, A. & Devoret, R. (1979) J. Mol. Biol., 131, 553-572. 14. Roberts, J. V¢. & Roberts, C. W. (1975) Proc. Natl. Acad. Sei. U~S.A., 72, 147-151. 15. Roberts, J. W., Roberts, C. W. & Mount, D. W. (1977) Proc. Natl. Acad. Sci. U.S.A., 74, 2283-2287. 16. Roberts, J. W., Roberts, C. W. ~ Craig, N. L. (1978) Proc. Natl. Acad. Sci. U.S.A., 75, 4714-4718. 17. Craig, N. L. & Roberts, J. W. (1980~) Nature, 283, 2,6-30. 18. Morand, P., Goze, A. ~ Devoret, R. (1977) Mol. Gen. Genet., 157, 69-82. 19. Castellazzi, M., Morand, P., George, J. & Buttin, G. (1977) Mol. Gen. Genet., 153, 297-310. 20. S,atta, G. ,~ Pardee, A. B. (1978) J. Bacteriol., 133, 1492-15~0. 21. Satta, G., Gndas, L. J. & Pardee, A. B. (1979) Mol. Gen. Genet., 168, 69-80. 22. Schwyzer, M. (19'77) in ¢ Early proteins of oncogenic DNA viruses >>, 63-68, INSERM Syrup. vol. 69. 23. Lacmmli, U. K. & Favre, M. (1973) J. Mol. Biol., 80, 575°599.
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24. Szybalski, W. & Iyer, V. M. (1967) in Antibiotics I. Mechanisms of action. (Gottlieb, S. & Shaw, P. D. eds), 2,10-245. Springer Verlag, New York. 25. Tomizawa, J. I. & Ogawa, T. (1967) J. Mol. Biol., 23, 247-2,63. 26. Pabo, C. O., Sauer, R. T., S~urtevant, J. M. & Ptashne, M. (1979) Proc. Natl. Acad. Sci. U.S.A., 76, 16081612. 27. Little, J. W., Edmiston, S. H., Pacelli, L. Z. & Mount, D. W. (1980) Proc. Natl. Acad. Sei. U S . A , 77, 32.25-3229. 28. Blumenthal, R. M. & Dennis, P. P. (1978) Mol. Gen. Genet., 165, 79-86. 29. Bass, I. A., Danilevskaya, O. N., Mekhedov, S. L , Fedoseeva, V. B. & Gorlenko, Z. M. (1979) Mot. Gen. Genet., 173, 104-107. 30. Goldberg, A. L. & John, A. C. (1976) Ann. Rev. Biochem., 4,5, 747-803. 31. Shinagawa, H., Mizuuchi, K. a E m m e r s o n , P. (1977) Mol. Gen. Genet., 156, 87-91. 32. Gndas, L. J. (1976) J. Mol. Biol., 104, 567-587. 33. Darby, V. ~ Holland', I. B. (1979) Mol. Gen. Genet., 176, 1~1-1,23. 34. Ogawa, T., Wabiko, H., Tsurimo[o, T., Horii, T., Masukata, H. & Ogawa, H. (1978) Cold Spring Hath. Syrup. quant. Biol., 43, 9~)9-915. 35. Either, G., Adler, B. & Frank, P. (1980) Stud. Biophys., 76, 71. 36. Bailone, A., Blanco, M. a Devoret, R. (1975) Mol. Gem Genet., 136, 291-3#7. 37. Maenhaut-Michel, G., Brandenburger, A. a Boiteux, S. (1978) Mol. Gen. Genet., 163, 293-29,9.
Induction of prophage ), does not require full induction of RecA protein synthesis ('). Patrice L. MOREAU, Mich61e FANICA and R a y m o n d DEVORET <>. (Re~a le 25-2-1980. Accept~ apr~s r e m a n i e m e n t le 28-~-1980).
Radiobioloffie Cellulaire, Laboratoire d'Enzymologie, C.N.R.S., 91190 Gi[-sur-Yvette, France.
Mots cl~s : prot6ine ReeA ; induction lysog~nique.
Key words : RecA protein ; ), repressor cleavage ; rifampiein ; mitomycin C.
R~sum6.
Summary.
Les bact6ries 1ysogbnes Escherichia coli K12 (X) sont induites par la mitomycine C alors que la vitesse de synthbse de la prot6ine RecA d'E. coli est s p ~ i f i q u e m e n t r~duite par une faible concentration de rifampicine (4 ~tg/ml). L'induction du prophage X a pu ~tre raise en 6vidence par la Iyse des bact6ries lysog~nes, par la production de phages ]ibres et par la destruction du r6presseur du prophage ~. La vitesse avec laquelle se produit le clivage du r6presseur et l'efficacit6 de ce clivaqe sont pratiquement inchang6es bien que la d6r6pression de la synth~se de la prot6ine RecA soit fortement inhib6e. Toutefois, la production de phages libres est diminu6e en pr6sence de rifampicine et la lyse des cellu|es un peu retard6e. On peut donc conclure de ces r6sultats que ches des bact~ries de type s a u v a g e (RecA÷), l'induction du prophage ~ n'exige pas pour se produire la d6r6pression complete de la synth~se de la prot6ine RecA.
