Sedimentation analysis of DNA degradation products resulting from the action of colicin E2 on Escherichia coli

320

BIOCHIMICAET BIOPHYSICAACTA

BBA 96566

S E D I M E N T A T I O N ANALYSIS OF DNA D E G R A D A T I O N PRODUCTS R E S U L T I N G FROM T H E ACTION OF COLICIN E2 ON E S C H E R I C H I A PETER

COL1

RINGROSE

Subdepartment o[ Chemical Microbiology, Department o[ Biochemistry, University o/ Cambridge, Cambridge (Great Britain) (Received March 5th, I97o)

SUMMARY I. The production and nature of DNA breakdown products resulting from colicin E2 action on Escherichia coli ROW has been investigated using colicin concentrations of lO3 and Io ~ molecules per bacterium. 2. Neutral and alkaline sucrose density gradients indicate that colicin E2induced DNA degradation occurs in three stages: (i) Stage I initiates the scission of single strands of the DNA duplex, (if) Stage I I follows after approx. 5 min with a limited endonucleolytic cleavage of the double strands of the DNA causing a reduction of the average molecular weight to between io 6 and lO7, (iii) Stage I I I involves a rapid exonucleolytic degradation of Stage II, and possibly Stage I fragments to an acid-soluble form about IO min after colicin E2 adsorption to the cell surface. 3. Stage I m a y be reversed on rapid removal of colicin E2 from the bacterial cell surface with trypsin, whereas Stage I I is irreversible and is shown to be the lethal process. 4- Isopycnic centrifugation in CsC1 and C%S0 4 gives results consistent with those obtained on sucrose density gradients and shows increasing density inhomogeneity with a decreasing size of DNA fragment. Denaturation of or protein association with DNA from colicin E2-treated cells can be discounted since there is no change in average density of the fragments. 5. Possible mechanisms of action of colicin E2 are discussed with reference to these results and the action of known deoxyribonucleases.

INTRODUCTION Colicins are highly specific antibacterial proteins produced by certain strains of Enterobacteria and each particular colicin brings about its own specific intracellular response. These antibiotics initiate a lethal sequence of events in sensitive bacteria b y adsorbing to specific cell surface receptor sites and disrupting membranemediated cellular control mechanisms 1. Colicin E2 has been shown to interfere with the control of DNA metabolism and to induce DNA breakdown in sensitive cells of Escherichia coll. This DNA degradation has been measured using the disappearance of radioactive DNA from the Abbreviation: BBOT, 2,5-bis-(5"-tert.-butylbenzoxazolyl-(2"))thiophene. Biochim. Biophys. Acta, 213 (197o) 320-334

PRODUCTS OF

DNA

DEGRADATION BY COLICIN

E2

321

cold acid-precipitable fraction 1,2 and a variety of time lags have been reported 3 depending on (a) the concentration of c01icin, (b) the sensitive strain of E. coli used and (c) the growth conditions employed prior to colicin attack. This lag has been interpreted as the time needed for the adsorbed colicin molecule to " t r a n s m i t " its lethal effect to its particular target. Genetic studies suggest that the enzymatic system involved in colicin-induced DNA degradation m a y be re]ated to that used in the excision-repair process occurring after ultraviolet irradiation 4. If this is the case, the first stage of colicin attack would be the breakdown of cellular DNA b y an endonuclease. The loss of radioactivity from labelled DNA present in an acid-insoluble precipitate m a y not therefore be a satisfactory method for following the initial events on addition of colicin E2 and it was decided to look for the appearance of single-and double-strand breaks in the DNA by analysis of lysates from colicin-treated cells on neutral and alkaline sucrose density gradients. Single-strand breaks were detected in the DNA within the first few minutes of colicin attack; these were followed by double-strand breaks and after a short lag exonucleolytic degradation was detected. Colicin E2 differs from other colicins in that its lethal action is not easily reversed b y trypsin 1,5,6. Reversal will occur with colicin E2 if trypsin is added within I0 rain of the addition of colicin or if the bacteria are pretreated with dinitrophenol 5. It has been found that the trypsin-induced reversal resulted from the repair of single-strand but not double-strand scissions induced b y the colicin in the cellular DNA.

MATERIALS AND METHODS

Bacterial strains The bacterial strain used for production of colicin E2 was E. coli K I 2 TR2 which received colicinogenic factor E2 from Shigella P9 and was kindly donated b y Professor P. Fredericq. The sensitive strain used was E. coli K I 2 R O W Stock No. CLI42 which was kindly donated b y Professor G. Meynell. Growth conditions The synthetic medium M 9 described b y ANDERSON7 was used for growth of both bacterial strains and contained 5o mM N a 2 H P Q , 20 mM NH4C1, 20 mM KH~P04, 20 mM glucose, o.I mM MgS04, I juM FeC13 and 3 g casamino acids (Difco) per l. The cells were grown under aerobic conditions at 37 ° . Production, puri/ication and assay o/colicin E2 Mitomycin C (final concn. 0. 4 #g/ml) was added to an exponentially growing culture of the colicinogenic strain when the cell density was 4" lOS bacteria/ml and this culture was incubated for a further 2 h at 37 °. The bacteria were harvested b y centrifugation and washed 3 times with I M NaC1 in IO mM phosphate buffer (pH 7.o) s. The saline washings were precipitated with (NH4)~SO 4 at 4 ° ~o saturation and the precipitate dissolved in IO mM phosphate buffer (pH 7.0). The protein solution was dialysed overnight against IO mM phosphate buffer and then mixed with preequilibrated DEAE-Sephadex at 4 ° for 30 rain and filtered. The colicin remained largely in the filtrate which was concentrated using Carbowax 20 M and dialysed overnight against 0.05 M phosphate buffer (pH 6.0). This was then applied to a Biochim. Biophys. Acta, 213 (197 o) 320-334

