The Escherichia coli B restriction endonuclease

BIOCHIMICA ET BIOPHYSICA ACTA

219

BBA 96322

THE ESCHERICHIA

COLI B RESTRICTION ENDONUCLEASE

DAISY ROULLAND-DUSSOIX AND H E R B E R T W. B O Y E R

Department o/Microbiology, University o/Cali/ornia, San Francls¢o Medical Center, San Francisco, Call/. 94122 (U.S.A.) (Received May 27th, 1969)

SUMMARY

I. The restriction endonuclease of Escherichia coli B has been purified IOOOfold from crude extracts. 2. It has been found to be similar to the K I 2 and P I restriction endonucleases s, i.e., it requires ATP, S-adenosyl-L-methionine and Mg ~+ and unmodified DNA for enzymatic activity and has an estimated large molecular weight (approx. 300 ooo daltons). 3. It introduces a limited number of double strand scissions in unmodified 2vir DNA, Escherichia coli chromosomal DNA and probably one double strand scission per fd replicative form DNA. The double strand scission in the unmodified fd replicative form DNA occurs b y a two-step mechanism. 4. Replicative form DNA generated from an fd m u t a n t which is only restricted by Escherichia coli B b y a factor of I - I O -~ (versus I . lO -4 for wild type fd) is also a substrate for the B restriction endonuclease. Cosedimentation of the endonucleasetreated wild type and m u t a n t replicative form DNA results in qualitatively identical patterns of DNA distribution.

INTRODUCTION

Escherichia coli strains K I 2 and B (among others) have a mechanism for rejecting non-homologous DNA, i.e., DNA originating in a cell of a different strain (for a review on the subject see ref. I). Phage, bacterial and episomal DNA, when transferred from E. coli K I 2 to E. coli B, or vice versa, are subject to host controlled restriction, or specific degradation b y the recipient cell 2. Several lines of evidence suggested that this enzymatic mechanism involves an endonuclease, recognizing and introducing double strand scissions at a limited number of sites (unique sequences of nucleotide base pairs) on a DNA molecule a-s. Other evidence suggested that the endonuclease did not recognize and carry out a double strand scission at these sites, if one or two bases in the site were methylatede, L The former has been confirmed with the purification and characterization of an endonuclease from E. coli K I 2 with the characteristics expected of the restriction endonuclease s. An enzymatic activity in extracts of E. coli B which specifically inactivated the infectivity of unmodified fd replicative form DNA has also been reported 9. We report here the purification of this activity of E. coli B, its characteristics and effect on several unmodified DNA substrates. Biochim. Biophys. Acta, 195 (1969) 219-229