In mitomycin C-treated ~ 1ysogens. even though the rate of synthesis of Reck protein w a s greatly reduced b y a l o w c o n c e n t r a t i o n of
Introduction. The R e c A gene in E s c h e r i c h i a colt controls genetic recombination [1] and various cellular responses to DNA damage, the so-called (( SOS phenomena >> ~2]. Not only do recA mutations affect recombination proficiency but lhey also prevent
* This paper is dedicated to the memory of Jacqueline George. © To whom all correspondence should be addressed.
rifcunpicin (4 ~tg/ml). i n d u c t i o n of p r o p h a q e
occurred readily as assessed b y (i) ceil lysis of the l y s o g e n s , (ii) p r o d u c t i o n of p r o g e n y p h a g e , a n d (iii) e x t e n s i v e c l e a v a g e of ~ r e p r e s s e r . T h e extent a n d the rate of c l e a v a g e of ~ repressor were not s i g n i f i c a n t l y a f f e c t e d b y the l o w rate of s y n t h e s i s of R e c A p r o t e i n r e s u l t i n g f r o m ri. f a m p i c i n action. H o w e v e r , the y i e l d of p h a g e p r o g e n y w a s r e d u c e d a n d Iysis of the cells w a s sliqhtly d e l a y e d . W e c o n c l u d e that in RecA + b a c t e r i a , i n d u c t i o n of p r o p h a g e ~ d o e s not r e q u i r e full i n d u c t i o n of R e c A p r o t e i n syn-
thesis.
inducible error-prone repair (SOS repair), cell filamentation and p r o p h a g e ), induction from o c c u r r i n g [2]. The p r o d u c t of the recA gene [3] was first identified as protein X [4-8], whose rate of synthesis increases sharply when SOS functions are provoked [4, 5, 9~. Induction of prophage ), is one of the most dramatic cellular responses to DNA d a m a g e ; its m e c h a n i s m is presently the most amenable to molecular investigation. In a )~ lysogen, p r o p h a g e )~ is maintained in a dormant state by a repressor protein, the p r o d u c t of the p r o p h a g e cl gene [101.
688
P. L. Moreau and coll.
I n a m o n o l y s o g e n , w h e n all t h e )~ r e p r e s s o r m o l e c u l e s , p r e s e n t at a c o n c e n t r a t i o n of a b o u t 140 c o p i e s [11], a r e i n a c t i v a t e d [12, 13], a n d c l e a v e d i n t o t w o a l m o s t e q u a l f r a g m e n t s [14], p r o p h a g e ), is i n d u c e d t o d e v e l o p v e g e t a t i v e l y . P r o t e o l y t i c c l e a v a g e of ), r e p r e s s o r o c c u r s a l s o in vitro w i t h p u r i f i e d R e c A p r o t e i n , it also r e q u i r e s A T P a n d s i n g l e - s t r a n d e d p o l y n u c l e o t i d e m o l e c u l e s [15-17]. G e n e t i c d a t a [13, 18, 19] s u p p o r t t h e i d e a t h a t , in oivo, t h e i n a c t i v a t i o n of ), r e p r e s s o r r e s u l t s f r o m t h e e x p r e s s i o n of a RecA g e n e f u n c t i o n . It h a s b e e n w i d e l y
s u r m i s e d t h a t i n d u c t i o n of induction, as it m a y b e f o r i n d u c t i o n of o t h e r c e l l u l a r r e s p o n s e s t r i g g e r e d b y D N A d a m a g e , s u c h as cell f i l a m e n t a t i o n , i n h i b i t i o n of D N A d e g r a d a t i o n , o r i n d u c i b l e e r r o r - p r o n e r e p a i r [6, 7, 16, 20, 21]. W e report here evidence that full induction of ReeA p r o t e i n is n o t r e q u i r e d f o r i n d u c t i o n of p r o p h a g e ), i n w i l d - t y p e l y s o g e n s .
RecA p r o t e i n is r e q u i r e d f o r p r o p h a g e
excess) was not p r e c i p i t a t e d by a d d i t i o n of a second a n t i s e r u m b u t e h r o m a t o g r a p h e d on columns of protein A-Sepharose CL-4B ( P h a r m a e i a ) [22]. Samples were dispersed in ¢ final sample buffer [23], h e a t e d for 2 rain at 100°C a n d subjected to sodium dodeeyl sulfate p o l y a e r y l a m i d e gel eleetrophoresis (SDS-PAGE) [23] in o r d e r to separate labeled proteins. The r a d i o a c t i v i t y in 35S per slot was a d j u s t e d to be the same in each e x p e r i m e n t . After eleetrophoresis, gels were dried a n d placed in contact w i t h Kodirex X-ray films. On the a u t o r a d i o g r a m s , the optical density of the b a n d of interest was m e a s u r e d w i t h a double b e a m a u t o m a t i c recording m i e r o d e n s i t o m e t e r (Joyee-Loebl a n d Co.). U n d e r o u r e x p e r i m e n t a l conditions a l i n e a r r e l a t i o n s h i p w a s observed between t h e n u m b e r of 85S counts in RecA p r o t e i n (expressed b y the t r a n s d u c i n g phage )~precA÷) a n d the optical density of corresponding b a n d on a u t o r a d i o g r a m s . Thus, w h e n t h e cellular p r o t e i n s were pulse-labeled w i t h [35S] m e t h i o n i n e , the optieal density of the ReeA b a n d on a u t o r a d i o g r a m s was a m e a s u r e of the rate of synthesis of ReeA p r o t e i n at the t i m e of the pulse. As for the optical d e n s i t y of ). r e p r e s s o r b a n d , it eorrespond.ed to t h e a m o u n t of )~ repressor r e m a i n i n g in the lysogens at a given time of i n c u b a t i o n .