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CM-Sephadex column, equilibrated with the same buffer, and the colicin eluted with an increasing p H gradient formed b y mixing equal volumes of 0.05 M phosphate buffer (pH 6.0) and 0.05 M K2HPO 4. Colicin E2 activity was eluted at a p H value of 6.80 (ref. 8). The colicin was further characterised by chromatography on a hydroxylapatite column using a linear gradient of O.Ol-O.4O M phosphate buffer (pH 7.0). Colicin E2 was eluted at o.18 M phosphate s. No further purification was achieved so this stage was usually left out. The colicin preparation was homogeneous in the analytical ultracentrifuge (s°20,~ = 4.2), on polyacrylamide-gel electrophoresis and had an approximate molecular weight of 60 ooo on Sephadex G-2oo. The yield from 5 1 was in the order of 25 mg and this was found to have a specific activity of lO3 units per/~g using the method of HILL AND HOLLANDg; the number of units being the reciprocal of the highest dilution of colicin to give minimal growth inhibition of E, coli ROW seeded on agar. The minimum inhibitory concentration was o. I ng coliein E2 per ml at a cell density of lO 7 bacteria per ml, i.e. IOO molecules of colicin per cell. This preparation of colicin E2 was used throughout. Labelling o] cell D N A and treatment with colicin E2 An overnight culture of E. coli ROW was diluted ioo-fold with prewarmed M9 medium and incubated aerobically until the cell density was 3"lOS bacteria per ml. These exponentially growing cells were then diluted io-fold with M 9 medium containing 200 #g deoxyadenosine per ml (to facilitate thymine uptake 1°) and IO #C EMe-3Hlthymine per ml (specific activity 2 C/mmole). Incubation was continued for a further 2-2. 5 h until the cell density was again 3"lOS bacteria per ml. The labelled cells were then diluted io-fold with prewarmed M 9 medium containing IOO ~g unlabelled thymine per ml and colicin E2 at either 3 or 30 ng/ml (i.e. lO s or lO4 colicin molecules per bacterium). Dilution of the purified colicin was performed in the presence of I m g bovine serum albumin per ml to prevent denaturation. The final dilution was carried out immediately before addition of the labelled bacteria and the final concentration of bovine serum albumin was I o # g / m l . At intervals after addition of colicin E2 to the labelled bacteria, 5-ml aliquots were diluted with 15 ml ice-cold lysis medium (o.15 M NaCl-o.i M E D T A (pH 8.0)) containing 2 mM dinitrophenol. The diluted bacteria were harvested by centrifugation, washed, resuspended in i ml lysis medium and then treated with 200/~g lysozyme per ml at 20 ° for 15 rain followed b y 0.5 % (w/v) sodium dodecyl sulphate and ioo/~g pronase per ml for 30 rain at 4 o°, to free the DNA from membrane and protein complexes and to eliminate any deoxyribonuclease activity 11. 0.2 ml DNA preparation was layered directly onto a sucrose gradient, the amount of sample corresponding to about o. 4 #g DNA (4° ooo counts/rain). The total radioactivity present in the DNA fraction was determined by removing o.5-ml samples at intervals after colicin E2 treatment and mixing with ice-cold IO °/o (w/v) trichloroacetic acid. After 30 min at 4 ° the precipitates were collected on Oxoid membrane filters and washed twice with 2.5 ml cold 5 % (w/v) trichloroacetic acid and twice with 5 ml 1 % (v/v) acetic acid. The membrane filters were dried at 80 ° for 30 rain and the radioactivity measured in a Packard Tri-carb scintillation spectrometer using a toluene base scintillation fluid containing 4 g/1 of 2,5bis-(5'-tert.-butylbenzoxazolyl-(2')) thiophene (BBOT). Sedimentation analysis on sucrose density gradients 5-20 % (w/v) sucrose density gradients were made up in o.I M NaCl-o.oi M Biochim. Biophys. Acta, 213 (197o) 32o-334

DNA

PRODUCTS OF

DEGRADATION BY COLICIN

E2

323

Tris-o.oi M E D T A buffer (pH 8.0) 12 for neutral gradients and in 0. 9 M NaCl-o.I M N a O H ~3 for alkaline gradients. The neutral gradient had a volume of 4.8 ml on which was layered 0.2 ml cell lysate, whilst the alkaline gradient had a volume of 4.7 ml on which was layered o.I ml 0.5 M N a O H followed by 0.2 ml cell lysate. After preparation, the alkaline gradients were allowed to stand for 3 ° rain at 20 ° prior to centrifugation in order to allow denaturation to take placO 4. The gradients were centrifuged for 1.5-3 h at 4 ° ooo rev./min at 20 ° in a Spinco Model-L2 HV ultracentrifuge using a SW-5o r o t o r . / o - d r o p samples (approx. o.15 ml) were collected from a pinhole punched in the bottom of the centrifuge tube. The samples were mixed with 0.3 ml water and 3 ml Triton X-Ioo/BBOT-toluene mixture (I :2) and their radioactivity determined using a Packard Tri-Carb scintillation spectrometer. This method gave identical results to one involving precipitation of the cold acid-insoluble fractions from the samples. The radioactivity was shown to be associated with DNA b y sensitivity to deoxyribonuclease, resistance to NaOH and buoyant density in CsC1. Recovery of labelled material from the gradients varied between 75 and 85 % of the input radioactivity (Table I).

Calculation o/ sedimentation coe/ficients and nwlecular weights o] the DNA ]ragments The sedimentation coefficient of the 3H-labelled DNA fragments was calculated using the formula of NOMURA et al. 15. The values obtained were checked using markers of known sedimentation coefficient: I14C]thymidine-1abelled T 4 phage DNA and its fragments 1° having sedimentation values of 63, 43 and 3o S (ref. 17); [3Hlthymine labelled colicinogenic factor E2 DNA (25 S)lS; and 5o and 3o S ribosomal subunits from sonicated E. coli labelled with I3H]uridine (Fig. i). The T 4 DNA fragments and colicinogenic factor DNA were centrifuged on neutral sucrose gradients as described above. The ribosomal subunits were centrifuged under conditions similar to those described in the preceding section except that the sucrose was made up in IO mM Tris-25 mM KCI-o.I mM magnesium acetate buffer (pH 7.o).

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S ribosome S)

S ribosome

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Tube No.

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36

Fig. I . S e d i m e n t a t i o n coefficient c a l i b r a t i o n of sucrose d e n s i t y g r a d i e n t fractions. T h e sediment a t i o n coefficients s h o w n are f o r sucrose d e n s i t y g r a d i e n t s p r e p a r e d as described ill MATERIALS

AND METHODS a n d centrifuged for 90 min at 4 ° ooo r e v . / m i n at 20 ° using a SW-5o rotor. The filled circles are the theoretical p o i n t s and t h e open circles are empirical points obtained using T~ p h a g e fragments, E2 colicinogenic factor D N A and E. coli ribosomal subunits. The n u m b e r of fractions has been corrected for the presence of sodium dodecyl sulphate.