220

D. ROULLAND-DUSSOIX, H. W. BOYER

MATERIALS AND METHODS

Media Tryptone broth is (g/l): Bacto-tryptone (IO.O) and NaC1 (5.o). fd medium is (g/l): Tris (I2.I), NaC1 (5.6), NH4C1 (I.O), N a 2 H P O 4 . 7 H 2 0 (I.6), and KH2PO 4 (o.45), MgSO4 (2.5), D-glucose (6), Casamino acids (IO), and 6.6 ml of 12 M HC1. L-bloth is (g/l): Bacto-tryptone (IO), yeast extract (5) and NaC1 (IO). The medium described by ROBLIN1° was used for labeling DNA with 32p. Preparation o! labeled nucleic acid Phage ~ DNA. Bacterial hosts were grown to log phase with vigorous aeration in 200 ml of tryptone broth. For 3H-labeled 2 DNA, 2-deoxyadenosine (250/~g/ml) was added 15 rain prior to the addition of 2 mC of [3H]thymidine (specific activity, 6. 7 mC/mmole, New England Nuclear) and infection was initiated 30 rain later. For 32P-labeled DNA, 2 mC of H a 3~po 4 (carrier free) (New England Nuclear) were added 30 min prior to infection. The cultures were infected to give a multiplicity of infection of I.O with 2vir and incubated until lysis occurred. Lysogenic cultures were induced b y ultraviolet irradiation, resuspended in medium containing E3H]thyrnidine or H332pQ and aerated until lysis. The labeled phage stocks were purified by low and high speed centrifugations and by zone centrifugation in a multi-step sucrose gradient. The sedimented phage band was collected b y aspiration and dialyzed against 3 2-1 vol. of 0.05 M Tris buffer (pH 7.5), o.I mM E D T A (Tris-EDTA buffer). The DNA was extracted 3 times with buffered phenol and the DNA dialyzed against 3 2-1 vol. of T r i s - E D T A buffer over 48 h at 4 °. The concentrations of DNA were determined with a Beckman DU Spectrophotometer (A2eo 6.6 = 330 #g DNA/ml). Bacterial DNA. A ioo-ml L-broth culture of E. coli (r~m~) with I mC of [3H]thymidine and 2-deoxyadenosine (25 ° #g/ml) was grown to saturation and the DNA extracted b y a procedure described by LARK11. ]d replicative ]orm DNA. Bacterial cultures (HfrH with K or B restriction and modification properties) were grown in 500 ml of fd medium with I mC of E3H]thymidine and 2-deoxyadenosine (250 #g/ml) or IOO ml of a defined medium 1° with 2 mC H33zpo 4. The radioisotopes were added about 15 min prior to the infection of the culture with fd phage. At a cell density of about 5" lO8 cells/ml, chloramphenicol was added to the culture (final concentration of 30/~g/ml) and after a 3-min incubation, the culture was infected with the phage fd at a multiplicity of about 20. The culture was incubated for an additional 75 rain at 37 °, and the cells were harvested by centrifugation and washed in NaC1-EDTA buffer (0.6 M NaC1, 25 mM EDTA, p H 8.0) The labeled replicative form DNA was purified by a procedure modified after JANSZ et al. 1~. After lysis with sodium dodecyl sulfate, the p H of the suspension (ioo ml) was adjusted to 12.5 with I M NaOH and after 3 rain adjusted to p H 8.0 with I M HC1. Debris was eliminated b y centrifugation and the supernatant concentrated to about 5-1o ml b y flash evaporation. Aliquots (2.5 ml) were applied to a column (3 c m × 5 o cm) of agarose (A 5 o m , Bio Rad) equilibrated with o.I M K2HPO 4KH2PO 4 (pH 6.8), 0.6 M NaC1. The single strand DNA and replicative form eluted together. The fractions containing the radioactivity were filtered under very low pressure through Millipore nitrocellulose filters (HA, 0.45 #, washed first in 95 % ethanol and then in o.I M K2HPO4-KH2PO 4 (pH 6.8), 0.6 M NaC1).The filtrates were nm

t3iochim. Biophys. Acta, 195 (1969) 219-229

E. coli B RESTRICTIONENDONUCLEASE

221

combined and concentrated b y flash evaporation and dialyzed against 3 2-1 vol. of T r i s - E D T A buffer. Polio replicative [orm RNA. asP-labeled polio replicative form RNA was a generous gift from J. M. Bishop.