Materials and Methods. Results. Strains and growth conditions. Escheriehia colt K12 C600 0,) and C600 (Xind-) lysogens were grown in, unless otherwise specified, e n r i c h e d YM9 m e d i u m eont a i n i n g per l i t e r of w a t e r : Na2I-IPOo 7.0 g ; KI-I~P04, 3.0 g ; NH4C1, 1.0 g ; NaC], 5.0 g ; MgCl.o 0.5 m m o l ; CaCti, 0.1 mmol ; MgSO4, 1 m M ; glucose, 2.0 g ; t h i a mine, 1 m g ; 14 essential L-amino acids, 50 m g each, t h e s u l f u r e o n t a i n i n g a m i n o acids b e i n g excluded. Low s u l f u r m e d i u m w a s b a s i c a l l y enriched YM9 m e d i u m w i t h o n l y 50 ~tM MgSO~. All cultures were grown or i n c u b a t e d w i t h drugs, at 37~C. The t u r b i d i t y of the c u l t u r e s was m o n i t o r e d w i t h a Lange p h o t o c o l o r i m e t e r ; a t u r b i d i t y v a l u e of 0.15 corresponded to a cell c o n c e n t r a t i o n of 2 × 108 b a c t e r i a per ml.
Chemicals. R i f a m p i e i n fRIF) (Boehringer) and chlora m p h e n i e o l (CAP) (Sigma) were dissolved in 0.4 per cent of d i m e t h y l sulfoxide (DMSO). Mitomycin C (MMC) (Sigma) was dlssolved in distilled water. L-[85S] m e t h i o n i n e (specific activity 5,0.0 C i / m m o l ) a n d carrierfree H235SO4 (specific activity 25 m C i / m l ) were o b t a i n e d f r o m New E n g l a n d Nuclear. Titration of RecA protein and of ~ reprcssor. RecA p r o t e i n was t i t r a t e d in whole cell e x t r a c t s as described b y Gndas ,~ Pardee [5]. RecA p r o t e i n was identified on a u t o r a d i o g r a m s b y c o m p a r i n g its m o b i l i t y to t h a t of ReeA protein m a d e b y t h e t r a n s d u c i n g phage ~precA ÷ [3] ; following infection of h e a v i l y UV-irradiated C600 (~inds) lysogens b y ~precA ÷, ReeA protein expressed b y the t r a n s d u c i n g phage was the only protein labeled d u r i n g i n c u b a t i o n w i t h [snS] m e t h i o n i n e . Cleavage of )~ repressor was m e a s u r e d b y i m m u n o precipitation e s s e n t i a l l y as described b y Roberts & Roberts [14] except t h a t the soluble complex ~ repress o r - a n t i b o d y (anti-)~ repressor s e r u m was a l w a y s in
BIOCHIMIE, 1980, 62, n ° 10.
1. R i f a m p i c i n decreases RecA protein synthesis induced by m i t o m y c i n C. M i t o m y c i n (MMC), a D!NA a l k y l a t i n g a n d c r o s s l i n k i n g a g e n t [241, i n h i b i t s DNA r e p l i c a t i o n ; it is a p o t e n t i n d u c e r of p r o p h a g e k d e v e l o p m e n t as w e l l as of R e c A p r o t e i n s y n t h e s i s . MMC t r e a t m e n t of E. colt is k n o w n to c a u s e i n d u c i n g e f f e c t s s i m i l a r to U V - i r r a d i a t i o n [9, 12, 14, 25]. R i f a m p i c i n ( R / F ) , at a h i g h c o n c e n t r a t i o n (80 ~ g / m l ) , i n h i b i t s R N A p o l y m e r a s e r a t h e r s p e c i f i c a l l y . Most n o t a b l y , R I F , at a l o w c o n c e n t r a t i o n (4 ~ g / m l ) , h a s b e e n r e p o r t e d to p r e v e n t s e l e c t i v e l y t h e s y n t h e s i s of RecA protein induced by nalidixic acid treatment
[20, 21]. F i r s t , w e c h e c k e d to see w h e t h e r o r n o t R I F at a c o n c e n t r a t i o n of 4 ~ g / m l p r e v e n t e d R e e A protein synthesis from being fully induced by MMC t r e a t m e n t . C600 ('~ind-) l y s o g e n s w e r e t h e r e fore incubated with RIF ~rst and, 5 min later, with MMC or, as a c o n t r o l , w i t h MMC a l o n e . At v a r i o u s t i m e s of i n c u b a t i o n , t h e c e l l u l a r p r o t e i n s w e r e p u l s e l a b e l e d f o r 3 m i n w i t h [35S l m e t h i o n i n e . T h e cellular proteins were separated by SDS-PAGE (fig. 1), a n d a u t o r a d i o g r a m s w e r e s u b j e c t e d to s c a n n i n g d e n s i t o m e t r y t o q u a n t i t a t e t h e r a t e of s y n t h e s i s of R e c A p r o t e i n (fig. 2). It is c l e a r t h a t i n d u c t i o n of R e c A p r o t e i n b y MMC t r e a t m e n t w a s g r e a t l y r e d u c e d b y t h e p r e s e n c e of R I F i n t h e
r e p r e s s o r cleavage.