Biochim. Biophys. Acla, 2I 3 (197 o) 320-334

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The sucrose density gradients were shown to be linear and the distance moved by a DNA sample was assumed to be directly proportional to the number of drops between the peak m a x i m u m and the meniscus since the difference in viscosity between 5 and 2o % sucrose made a variation of less than 3 % in drop size. The number of drops collected per gradient was 3oo-t-5 after correction for the effect of sodium dodecyl sulphate in the last few fractions. The molecular weights (weight average) of the DNA fragments were calculated from their sedimentation coefficients la,17A9,2°. CsCl and Cs2SO a isopycnic centri/ugation Sodium dodecyl sulphate forms a precipitate with CsC1 and was therefore replaced b y 1 % (w/v) sarkosyl solution in the preparation of all-labelled cell lysate for CsC1 and Cs2SQ equilibrium centrifugation. The lysate was mixed with a solution of CsC1 or Cs2S04in o.oi M Tris buffer (pH 8.o) containing o.oi M E D T A for neutral gradients or in o.oi M Tris-o.I M K2HPO ~ buffer (pH 12.5) containing o.oi M E D T A for alkaline gradients a. The final densities were adjusted to 1.71oo g/ml for CsC1 or 1.425o g/ml for Cs2SO4 b y the refractive index method described by SZVBALSKI~2. 4 ml of the mixture containing approx. I/~g all-labelled DNA were overlayered with I ml liquid paraffin and centrifuged to equilibrium (45-6o h) at 32 ooo or 44 ooo rev./min at 2o ° in a Spinco Model-L2 HV ultracentrifuge using the SW-5o rotor. The contents of the tubes were fractionated to give 5o ×5 drop samples and the radioactivity in the DNA determined by either acid precipitation as described above or by dilution of each sample with I ml distilled water and mixing with 7 ml Triton/ BBOT 4oluene liquid scintillation fluid. There was no difference between the two methods. Materials [Me-aHlThymine (2 C/mmole), [2-14Clthymidine (56.2 mC/mmole) and [5-3H1 uridine (5 C/mmole) were obtained from The Radiochemical Centre, Amersham. Unlabelled thymine, deoxyadenosine and mitomycin C were obtained from Sigma Chemical Co. Sarkosyl NL 35 o//owas a kind gift from Geigy (UK), Manchester. CsC1 and CsS04 were obtained from B.D.H. Chemicals, Poole, Great Britain. BBOT was a CIBA product. Egg white lysozyme was obtained from Armour Pharmaceutical Co., Eastbourne, Great Britain. Deoxyribonuclease ("electrophoretically purified") was obtained from Worthington Biochemical Corp., N.J. Trypsin (pancreatic) was obtaind from B.D.H. Poole, Great Britain and Pronase Grade B was obtained from Calbiochem. All other chemicals were A.R. grade.

RESULTS

initially it was intended to investigate the physical integrity of DNA from colicin-treated cells b y lysing spheroplasts directly on top of the sucrose density gradient so as to cause minimum shear of the released DNA. However, after E. coli had been incubated in the presence of colicin E2 for a few minutes it proved extremely difficult to form spheroplasts; similar effects with other antibacterial agents have been observed b y CUNDLIFFE23. This was probably the result of changes in the cell Biochim. Biophys. Acta, 213 (197o) 320-334

PRODUCTS OF D N A DEGRADATION BY COLICIN E2

325

envelope a n d m a y be r e l a t e d to the a p p e a r a n c e of long filamentous forms of E. coli (approx. I0 ff in length) after p r o l o n g e d colicin t r e a t m e n t 2 (P. RINGROSE, u n p u b lished observations). The a l t e r n a t i v e a p p r o a c h of t r e a t i n g p r e f o r m e d E. coli spher o p l a s t s with colicin was also unsuccessful owing to the r e d u c e d s e n s i t i v i t y at t h e high sucrose c o n c e n t r a t i o n s used a n d the implicit d a m a g e to the b a c t e r i a l cell envelope (see S M A R D A 24 a n d ] 3 E P P U A N D ARIMA25).However, S M A R D A A N D T A U B E N E C K 26 have more r e c e n t l y r e p o r t e d an increased colicin s e n s i v i t y of E. coli L forms. I t was therefore decided to use c o n v e n t i o n a l m e t h o d s of cell lysis. These prod u c e d h i g h l y reproducible s e d i m e n t a t i o n coefficients of 4 ° a n d 47 S for the control D N A in n e u t r a l a n d alkaline gradients, b o t h of which correspond to an original d o u b l e - s t r a n d e d D N A of m o l e c u l a r weight 5"Io7 (ref. i3) a n d agree w i t h t h e values o b t a i n e d b y SAKABE AND OKAZAK127,SMITH AND HANAWALT12 a n d NOMURA et al. 2s. U n d e r the ionic conditions used, s i n g l e - s t r a n d e d D N A (denatured) has a slightly higher s e d i m e n t a t i o n coefficient t h a n the d o u b l e - s t r a n d e d D N A (native), see STUDIERla. The a m o u n t of D N A used per g r a d i e n t was a l w a y s less t h a n I fig so as to eliminate c o n c e n t r a t i o n effects due to aggregation, D N A / w a l l i n t e r a c t i o n in the centrifuge t u b e a n d r o t o r speed la,17.

(a) Analysis o~ cellular D N A after colicin action (i) Sucrose density gradients. Cultures of E. coli R O W prelabelled w i t h [3H]t h y m i n e were t r e a t e d with colicin E2, s a m p l e d a t i n t e r v a l s a n d after lysis the D N A was a n a l y s e d using b o t h n e u t r a l a n d alkaline sucrose d e n s i t y gradients. The results are shown in Figs. 2a-2c. The s e d i m e n t a t i o n coefficients of b o t h n a t i v e a n d d e n a t u r e d o 0*27