Assay/or restriction endonuclease o] E. coli B The reaction mixture contained the following in a total volume of IOO#1: Tris buffer (pH 7.6), 9#moles; MgC12, 0.2/~moles; S-adenosyl-L-methionine, 0.050 /,moles; ATP, 0.050 #mole; ~ L3~PJDNA with B modification, 2.5 nmoles a n d ~ EaHIDNA with no modification, 2.5 nmoles. The reaction mixtures were incubated with the appropriate amounts of extract at 37 ° for variable times, and the reaction was stopped b y adding 2 amoles of E D T A and IO #1 of 20 °/o sodium dodecyl sulfate. The reaction mixture was layered on 5-20 % linear sucrose gradients and centrifuged in a Beckman model L2-65 preparative ultracentrifuge. (SW 5o, SW 65 or SW 56 rotors were used for routine assays.) After centrifugation the tubes were punctured at the bottom and 9-drop fractions were collected on W h a t m a n filter paper No. I, cut into 2.5-cm squares and dried with an infrared lamp. The papers were either washed with cold 2 M HC1, dried and counted or counted directly without washing. No significant difference was found between the two methods. The papers were counted in vials with io ml of scintillation mixture (8 pints of toluene; 16o ml of New England Nuclear Liquifluor containing IOO g 2,5-diphenyloxazole plus 1.25 g 1,4-bis-(5-phenyloxazolyl-2)benzene in toluene) in a Beckman LS2oo liquid scintillation counter. The activity of the enzyme is expressed in terms of the amount of unmodified DNA degraded to a form sedimenting slower than modified DNA. One unit is equal to the degradation of i . lO -15 mole of unmodified DNA per h. Although we could observe a differential inactivation of unmodified and modified infectious fd replicative form DNA, the procedure in our hands was not reliable even with our most purified preparation. Therefore, the zone sedimentation assay was used routinely. With the SW 56 6-place rotor we managed 18-24 assays per day.

Preparation o/ extracts. A derivative of E. coli ILOO, obtained from H. Hoffman-Berling, deficient in endonuclease I activity and containing the restriction and modification alleles of E. coli B was used for the purification of the restriction endonuclease of E. coli B. Cultures of this derivative were prepared in lO-12 1 of L-broth contained in a New Brunswick Fermenter. The culture was incubated at 37 ° with constant agitation (500 rev./min) and aeration (8 l/rain) until late log phase and the culture was harvested b y continuous flow centrifugation (22 ooo ×g) in a Servail Refrigerated Centrifuge. Yields were usually 60-70 g of wet packed cells. The cells were resuspended in extract buffer (o.oi M K2HPO4-KH2PO 4 (pH 7.o), 2 mM EDTA, 7 mM fl-mercaptoethanol) and centrifuged at low speed (5800 × g for IO mill). The washed cell paste was ground with aluminum hydroxide powder (Alcoa) at double the mass of the cell paste. The ground cells were resuspended in IOOOml of extract buffer with the aid of a refrigerated Waring blender. All subsequent operations were carried out at 4*. The extract was cleared of debris and alumina b y centrifugation at low speed, and the supernatant was our crude extract fraction. Biochim. Biophys. Acta, 195 (1969) 219-229

222

D. ROULLAND-DUSSOIX,

H. W. BOYER

Purification o/ the E. coli B restriction endonuclease Streptomycin sulfate (5 %) was added slowly to the crude extract to a final concentration of 0.375 g/ml and centrifuged at low speed (5000 ×g for IO min) to remove the precipitate. An additional 0.375 g/ml was added and stirred slowly overnight and the precipitate removed b y a similar low speed centrifugation. The supernatant was treated with 31 g of solid (NH4)2S Q per IOO ml and centrifuged at high speed (12 ooo ×g for 20 min). The precipitate was resuspended in approx. 75 ml of extract buffer and dialyzed against a total of 16 1 of extract buffer. The dialyzed preparation was applied to a DEAE-cellulose column (3 cm ×4o cm) equilibrated with the extract buffer and eluted with an 0.0-0.5 M NaC1 linear gradient. Tile restriction endonuclease activity eluted at about 0.2 M NaC1. The fractions containing the activity were pooled, dialyzed against extract buffer, p H 7.0, and concentrated to a small volume b y dialysis against dry Sephadex G-I5O. This concentrated fraction was layered on a Sephadex G-2oo column equilibrated with extract buffer, p H 7.0. The restriction endonuclease activity eluted about one fraction behind the void volume. The three tubes at the peak were pooled and applied to a phosphocellulose column (3 c m × 3 5 cm) equilibrated with extract buffer and eluted with a linear gradient of o-I.O M NaC1. The activity eluted at about 0.4-0. 5 M NaC1 and the peak tubes were pooled and dialyzed against extract buffer. The preparation contained about IO/,g protein/ml in a total volume of 15 ml and represented about a iooo-fold purification (see Table I). Application of a 5o-#1 sample to a 5 % acrylamide gel (pH 8.0 buffer) yielded about 8-1o discrete bands. This preparation was free of detectable endonuclease I (ref. 14) or I I (ref. 15) activities, DNA polymerase activity and very little or no exonuclease activity. TA13LE