PrecA synthesis and
689
b y R I F i n d e p e n d e n t l y of t h e p r e s e n c e of MMC (fig. 1) b u t w a s not a f f e c t e d b y C ~ P ( d a t a n o t shown).
i n c u b a t i o n m e d i u m (fig. 1). T h e r e l a t i v e r a t e of s y n t h e s i s of R e c A p r o t e i n i n c r e a s e d r o u g h l y l i n e a r l y f o r 3,0 m i n f r o m I to 4.'5 w i t h o u t R I F , a n d f r o m 1 to 1.5 w i t h R I F (fig. 2). At 30 m i n of i n c u bation, t h e r a t e of s y n t h e s i s of R e c A p r o t e i n i n d u c e d b y MMC w a s t h e r e f o r e r e d u c e d b y R I F by about 77 p e r cent. In c o n t r a s t , the o v e r a l l r a t e of s y n t h e s i s of c e l l u l a r p r o t e i n s w a s r e d u c e d o n l y by 10 p e r c e n t at t h e s a m e c o n c e n t r a t i o n of R I F (data not s h o w n ) .
2. I n d u c e d ~ lysogens lyse in the presence of RIF. Satta et el. [213 h a v e r e p o r t e d t h a t R I F at a c o n c e n t r a t i o n of 4 ~ g / m l r e d u c e d b a c t e r i o p h a g e P1 <> in E. colt B / r by l o w e r i n g t h e i n d u c e d s y n t h e s i s of R e c A p r o t e i n . In c o n t r a s t ,
m recA
rain
0
10
20
30
40
40
0
10
20
30
40
40
MMC
--
÷
÷
÷
÷
--
--
4.
.Ik
41.
4t-
--
RIF
FxG. 1. - - Induction of ReeA protein synthesis. C600 O~ind-) lysogens grown in enriched YM9 medium up to 0.15 Lange units were incubated, or not, with 4 ~ g / m l RIF for 5 min before addition of 5 ~g/ml MMC. As a function of the time of incubation after the addition of MMC, 2@0-~1 samples were withdrawn and incubated further with 10 ~Ci/ml [35S] methionine for 3 rain and quickly frozen after the addition of cold YM9. Labeled cellular proteins were then analysed by SDS-PAGE (on a 12 per cent acrylamide gel) and autoradiography. The radioactivity in 35S per slot was adjusted to 10~ epm. Below each lane on the autoradiogram are indicated the times of incubation at which 3-min pulses were started. In the first slot at the left was an extract of heavily UV-irradiated C600, (kind') lysogens infected with phage )~precA+ and incubated with 10 ~Ci/ml [sss] methioninc for 30 min ; the sample contained 7 X 103 epm. The molecular weight of the ReeA protein was about 4~),000.
When chloramphenicol (CAP) ~vas u s e d at 1.4 ~ g / m l , a c o n c e n t r a t i o n t h a t m i m i c k e d R I F effects on c e l l g r o w t h a n d p r o t e i n s y n t h e s e s ( d a t a n o t s h o w n ) , it d i d n o t affect t h e i n d u c t i o n of R e c A p r o t e i n s y n t h e s i s b y MMC (fig. 2). T h e s y n t h e s i s of a n o t h e r p r o t e i n , n o t yet i d e n tified, of a p p r o x i m a t e l y 50 Kd, w a s also i n h i b i t e d
BIOCHIMIE,
1980,
62, n °
10.
w e o b s e r v e d full cell l y s i s of k l y s o g e n s i n c u b a t e d w i t h R,IF a n d MMC, C ~ P a n d MMC, o r nvith MMC a l o n e (fig. 3). A l t h o u g h in t h e first c u l t u r e ( R I F + MMC) cell lysis w a s s l i g h t l y d e l a y e d a n d t h e final p h a g e t i t e r w a s 2.5-fold l o w e r t h a n in t h e c o n t r o l c u l t u r e , t h e r e is n o d o u b t t h a t p r o p h a g e k w a s fully i n d u c e d in all t h e t r e a t e d lysogens.
P. L. Moreau a n d coll.
690
T o a c c o u n t f o r t h e effect of R I F o n p h a g e k production, we must consider two alternative m e c h a n i s m s . E i t h e r t h e i n a c t i v a t i o n of k r e p r e s s o r w a s d e l a y e d a n d , c o n s e q u e n t l y , t h e o n s e t of p h a g e
-
incubated
f o r d i f f e r e n t t i m e s w i t h MMC.