b

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0 2

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10

20

30

10

20

30

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Fig. 2. a and b. Sedimentation analysis of DNA from [all]thymine-labelled cells of E. coli after treatment with colicin E2. The labelled bacteria were incubated in the presence of colicin E2 {3 ng/ml) [for o rain ( • - • ), 2 min ( &- • ) , 7 min ( O - 0 ) , 15 min ( • - •), 30 and 60 rain (V?-[-])], harvested by centrifugation, lysed and centrifuged for 90 min at 4° ooo rev./min through neutral (a) and alkaline (b) sucrose density gradients. 3°- and 6•-rain peaks differ only in magnitude, c. DNA profiles in alkaline sucrose from control bacteria ( • - O ) and bacteria incubated for 15 rain (0-(2)) in the presence of colicin E2. The conditions were the same as for b except that the DNA sample from the colicin-treated bacteria was centrifuged for 3 h. D N A were p r o g r e s s i v e l y r e d u c e d on i n c u b a t i o n w i t h colicin E2 over a period of 3o min, b u t there was a difference in t h a t t h e r e d u c t i o n in the case of t h e d e n a t u r e d D N A was d e t e c t e d a f t e r 2 min of i n c u b a t i o n , whereas no differences from t h e c o n t r o l s a m p l e were o b s e r v e d in t h e n a t i v e D N A u n t i l 7 rain a f t e r t h e a d d i t i o n of colicin E2. This alkaline shift was more p r o n o u n c e d a t higher colicin c o n c e n t r a t i o n s (30 ng/ ml) Fig. 3. The difference of results on t h e two t y p e s of g r a d i e n t s after 2 rain indicates the presence of single-strand b u t not d o u b l e - s t r a n d scissions in t h e D N A helix.

Biochim. Biophys. Acta, 213 (197o) 320--334

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Fig. 3. A l k a l i n e sucrose d e n s i t y g r a d i e n t profiles of D N A from cells of E. coli a f t e r t r e a t m e n t w i t h colicin E2 (3 ° n g / m l ) . [ 3 H ] T h y m i n e - l a b e l l e d cells of E. coli were i n c u b a t e d in t h e p r e s e n c e of colicin E 2 for o miD ( 0 - - - O ) , 5 m i d ( A - A ) , i o miD ((2) O ) , 20 miD (m-m), 3 ° a n d 60 miD (U]-D). The c o n t r o l D N A was c e n t r i f u g e d for 90 miD a t 4 ° ooo r e v . / m i n , w h e r e a s t h e D N A s a m ples from c o l i c i n - t r e a t e d b a c t e r i a were c e n t r i f u g e d for 3 h. 3 °- a n d 6D-miD p e a k s differ o n l y in magnitude.

After 5-IO miD of incubation the sedimentation coefficient of the native DNA from colicin-treated bacteria was progressively reduced but was still greater than that of the denatured DNA from the same bacteria after 60 miD. The recovery of radioactivity in the DNA preparation after centrifugation is shown in Table I. Radioactivity was not lost from the DNA during the first 15 miD of incubation of the bacteria with colicin E2, yet it is clear from Fig. 2c that there was an appreciable inhomogeneity in the DNA strands which manifested itself as an increase in peak width. The possibility that this was the result of increased exposure to alkaline conditions (3 h) was eliminated by exposing control DNA to alkaline conditions for 9 ° mid prk)r to centrifugation. No increase in peak width was observed. TABLE I RELATIONSHIP

EET\VEEN

ACID-PRECIPlTABLE

ALKALINE SUCROSE DENSITY

Colicin E2 (3 ng]ml) incubation time (miD)

o 2 7 15 3° 60

.4cid-precipitable counts in 0.2 ml lysate be/ore centri/ugation*

40 4° 4° 38 3o 23

C O U N T S A N D TOTAL P E A K C O U N T S IN N E U T R A L

AND

GRADIENTS

13o 5oo iio 9 lo 12o 640

Control counts/or column 2 (%)**

IOO IOI IOO 97 75 59

Total counts recoveredin main peak after centri/ugation*** pH 8

pH 22

32 32 31 32 24 19

33 31 31 32 23 18

89o 13o 580 09 ° 13o 59 °

OlO 2o0 63o OlO 870 700

Recovery (%)

Control counts /or column 4

83 78 79 83 80 81

IOO 96 95 97 73 58

* C a l c u l a t e d from cold t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e m a t e r i a l in 0.5 ml of t h e o r i g i n a l cell s u s p e n s i o n before lysis (MATERIALSAND METHODS). ** R e s u l t s p l o t t e d in Fig. 5. *'* Profiles from w h i c h t h e s e v a l u e s are d e r i v e d are s h o w n in Fig. 2.

The sedinlentation coefficients of the DNA preparations were calculated from the positions of the peaks in Figs. 2 and 3 using the formula of NOMURAet al. 15 (Fig. I) and from these were derived the molecular weights of the DNA's 13 (Fig. 4)- The roDBiochim. Biophys. Aeta, 213 (197 o) 320-334

PRODUCTS OF

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32 7

lecular weights of the single-stranded DNA prepared from cells incubated in the presence of colicin E2 at a concentration of either 3 or 3o ng/ml fell rapidly with no detectable lag, whereas reduction of the molecular weight of native DNA occurred only after a lag of 5-IO min (Fig. 4). The concentration of colicin E2 in the incubation medium not only influenced the rate of DNA degradation but also its extent. The molecular weight of the DNA isolated from cells treated with colicin for 3 ° min at a concentration of 3o ng/ml had a molecular weight under neutral conditions of 2 - I o 6 compared with lO 7 for DNA from cells incubated in the presence of 3 ng colicin per ml. Although there was this difference in molecular weights, the number of singlestrand scissions per duplex fragment was between i and 3 for both colicin concentrations, and the whole process of single- and double-strand snipping was largely over by 3o rain.

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Fig. 4. Decrease in m o l e c u l a r w e i g h t of D N A from cells of E. coli after t r e a t m e n t w i t h colicin E2 [3HJThymine-labelled cells of E. coli were t r e a t e d w i t h colicin E2 a t c o n c e n t r a t i o n s of (a) 3 ° ng/ ml a n d (b) 3 ng/ml. T h e b a c t e r i a were lysed at i n t e r v a l s a n d t h e s e d i m e n t a t i o n coefficients of single- a n d d o u b l e - s t r a n d e d D N A o b t a i n e d b y c e n t r i f u g a t i o n in alkaline a n d n e u t r a l sucrose dens i t y g r a d i e n t s (Figs. 2 a n d 3). T h e m o l e c u l a r w e i g h t s of single-, ( Q - O ) a n d double- ( 0 - 0 ) s t r a n d e d D N A were c a l c u l a t e d f r o m t h e i r s e d i m e n t a t i o n coefficients u s i n g t h e f o r m u l a e of Studier: s°20,w = 0.0528 M °.4°0 (neutral g r a d i e n t ) a n d s°~0,~ = 0.0882 M 0.s46 (alkaline g r a d i e n t s ) .

(ii) Isopycnic centri/ugation. It was of interest at this stage to compare molecular weights computed from sedimentation through a sucrose density gradient with peak widths obtained after isopycnic centrifugation in either CsC1 or Cs2SO 4 using the method of THOMAS AND BERNS29. The results of such an experiment are shown in Figs. 5 and 6. Incubation of the bacteria in the presence of colicin E2 did not result in a change in the position of the DNA peaks, but after a 3o-min exposure to colicin there was a dramatic increase in peak width consistent with the observation that peak variance is indirectly proportional to molecular weight 3°. The peak a s y m m e t r y or skewness was probably the result of a heterogeneity in molecular weight and density of the DNA preparations since similar results have been reported for small phage fragments 3a and sonicated calf thymus and bacterial DNA's ~z,~a.The peak a s y m m e t r y was more pronounced in C%SO 4 than in CsCI and its increase with the length of duraBiochim. Biophys. Acta, 213 (197 o) 320-334

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Fig. 5. Cs~SO4 isopycnic c e n t r i f u g a t i o n of D N A isolated f r o m [3FI]thymine-labelled cells of E. coli after t r e a t m e n t w i t h colicin E2. T h e labelled b a c t e r i a were i n c u b a t e d in t h e presence of colicin E z (3 ng/ml) for o m i n ( O - O ) , 7 m i n ( O - Q ) , 15 m i n (/X-Dk) a n d 3 ° rain ( l - I I ) , h a r v e s t e d b y e e n t r i f u g a t i o n , lysed a n d c e n t r i f u g e d to e q u i l i b r i u m a t 32 ooo r e v . / m i n a t 20 ° in CsoSO 4 at a d e n s i t y of 1.425o g/ml. T h e arrows indicate t h e b u o y a n t d e n s i t y positions of m a r k e r D N A ' s from T 4 phage, Micrococcus lysodeikticus (NIL) a n d E. coli (the s y m b o l s d N a n d N N r e p r e s e n t h e a t - d e n a t u r e d a n d n a t i v e D N A ) a n d also c o m p l e t e T 4 phage.

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20 30 40 50 60 Molecuk3r "w~ght xlO-6

70

Fig. 6. D e t e r m i n a t i o n of 13 v a l u e s of D N A p e a k s after isopycnic c e n t r i f u g a t i o n . T h e p e a k w i d t h (w) s q u a r e d is p l o t t e d a g a i n s t t h e log of t h e fractional p e a k h e i g h t (Y/Yo) u s i n g t h e p e a k s o b t a i n e d after isopycnic e e n t r i f u g a t i o n as described in Fig. 5- fl is a c o n s t a n t for t h e g r a d i e n t c o n d i t i o n s u s e d 2. a n d w a s f o u n d to be 0.36. lO 4 for Cs.~SO, for 32 ooo r e v . / m i n u s i n g a S W - 5 o rotor. T h e /~ v a l u e is g i v e n b y t h e slope of t h e plot: In Y/Yo T h e s t r a i g h t line plots are g i v e n b y D N A ' s f r o m cells of E. coli a f t e r i n c u b a t i o n w i t h colicin E2 (3 n g / m l ) for o rain ( V - V ) , 7 rain ( O - O ) , 15 m i n ( A - - a ) a n d 3o m i n ( O - O ) - b. R e l a t i o n s h i p b e t w e e n B v a l u e s a n d m o l e c u l a r weights. T h e B v a l u e s o b t a i n e d as described in a are p l o t t e d a g a i n s t t h e i r c o r r e s p o n d i n g molecular w e i g h t s calculated f r o m sucrose d e n s i t y c e n t r i f u g a t i o n (MATERIALS AND METttODS). T h e t i m e v a l u e s g i v e n a b o v e t h e figure indicate t h e degree of colicin E2 t r e a t m e n t of t h e D N A samples. T4 D N A half f r a g m e n t is used as a s t a n d a r d .

Biochim. Biophys. Acta, 213 (197o) 32o-334

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E2

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tion of colicin treatment was not simply the result of a decrease in molecular weight of the isolated DNA fragments, since colicinogenic factor E2 has a molecular weight similar to DNA from E. coli incubated in the presence of colicin E2 for 30 rain, but does not show a comparable skewness under similar conditions. The B values were determined by plotting the log of the fractional peak height against the peak width (squared) as described in the legend to Fig. 6 and these were related to the weightaverage molecular weights of the DNA preparations determined b y sedimentation (Fig. 6b). The linear relationship shown in Fig. 6b provides a useful confirmation of the relative molecular weights assigned to particular DNA fragments isolated from colicin-treated cells. Isopycnic centrifugation also detects any change in density of the DNA brought about by denaturation or protein interaction; for example heat-denatured DNA increases in density more in Cs2SO 4 than in CsC1, whereas with ultraviolet-damaged DNA the opposite is true 35. Protein interaction, however, results in a decrease of the buoyant density as shown in Fig. 5 with T 4 DNA and T 4 phage. The DNA fragments from bacteria incubated with colicin E2 showed no detectable shift in mean density in neutral or alkaline CsC1 or Cs2SO 4 gradients, with or without pronase treatment, and this would appear to eliminate the possibility that colicin E2 acts b y binding irreversibly to DNA or specifically denatures DNA of sensitive bacteria in vivo. (iii) Exonuclease action. DNA breakdown in sensitive strains of E. coli following addition of colicin E2 has in the past been followed b y measuring the disappearance of radioactivity from the cold acid-precipitable fraction of prelabelled bacteria 1,2 and a typical progress curve is shown in Fig. 7. The DNA fragments present after a 3o-min incubation in the presence of colicin E2 under the conditions described in Section a (i) have molecular weights in the range 2" IO6-IOv and no fragments of DNA with molecular weights between this value and that of acid-soluble fragments (5-1o 3) have been detected. This observation must indicate that once the exonuclease has attached itself to a particular piece of DNA the breakdown is extremely rapid (see ref. 35). The degree of DNA degradation induced b y colicin E2 can be increased to a m a x i m u m of 70-75 % (i.e. 25-30 O//o DNA remaining in the cold acid-insoluble fraction) after 60 min with an appropriate increase in colicin concentration. The degrees of degradation shown in Fig. 7 are consistent with the results of HOLLAND2 and NOMURA1 at similar colicin concentrations.