Samples were t h e n lysed a n d labeled k repressor was titrated i n the crude cell extracts (figs 4 a n d 5). F i g u r e 5 s h o w s t h a t t h e d e c a y of k r e p r e s s o r was deIayed by approximately 3 rain when the l y s o g e n s w e r e i n c u b a t e d w i t h R I F a n d MMC, as c o m p a r e d t o l y s o g e n s i n c u b a t e d w i t h MMC a l o n e . When the lysogens were incubated with CAP and MMC, t h e d e c a y of k r e p r e s s o r w a s a l i t t l e m o r e rapid than in the control culture. In any case, a ,5 t o 10 r a i n l a g p e r i o d p r e c e d e d t h e d e c a y of k r e p r e s s o r a n d m o r e t h a n 90 p e r c e n t of k r e p r e s s o r ~vas d e s t r o y e d b y 40 m i n of i n c u b a t i o n .
V
~5 ~4
further
•
o_
~3
pfulml
~2
,, 107
0.5 _
x.x..X..-x (t.3) x"
I
I
I
I
I
I
0
10 20 3 0 . 4 0 50 60 minutes Fla. 2. - - Time course of induction of RecA protein
synthesis. C600 (kind-) lysogens were pulse-labeled w i t h lasS] m e t h i o n i n e (as described in fig. 1) as a f u n c t i o n of the time of i n c u b a t i o n w i t h RIF a n d M'~C (A)., CAP a n d MM~C (V), D~SO a n d MMC (O), or MMC alone (O). The relative rate of s y n t h e s i s of RecA p r o t e i n w a s calculated as the r a t i o of the optical density (on a u t o r a d i o g r a m s ) of the b a n d at a given t i m e of i n c u b a t i o n with MM,C over t h a t observed before MMC addition. The b a s a l rate of RecA p r o t e i n s y n t h e s i s was not significantly different w h e t h e r the b a c t e r i a were prei n c u b a t e d or not w i t h RIF or CAP.
d e v e l o p m e n t , o r t h e i n a c t i v a t i o n of k r e p r e s s o r was complete but not the repair of the MMC-damaged prophage whose replication, and therefore maturation into progeny phage might have been hampered.
0.1 ./
\\\^(t,5oo) v A3.7oo)
"o-
0.05-
1(3,9001 I
I
I
0
60
120 180
minutes FIO. 3. - - Cell lysis produced by phage "h deoelopment. G600 0,) lysogens were g r o w n in enriched YM9 med i u m up to 0.15 Lunge units. The culture was split into 4 samples : (a) was t r e a t e d w i t h 4 ~ g / m l RIF, (b) w i t h 1.4 I~g/ml CAP, (c) a n d (d) were left u n t r e a t e d . The f o u r samples were i n c u b a t e d for 5 m i n a n d t h e n 5 ~ g / m l MMC were added (arrow) to (a), (b) a n d (e), b u t not to (d). At v a r i o u s t i m e s a f t e r MMC a d d i t i o n (zero time), the t u r b i d i t y of the cultures was m e a s u r e d (values h i g h e r t h a n 0.3 Lange u n i t s are u n d e r e s t i mated). After i n c u b a t i o n for 180 min, the t i t e r of phage released is indicated in p a r e n t h e s e s on the graph, a : RIF + MM,C ( A ) ; b : CAP + M M C ( V ) ; c : MMC (0) ; d : u n t r e a t e d (X).
3. R I F does n o t a f f e c t the rate of cleavage
of ~ repressor. To differentiate b e t w e e n the two m e c h a n i s m s e n u n c i a t e d above, w e m e a s u r e d the time-course of i n a c t i v a t i o n b y cleavage of k r e p r e s s o r [14]. C600 0,) lysogens w e r e first i n c u b a t e d for 70 rain (time for 1.5 cellular divisions) w i t h H 2 35504 ; after a 5-min chase b y cold MgSO 4, in the p r e s e n c e or the absence of R I F or CAP, the lysogens were BIOCHIMIE, 1980, 62, n ° 10.
U p o n i n c u b a t i o n of l y s o g e n s w i t h MMC, i n t h e p r e s e n c e o r t h e a b s e n c e of R I F (fig. 4) o r C A P ( d a t a n o t s h o w n ) , t h e k r e p r e s s o r m o n o m e r (R) was transformed into characteristic cleavage fragments (R'), which are approximately one-half the size of k r e p r e s s o r [14, 26]. T h e a m o u n t of R' f r a g m e n t s i n c r e a s e d f r o m 10 to 30 r a i n i n c u b a t i o n time and declined rapidly thereafter. Note that
PrecA synthesis and ~ repressor cleavage. t h e a m o u n t of g r a m s (fig. 4) sence of RIF. lysogens with
c a n r e a c h 3 p e r c e n t of t h e t o t a l o f t h e c e l l u l a r p r o t e i n s a f t e r 5,0 r a i n o f i n c u b a t i o n [5]. S u c h t r e a t m e n t s i n d u c e also t h e s o - c a l l e d SOS f u n c t i o n s , s u c h as c e l l f i l a m e n t a t i o n , i n h i b i t i t o n o f DNA
R ' f r a g m e n t s s e e n on t h e a u t o r a d i o was slightly increased by the preA f t e r 10 m i n of i n c u b a t i o n o f t h e R I F a n d MMC, R' f r a g m e n t s w e r e
mitt M M C
0
10
--
÷
20
30
40
÷
+
+
691
40 -
0 --
10 2 0 3 0 4 0 4 0 +
+
+
+
-
RIF
Fro. 4. - - Cleavage of ~ repressor. C600 00 lysogens were grown in low sulfur m e d i u m containing 0.4 m C i / m l HeaSSO~ f r o m 0.05 to 0.15 Lange units. The culture was then split into two samples : one h a l f was treated with RIF and 10 mM cold M'gSO4, to the other h a l f cold MgS04 was added. The two cultures were incubated for a f u r t h e r 5 min, centrifuged and t h e n resuspended in w a r m enriched YM9 m e d i u m containing RIF and MMC, or MMC alone, as indicated on the autoradiogram. As a function of the time of incubation w i t h MMC, 1-ml samples were removed and processed for )~ repressor precipitation by an a n t i - r e p r e s s o r serum. Purified labeled p r o t e i n s were analysed by SDS-PAGE (on a 15 per cent acrylamide gel) and autoradiography. The radioactivity in 85S per slot was a d j u s t e d to 6 × 103 cpm. Below each lane on the autoradiogram arc indicated the t i m e s at which the samples were removed and quickly frozen. The molecular weight of the ~. rcpressor m o n o m e r (R) and of its f r a g m e n t (R') were ahout 27,000 and 14,000 respectively.