(b) Lethal process in colicin-induced cell death It was apparent from the studies reported so far that DNA degradation occurring in the presence of colicin E2 can be divided into three stages: Stages I and I I being the single- and double-strand endonuclease steps and Stage I I I the exonuclease step. It was therefore necessary to determine which was the "lethal step" after which the effects of colicin E2 could not be reversed by the addition of trypsin. The method for trypsin treatment was similar to that described b y REYNOLDS AND REEVES5 and is given in the legend to Fig. 8. The shape of the graph of the viability of cells incubated with only colicin against time indicates the rate of adsorption of colicin molecules to the cell surface. However, if at the times indicated the cells were treated with trypsin before dilution, then the survival curve differed from the control in that there was apparently a period during which trypsin reversed the effect of colicin E2. A comparison of these survival curves with those showing the variation of the molecular Biochim. Biophys. Acta, 213 (i97 o) 32o--334

P. RINGROSE

33 °

100~

I0C

.~ 8C

10

h

io

6c

4c

10

20 30 40 Time (rain)

50

60

oj \, 0

5 10 15 Time (mln)

2'0

Fig. 7. I n d u c t i o n of D N A b r e a k d o w n b y coticin E2 and the effect of trypsin. [3H]Thyminelabelled cells of E. coli were incubated in the presence of colicin E2 at a concentration of 3 n g / m l ( O - 0 ) and 3 ° ng/ml ( 0 - 0 ) . Samples were t a k e n at intervals and the labelled nucleic acid fraction precipitated with trichloroacetic acid at 4 °. The precipitates were collected on Oxoid m e m b r a n e filters and the radioactivity expressed as a percentage of the u n t r e a t e d control. I n addition the cells incubated in the presence of 3 ng colicin E2 per ml were treated with t r y p s i n (2 mg/inl) after 2 min ( D - D ) and 5 rain ( I - I ) and samples were t a k e n as above. Fig. 8. Effect of t r y p s i n on the viability of E. coli treated with colicin E2. E x p o n e n t i a l l y growing cells of E. coli at a density of 3" I°v bacteria per ml were t r e a t e d with colicin E2 (3 ng/ml) and serially diluted in M9 m e d i u m at intervals to p r e v e n t f u r t h e r adsorption of colicin molecules and hence determine the n u m b e r of bacteria still able to produce colonies on agar plates. A p p r o p r i a t e dilutions were plated out in triplicate ( O - C ) ) . Similar colicin-treated samples were incubated with t r y p s i n (2 mg/ml) and 2,4-dinitrophenol (2 raM) for 20 min at 37 ° and t h e n serially diluted and plated o u t as before (see refs. 5 and 6) ( 0 - 0 ) .

weight of native DNA from coliein E2-treated bacteria (Fig. 4) would indicate that once double-strand breaks occurred in the DNA, the lethal effect could not be reversed by trypsin.

(c) Trypsin-induced repair o/colicin E2-damaged DNA If the lethal process in colicin-induced cell death is the production of doublestrand breaks, then it must be possible for cells to repair single-strand lesions produced initially. Addition of trypsin during the first IO rain to a culture of colicintreated cells reduced the extent of DNA degradation (Fig. 7) and, if added 2 rain after the addition of coliein, completely prevented any exonuelease activity. The repair process in the presence of trypsin was followed by analysing the cellular DNA using alkaline sucrose density gradient eentrifugation (Fig. 9). A concentration of 3 ug colicin E2 per ml was used as at this concentration there was a longer time lag before the appearance of double-strand breaks in the DNA. The high trypsin concentrations used were similar to those prescribed by REYNOLDS AND R E E V E S 5'6. There was a lag between the addition of trypsin and the cessation of eolicin E2 activity, and if eolicin action was not stopped before the appearance of doublestrand breaks then the exonuclease activity was higher. When trypsin was added 2 min after the addition of colicin E2 to E. coli, the sedimentation coefficient fell for a further 5-1o rain although at a reduced rate compared with the colicin-treated control and did not begin to increase until about 20 rain after the addition of trypsin; by 50 rain the sedimentation coefficient of the DNA was similar to the initial value. Addition of trypsin to the culture after 5 min incubation in the presence of coliein E2 did not completely inhibit Stage II of colicininduced DNA degradation. There was an immediate reduction in the rate of change BiochbT2. Biophys. Acta, 213 (197 o) 320-334

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331

5C

4C S2o,w

10

20 30 40 Time (mln)

50

Fig. 9. Effect of r e v e r s i n g colicin E2 a c t i v i t y w i t h t r y p s i n on t h e s e d i m e n t a t i o n coefficient of s i n g l e - s t r a n d e d D N A f r o m E. coll. [3H]Thymine-labelled cells of E. cull were i n c u b a t e d in t h e presence of colicin E2 (3 n g / m l ) . A f t e r 2 m i n (ll-li), 5 m i n ( D - D ) a n d io rain ( 0 - 0 ) t h e bacteria were t r e a t e d w i t h t r y p s i n (2 m g / m l ) . 0 - 0 , colicin-treated control. S a m p l e s were t a k e n a t i n t e r v a l s , lysed, t h e "~H-labelled D N A d e n a t u r e d a n d c e n t r i f u g e d on alkaline sucrose d e n s i t y gradients. T h e s e d i m e n t a t i o n coefficients were calculated f r o m t h e positions of t h e p e a k s a f t e r c e n t r i f u g a t i o n u s i n g Fig. I.

of the sedimentation coefficient followed b y a period of no apparent change, probably a result of competition between degradative and repair processes occurring within the cell, but after 3o min there was a further fall in the sedimentation coefficient. Trypsin treatment after a Io-min incubation with colicin E2 had virtually no effect on DNA degradation. Comparable samples were analysed on neutral sucrose density gradients and double-strand breaks were found in the preparations from the 5-min trypsin treatment but not the 2-min treatment. It would appear probable that once double-strand cleavages have occurred, recovery is impossible and even in the absence of colicin E2 an exonucleolytic process is initiated. DISCUSSION

Stages o! DNA degradation The results reported show that there are at least three distinct stages in colicin E2-initiated degradation of E. eoli DNA. Stage I is the attack b y an endonuclease which specifically snips single strands of the DNA duplex. Stage I I involves breakdown of DNA into double-stranded fragments the molecular weight of which depends on the concentration of colicin used. These two stages overlap to some extent, although Stage I is usually observed in isolation during the first few minutes. Stage I I I involves a rapid exovucleolytic attack on the Stage I I and possibly Stage I fragments producing cold acid-soluble products without any observable intermediates. Exonuclease activity is detected after a lag of IO rain and is largely over by 45-6o rain, similar to T 4 phage-induced degradation of host DNA where there is a lag of about 5 rain and the whole process is over b y 30 min after infection ~5. However, the percentage of residual DNA left after this stage is only I 2 - I 5 % with T, phage ~5, compared with 25-3o % at high colicin concentrations. This initial lag phase with Biochim. Biophys. Acta, 213 (197 o) 320-334