m o r e a b u n d a n t t h a n w i t h o u t R~F, a n d t h e i r d i s a p p e a r a n c e w a s also d e l a y e d ; n o s i m i l a r a f f e c t w a s o b s e r v e d i n t h e p r e s e n c e of CAP ( d a t a n o t s h o w n ) .
Discussion. U p o n t r e a t m e n t of E. colt w i t h D N A d a m a g i n g a g e n t s , t h e s y n t h e s i s of R e c A p r o t e i n is i n d u c e d [4, 5, 9, 27] ; t h e c o n e e n t r a t i o n of R e c A p r o t e i n
BIOCHIMIE, 1980, 62, n ~ 10.
degradation and prophage s i o n of w h i c h a p p e a r s to t r o l l e d [21. D o e s t h i s m e a n R e c A p r o t e i n is r e q u i r e d to t i o n s as h a s b e e n p r o p o s e d 16, 20, 211 ?
induction, the expresbe coordinately cont h a t full i n d u c t i o n o f p r o m o t e all SOS f u n c b y m a n y a u t h o r s [6, 7,
W e s h o w in t h i s p u b l i c a t i o n t h a t t h i s is n o t so f o r i n d u c t i o n of p r o p h a g e k. I n d e e d , i n MMCt r e a t e d l y s o g e n s , e v e n t h o u g h t h e r a t e of s y n t h e s i s of RecA p r o t e i n was quite specifically r e d u c e d by a l o w c o n c e n t r a t i o n of R I F , i n d u c t i o n of p r o p h a g e ), o c c u r r e d r e a d i l y as a s s e s s e d b y : (i) cell
P. L. M o r e a u a n d coll.
692
lysis of the lysogens, (it) p r o d u c t i o n of p r o g e n y phage, and (iii) e x t e n s i v e c l e a v a g e of ~ repressor. In p a r t i c u l a r , the rate and the extent of cleavage of X r e p r e s s o r w e r e not significantly affected by the d e c r e a s e d rate of synthesis of RecA p r o t e i n
I 0 0 '_-~ .-a
7"V~X
N o t w i t h s t a n d i n g , it is c l e a r that c l e a v a g e of r e p r e s s o r , w h i c h is t h o u g h t to be t h e t e r m i n a l c e l l u l a r event l e a d i n g to p r o p h a g e )~ i n d u c t i o n , did not r e q u i r e full i n d u c t i o n of ReeA p r o t e i n .
0'3
[ a.) L.
~
10
e-.-
._~ --
A k..)
I
i
i
I
I
0 10 20 30 40 minutes FIG. 5. Time coarse of cleavage of )~ repressor. C600 00 bacteria were incubated as described in figure 4 with R~F and MMIC (A), CAP and M.MG , V', or with I~M!Calone (0). The amount of )~ repressor was measured on autoradiograms by densitometry of the repressor (R) band. The plotted values on the ordinate are expressed in percentage of )~ repressor in the control culture (RIF and GAP free) just before MMC addit i o n ; they represent the mean of two experiments. On the abscissa is indicated the time of incubation with MMC. -
-
resulting from R I F action. T h e lag p e r i o d before the d e c a y of )~ r e p r e s s o r was, h o w e v e r , slightly e x t e n d e d in the p r e s e n c e of R I F (fig. 5) and the d i s a p p e a r a n c e of ), r e p r e s s o r f r a g m e n t s (R') was slow (fig. 4) ; in contrast, the t i m e course of prod u c t i o n of R' r e p r e s s o r f r a g m e n t s w a s r a t h e r accel e r a t e d by R I F (fig. 4). T h e a p p a r e n t d i s c r e p a n c y b e t w e e n the t w o results m i g h t reflect the c o m p l e x c e l l u l a r effects of R I F at low c o n c e n t r a t i o n . It has been s h o w n that R I F at low c o n c e n t r a t i o n can stim u l a t e the t r a n s c r i p t i o n of some o p e r o n s and r e d u c e that of others s e l e c t i v e l y [28, 291. In our e x p e r i m e n t s , w e m i g h t h a v e o b s e r v e d the net result of v a r i o u s effects of RIF. R I F c l e a r l y reduced the i n d u c e d s y n t h e s i s of RecA p r o t e i n but might have also d e l a y e d the d e g r a d a t i o n of ),
BIOCHIMIE, 1980, 62, n ° 10.