332

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colicin m a y be the result of exonuclease activity being initiated only when Stage I I DNA fragments are of a suitable size (about IOv daltons). Stage I is not the lethal process associated with coliein-induced cell death since the single-strand lesions are repairable if the adsorbed coliein molecules are removed with trypsin. However, Stage I I is irreversible and comparison of temporal changes in the molecular weights of double-stranded DNA fragments from colicin E2-treated cells with survival curves suggests that this is the lethal event. Possible nucleases involved

The appearance at the end of Stage I I of DNA fragments having molecular weights between 106 and 107 m a y be related to the finding of SZYBALSKI et al. 35 that there is a cytosine-rich cluster per 106--107 of E. coli DNA and that these m a y be the sites for initiation of transcription by RNA polymerase. Hence, Stages I and I I could involve a cytosine-specific nuclease of the type described b y KUTTER AND WIBERC ~6 and this m a y account for the apparent density heterogeneity of the DNA fragments reported on isopycnic centrifugation. This supposition would also explain why T 4 DNA present in colicin-treated cells of E. coli is relatively resistant to degradation 1, since here the cytosine is substituted by hydroxymethylcytosine and these clusters occur with less frequency in T 4 than in E. coli 3e,37. However, there is one maior objection to this comparison between phage- and eolicin-induced breakdown of DNA in that eolicin-induced DNA breakdown is independent of protein synthesis whereas phage-induced host DNA degradation is inhibited by chloramphenieol ~. In other words eolicin action involves an "activation" of nucleases already present in E. coli, whereas phage needs to initiate the synthesis of a new phage-specific protein. This T 4 coded protein m a y be involved directly in the initial attack on host DNA or may, however, activate cellular nucleases similar to colicin E2. Colicin E2 has no endo- or exonueleolytic in vifro 3s (P. RINGROSE, unpublisLed observations), and before discussing possible mechanisms whereby eolicin E2 initiates the degradation of E. coli DNA, it is worthwhile considering which of the deoxyribonucleases so far characterised m a y be involved. Endonuclease I was originally an attractive candidate for the initial stages of DNA degradation, since it is located in the region of the bacterial membrane and is inactivated by t R N A 39. But as there was no detectable degradation of RNA during the first hour of colicininduced DNA degradation (P. R1NGROSE,unpublished observations) and endonuclease I is specifically involved in a single-hit double-strand cleavage la,4°, it is unlikely that colicin E2 acts b y removing t R N A and activating this enzyme, Endonuclease I I (ref. 41) produces single-strand scissions in native DNA and is not inhibited b y tRNA. Together with endonuelease IV it appears to be involved in recombination of T 4 DNA 4~, and although it is synthesised on T 4 infection after host DNA degradation, a similar activity is found in normal E. coli a3. This observation, together with the finding that certain ultraviolet-sensitive and recombination-deficient mutants of E. coli are resistant to colicin E2 (see ref. 4) gives support to the hypothesis that endonuclease I I or a similar enzyme m a y be involved in Stage I of coliein-initiated breakdown of E. coli DNA. Endonuclease IV specifically attacks singlestranded DNA and is believed to fragment double-stranded DNA after endonuclease I I has cleaved single strands of the native DNA and these single-strand snips have been opened up by an exonuclease, thus revealing localised regions of single-stranded Biochim. Biophys. Acta, 213 (i97 o) 320-334

PRODUCTS OF

DNA

DEGRADATION BY COLICIN

E2

333

DNA in the duplex molecule 4~. The fragments produced are large and have been reported as 700 nucleotides long for fd phage and 104 nucleotides long (6. lO s daltons) for 2 phage *J. However, as mentioned earlier, a cytosine-specific nuclease is also possible in the initial stages. Other possibilities are the restriction enzymes that enable bacterial cells of one strain to destroy DNA from foreign strains. In this context colicin E2 m a y act by making native DNA appear foreign to its own cellular restriction mechanisms. Endonuclease I I I is a well-characterised example and functions in tile restriction of ;t phage 44. This enzyme is known to initiate single breaks and to be dependent on ATP, Mg 2+ and S-adenosylmethionine. It is well established that colicin E2-induced DNA degradation is inhibited by metabolic inhibitors such as dinitrophenol and colicin K (ref. i). The exonuclease involved in Stage I I I could be exonuclease IV (ref. 39) which is known to degrade oligonucleotides rapidly to mononucleotides whilst being relatively inactive on native or denatured DNA. Possible modes o/action/or colicin E2 Colicin E2 can initiate the breakdown of cell DNA either by activating a specific endonuclease :exonuclease system or b y causing a change in state of the DNA so that DNA is rendered susceptible to an already active nuclease system. The first possibility is in keeping with the membrane transmission system s,as which proposes that the colicin interferes with delicately balanced cellular control mechanisms, so that nucleases normally present in E. coli but only used under strict control in replication, recombination, repair and restriction become uncontrolled and rapidly destroy the cell DNA. The second possibility can encompass a variety of interactions of the colicin molecule with cellular DNA. Colicin E2 does not possess a n y endo- or exonucleolytic activity but could denature the double helix so that one of the repair nucleases was activated. Isopycnic centrifugation in CsC1 and C%SO 4 and optical rotatory dispersion measurements (P. RINGROSE, unpublished results) indicate that DNA is not denatured b y colicin E2 in vivo or in vitro. Possibilities such as the colicin molecule binding to specific regions of DNA as factor "a" presumably does to cytosine-rich clusters and causing local distortions akin to alkylation have not been ruled out and m a y explain the effect of colicin E2 on DNA melting (P. RI~GROSE, in preparation). It is also possible that colicin E2 interferes with DNA interaction with the membrane at the site of replication, where it is known that nuclease activity exists. However, all but the transmission system hypothesis and the last possibility suffer from the disadvantage that they all assume penetration of the cell by the colicin molecule or a hypothetical component, and this is difficult to reconcile with the trypsin recovery results and the results with [14Clcolicin45. ADDENDUM

After preparation of this manuscript the paper b y OBINATA AND MIZUNO4~ appeared in this journal and confirmed the results described above for the later stages of colicin E2 action. However, their results differed from those reported here in that they found a time lag in the appearance of single-strand breaks and no trypsin-induced repair of the DNA scissions. Biochim. Biophys. Acga, 213 (197 o) 32c~334