r e p r e s s o r f r a g m e n t s , w h i c h is n o r m a l l y o c c u r r i n g r a p i d l y in g r o w i n g cells [30]. A l t e r n a t i v e l y , R I F at l o w c o n c e n t r a t i o n m i g h t have a l l o w e d a h i g h e r synthesis of X r e p r e s s o r d u r i n g t h e 5-min chase p e r i o d before t h e a d d i t i o n of MMC, w h e r e a s CAP at l o w c o n c e n t r a t i o n m i g h t h a v e i n h i b i t e d the synthesis of k r e p r e s s o r , a n d t h e r e f o r e a c c e l e r a t e d the cleavage of k r e p r e s s o r (fig. 5) and thus i n d u c tion of p r o p h a g e )~ (fig. 3).
W e do not c l a i m that the basal level of RecA p r o t e i n is sufficient to c l e a v e )~ r e p r e s s o r s i n c e R I F did not c o m p l e t e l y abolish the i n d u c t i o n of RecA p r o t e i n synthesis. Moreover, our data do not e x c l u d e the p o s s i b i l i t y that a b a c k g r o u n d of cellular p r o t e i n s o t h e r t h a n RecA (of s i m i l a r m o l e c u l a r w e i g h t ) m i g h t h a v e led to u n d e r e s t i m a t e the i n c r e a s e in t h e r a t e of synthesis of RecA p r o t e i n . This is all the m o r e possible as the t i t r a t i o n of RecA p r o t e i n ~vas not p e r f o r m e d w i t h a techn i q u e as specific as that used for X r e p r e s s o r titration. Since RecA m o l e c u l e s are stable for at least I h [4, 33], the a c c u m u l a t i o n of RecA p r o t e i n induced by MMC m i g h t h a v e been u n d e r e s t i m a t e d in the p r e s e n c e of RIF. F u r t h e r m o r e , it is k n o w n that (i) de novo protein synthesis is r e q u i r e d for cleavage of )~ r e p r e s sor in MMC-induced lysogens [31], (it) lexA mutants, in w h i c h RecA p r o t e i n synthesis is p o o r l y i n d u c e d after DNA damage [32!, do not express SOS f u n c t i o n s [2], and (iii) the b e g i n n i n g of ~ r e p r e s s o r b r e a k d o w n is p r e c e d e d by a lag p e r i o d [12, see also fig. 5]. These facts and our results taken t o g e t h e r m a y simply m e a n that a small a c c u m u l a t i o n of RecA p r o t e i n is n e c e s s a r y to obtain full cleavage of k repressor. It m a y also be that the cellular events p r e c e d i n g the d e c a y of ~ r e p r e s s o r ore related to the occurr e n c e of DNA r e p a i r processes, w h i c h m a y lead to the a c t i v a t i o n of RecA p r o t e i n to cleave )~ r e p r e s sor [17]. T h a t RecA p r o t e i n must be a c t i v a t e d for the c l e a v a g e r e a c t i o n to o c c u r is s u p p o r t e d by v a r i o u s g e n e t i c and b i o c h e m i c a l data : (i) Although RecA p r o t e i n is p r o d u c e d at a high cellular c o n c e n t r a t i o n as a result of m u l t i p l e lexA m u t a t i o n s (lexA3 spr-51 or lexA3 tsl-1) or of amplification due to a plasmid, i n d u c t i o n of prop h a g e ), does not o c c u r constitutively, lhat is, ill
PrecA
synthesis
and
the a b s e n c e of DNA d a m a g e [6, 15, 34, 35]. Moreover, in some b a c t e r i a l mutants, such as i n f A , DNA damage i n d u c e s RecA p r o t e i n synthesis, w h e r e a s there is no p r o p h a g e ), i n d u c t i o n [9, 36, L. J., Gudas, pers. c o m m . ] . (it) D e n o v o RNA a n d p r o t e i n syntheses are r e q u i r e d in o r d e r for )~ r e p r e s s o r to be i n a c t i v a t e d in a ti[-1 m u t a n t at 42°C w h e r e a s the a m p l i f i c a t i o n of RecA p r o t e i n m o d i f i e d by the t i f m u t a t i o n [68] is not n e e d e d [~7]. Thus, in the case of the ti[ mutant, the a c t i v a t i o n only of the (RecA) Tif protein seems sufficient for p r o p h a g e k i n d u c t i o n . H o w e v e r , note that : (1) the ti[ mutant synthesizes RecA p r o t e i n at a faster rate than t h e w i l d type strain even at 30°C [3~], (2) purified T i f p r o t e i n is c o n s i d e r a b l y m o r e active t h a n the RecA + p r o t e i n as r e g a r d s cleavage of ), r e p r e s s o r [16]. W h a t applies to the ti[-1 m u t a n d does not a p p l y necessarily to w i l d t y p e b a c t e r i a c o n s i d e r e d in our experiments. (iii) Cleavage of ), r e p r e s s o r r e q u i r e s , a p a r t f r o m the p u r i f i e d ( R e e A ) T i f p r o t e i n , s i n g l e - s t r a n d e d DNA a