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ACKNOWLEDGEMENTS

The author is indebted to the members of the Subdepartment of Chemical Microbiology for instructive criticism, and especially to Dr. D. Kerridge for his assistance in the preparation of the manuscript. This work was carried out during the tenure of a Medical Research Council Scholarship for training in research methods. REFERENCES i M. NOMURA, Cold Spring Harbor, Syrup. (2uant. Biol., 28 (1963) 315 . 2 I. B. HOLLAND, J. Mol. Biol., 31 (1968) 267. 3 K. NOSE AND D. MIZUNO, J. Biochem. Tokyo, 64 (1968) i. 4 E. J. THRELFALL AND l. I~. HOLLAND, J. Gen. ~/Iicrobiol., 53 (1968) v. 5 B. L. REYNOLDS AND P. R. REEVES, Biochem. Biophys. Res. Commun., i i (1963) 14o. 6 B. L. REYNOLDS AND P. l{. REEVES, J. Bacteriol., i o o 11969) 3Ol. 7 H. ANDERSON, Proc. Natl. Acad. Sci. U.S., 32 (1946) 12o. 8 H. I{. HERSCHMAN AND D. R. HELSINKI, fir. Biol. Chem., 242 (1967) 5360. 9 C. HILL AND I. B. HOLLAND, J . Bacteriol., 94 (1967) 677. i o R. P. BOYCE AND I{. B. SETLOW, Biochim. Biophys. Acta, 61 (1962) 618. I I J. MARMUR, J. 1Viol. Biol., 3 (1961) 2o8. 12 D. V~r. SMITH AND P. C. HANAVv'ALT, Bioehim. Biophys, Acta, 149 / i 9 6 7 ) 519. 13 F. W. STUDIER, J. Mol. Biol., I I (1965) 373. 14 R. A. ,'V[CGRATH AND R. \ ¥ . WILLIAMS, Biophys. J., 7 (1967) 309 • 15 M. NOMURA, B. D. HALL AND S. SPIEGELMAN, J. Mol. Biol., 2 (196o) 306. 16 C. A. THOMAS AND J. A. BELSON, in G. L. CANTONI AND D. ~1. DAVIES, Procedures in Nucleic Acid Research, H a r p e r a n d Row, N e w Y o r k - L o n d o n , 1966, p. 553. 17 E. BURGI AND A. D. HERSCHEY, Biophys. J., 3 (1963) 309. i 8 M. BAZARAL AND D. 11. HELSINKI, J. Mol. Biol., 36 (i968) 185. 19 P. DOTY, B. MCGILL AND S. ]:{ICE, Proe. Nail. Aead. Sci. U.S., 44 (1958) 4342o L. RUBENS'rEIN, C. A. THOMAS AND A. D. HERSHEY, Proc. Natl. Acad. Sci. U.S., 47 (1961) I 113. 21 D. V¢. SMITH AND P. C. HANA~VALT, .]. Mol. Biol., 46 (I969) 5722 W. SZYBALSKI, in L. GROSSMAN AND K. MOLDAVE, Methods in Enzymology, Vol. X I I , Aca d e m i c Press, New Y o r k - L o n d o n , 1968, p. 33o. 23 E. CUNDLIFFE, Mol. Pharmacol., 3 (1967) 4 °1. 24 J. SMARDA, Zentr. Bakteriol. Parasitenk. Abt. I. Orig., 196 (1965) 24o. 25 T. BEPPU AND IQ ARIMA, ]. Bacteriol., 93 (1967) 8o. 26 J. SMARDA AND [7. TAUBENECK, J. Oe~. 2~Iicrobiol., 52 (1968) I 6 i . 27 1,2. SAKABE AND I)t. OKAZAKI, Biochim. Biophys. Acta, 129 (1966) 654. 28 M. NOMURA, K. MATSUBARA, 11. OKAMOTO AND I{. FUKIMURA, ,[. Mol. Biol., 5 (1962) 535. 29 C. A. THOSIAS AND I(. I. BERNS, J. Mol. Biol., 3 ( I 9 6 I ) 277. 30 M. MESELSON, F. ~V. STAHL AND J. XrlNOGRAD, Proc. Natl. Mead. Sci. U.S., 43 (1957) 58131 C. A. THOMAS AND T. C. PINKERTO~, J. 3,I01. Biol., 5 (1962) 35632 N. SUEOKA, Proc. Natl. Acad. Sci. U.S., 45 (1959) 148o. 33 P. DOTY, J. MARS~UR AND N. SUEOKA, Structure and Function o/Genetic Elements, Brookhaven Syrup. Biol., 12 (1959) 1. 34 Z. OPARA-I(UBINSKA, Z. KURYLO-BOROWSKA AND \V. SZYBALSKI, Biochim. Biophys. Acta, 27 (1963) 298. 35 E. M. KUTTER AND J. S. WIBERG, J. Mol. Biol., 38 (1968) 395. 36 "~V. SZY13ALSKI, H. [4~UBINSKI AND ~¥. SHELDRICK. Cold Spring Harbor Syrup. ()uant. Biol., 31 (I966) I23. 37 [(. TAYLOR, Z. HRADECNA AND \V. SZYBALSKI, Proc. Natl. Acad. Sci. U.S., 57 (1967) i618. 3 8 .'V[. NOMURA, Proc. Natl. Acad. Sci. U.S,. 52 (1964) 1514. 39 C. C. I{ICHARDSON, Ann. t?ev. Biockem., 38 (1969) 795. 4 ° E. MELGAR AND D. A. GOLDTItWAIT, J. Biol. Chem., 243 (1968) 44o9. 4 t J. HURWlTZ, A. BECKER, M. L. GEI;TER AND M. GOLD, J. Cellular Comp. Physiol., 7 o, SuppI I (1967) 181. 42 P. SADOU,SI(I, T~. (~INSBERG, A. YUDELEVITCH, L. FEINER AND J. HURV¢ITZ, Cold Sprin,~ Harbor Symp. CHant. Biol., 33 (1968) 165. 43 E. C. FRIEDBERG AND D. A. GOLDTHWAIT, Federation Proc., 23 (1964) 94 o. 44 M. MESELSON AND R. YUA~, Nature, 217 (1968) IIIO . 45 A. MAEDA AND M. NOMURA, .,l. Bacteriol., 91 (1966) 685. 46 M. OBINATA AND I). ~]~IZUNO, Biochim. Biophys. Acta, 199 (197 o) 33 TM

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