n d also A T P [17]. If a c t i v a t i o n of R e c A p r o t e i n is r e q u i r e d for cleavage of p h a g e k r e p r e s s o r , our results i n d i c a t e that only a small (if any) i n c r e a s e o v e r the b a c k g r o u n d l e v e l of RecA p r o t e i n is n e c e s s a r y to p r o m o t e total ), r e p r e s s o r cleavage. T h i s m a y be i n t e r p r e t e d as i n d i c a t i n g that the m o l e c u l a r signals that cleaves ~ r e p r e s s o r , the m o l e c u l a r signals activating ReeA protein, whatever their nature [17], are p r o d u c e d in l i m i t e d a m o u n t s after a DNA d a m a g i n g treatment. This h y p o t h e s i s can a c c o u n t in a s i m p l e w a y for the a p p a r e n t paradox that t h o u s a n d s of ReeA m o l e c u l e s must take 40 rain (about a g e n e r a t i o n time) to cleave a hund r e d or so m o l e c u l e s of k r e p r e s s o r in an i n d u c e d k lysogen. A f r a c t i o n only of i n d u c e d RecA molecules m a y act as a p r o t e a s e [13, 34], in s u c h a w a y that a r e d u c t i o n in the total level of ReeA p r o t e i n w i l l not affect p r o p h a g e ), i n d u c t i o n , even t h o u g h o t h e r c e l l u l a r p r o c e s s e s , s u c h as DN,A r e p a i r , m a y then be i n h i b i t e d . A l t e r n a t i v e l y , in an i n d u c e d lysogen, t h e r e m a y be an a b u n d a n c e of active RecA p r o t e i n but a l i m i t e d a m o u n t of ), r e p r e s s o r susceptible to cleavage at a given time. E x p e r i m e n t s p e r f o r m e d in o u r l a b o r a t o r y h a v e s h o w n that u p o n UV-irradial i o n of k lysogens, the rate of i n a c t i v a t i o n of r e p r e s s o r did not c h a n g e in spite of a 5-fold i n c r e a s e due to a p l a s m i d in the c e l l u l a r level of k r e p r e s s o r ; t h e i n a c t i v a t i o n system, h o w e v e r , d i d not a p p e a r to be s a t u r a t e d [13]. One i n t e r p r e t a t i o n of this result is that t h e r e are t w o f o r m s of )~
BIOCHIMIE, 1980, 62, n o 10.
)~ r e p r e s s o r
cleavage.
693
repressor, cleavable and non cleavable, and that t h e r e is a d y n a m i c e q u i l i b r i u m b e t w e e n these t w o forms !113, J. W. Roberts, pers. c o m m . ] . T h e speed of g e n e r a t i o n of c l e a v a b l e (e.g., m o n o m e r i c ) f o r m s d r a w n f r o m a pool of non c l e a v a b l e (e.g., o l i g o m e r i c [26]) forms of k r e p r e s s o r m a y be t h e l i m i t i n g factor of the cleavage r e a c t i o n a n d m a y r e n d e r it i n d e p e n d e n t of the cellular level of RecA p r o t e i n w i t h i n a r e l a t i v e l y b r o a d range. Finally, we cannot rule out the p o s s i b i l i t y that RecA p r o t e i n might not be itself the r e p r e s s o r c l e a v i n g e n z y m e but m i g h t be the r e q u i r e m e n t for a n o t h e r u n k n o w n i n d u c i b l e p r o t e a s e to cleave k r e p r e s s o r . The p r o t e a s e m i g h t be p r e s e n t in t r a c e a m o u n t s associated w i t h purified RecA p r o t e i n I17]. W h a t e v e r the exact role or f u n c t i o n of RecA p r o t e i n in p r o p h a g e )~ i n d u c t i o n is, w h e n t h e cleavage of ), r e p r e s s o r starts, t h e r e is no a p p a r e n t c o r r e l a t i o n b e t w e e n the rate of c l e a v a g e of k r e p r e s s o r and the rate of synthesis of RecA p r o tein. Recently, a lack of c o r r e l a t i o n has also been o b s e r v e d b e t w e e n the synthesis of RecA p r o t e i n and a n o t h e r SOS function, n a m e l y t h e i n h i b i t i o n of cell d i v i s i o n [33]. Note added in p r o o f : While this paper was in press, a paper by Balueh et al. (198ff) was published whose title is : Induction of prophage )~without amplification of ReeA protein, Mol. Gen. Genet., 178, 317-323.
Acknowledgements.
W e are grateful to M. K a m i n s k i and P. Liacopoulos f o r their help in the preparation of the anli-repressor sera. W e t h a n k D. P a n t a l o n i f o r advice, M. O. Mossd for assistance w i t h the d e n s i t o m e t e r and J. Nappd f o r typing the manuscript. This work was aided by grants f r o m E u r a t o m and Fondation pour la Recherche M~dicale.
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