I

P U R I F I C A T I O N OF t3 R E S T R I C T I O N E N D O N U C L E A S E

Step

Streptomycin supernatant (NH4)2SO4 DEAE-cellulose Sepkadex G-2oo Phosphocellulose

Volume (ml)

Total mg protein

9oo I5o 155 32 I5

36oo 21oo 155 6.1 0. 5

Total units

-3 75o 3 515 i 122 800

ooo ooo ooo ooo

Recovery (%)

Speci[ic activity

-ioo 94 3° 20

-i 22 182 i 600

785 800 ooo ooo

RESULTS

Requirements/or the reaction Fig. I represents the results of a typical assay for restriction endonuclease activity. I t is clear that the ~ DNA originating in the E. coli C strain is degraded to fragments about 1/2 the size of the ~ DNA originating in E. coli B which is not degraded by the enzyme. Molecular weights of the DNA fragments were estimated b y the formula of BURGI AND HERSHEY16. The molecular weight of the ~ DNA used here (~tcb2 DNA = 27 • Io s daltons) 17 was the standard marker for DNA molecular weight estimates. The degradation appears to be limited since a doubling of the Biochim. Biophys. Acta,

195

(1969)

219-229

E. coli B RESTRICTIONENDONUCLEASE A

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Fig. I. ;t [DHIDNA f rom E . coli C (2.o • lO -14 moles) a n d ;t Ls~PIDNA f r o m E . coli ]3 (z.o • IO -14 moles) were i n c u b a t e d w i t h o u t (A) a n d w i t h (B) t h e r e s t r i c t i o n e n d o n u c l e a s e of E. coli t3 for 3 ° miD a t 37 ° u n d e r c o n d i t i o n s d e s c r i b e d in MATERIALS AND METHODS. T h e r e a c t i o n s w e re s t o p p e d b y t h e a d d i t i o n of IO/~1 of s o d i u m d o d e c y l s u l f a t e (20 %) a n d l a y e r e d on 4.5-ml 5-20 % l i n e a r s u c r o s e g r a d i e n t s (0.o 5 M Tris (pH 7.5), o . i mM E D T A , o . i M NaC1) a n d c e n t r i f u g e d a t 55 ooo r e v . / m i n in an S W 56 r o t o r for 12o miD a t IO °. T h e g r a d i e n t s w e re f r a c t i o n a t e d a n d p r o c e s s e d as d e s c r i b e d in MATERIALS AND METHODS. O - - - O , SH; O - @ , ~ P . Fig. 2. ~ [SH_DDNA f r o m E . coli C (2.o. IO -]4 moles) a n d $ [DZP]DNA from E . coli B (2.0. lO -14 moles) were i n c u b a t e d w i t h (A) 4 ° a n d (]3) 80 u n i t s of B r e s t r i c t i o n e n d o n u c l e a s e for 3 ° miD a t 37 °. T h e D N A w a s s u b j e c t e d to zone s e d i m e n t a t i o n ill an S W 65 r o t o r for 8o miD a t 60 ooo r e v . / m i n , i o °, a n d t h e g r a d i e n t s w e r e f r a c t i o n a t e d as d e s c r i b e d in MATERIALS AND METHODS. The ~ [s~P]DNA f r o m E . coli B (27 • lO 6 d a l t o n s ) s e r v e d as a reference m a r k e r to d e t e r m i n e t h e f r a c t i o n corres p o n d i n g to 15 • lO s a n d 5 " lOS d a l t o n s . 0 - 0 , a l l ; (2)- - - © , s2p.

amount of restriction endonuclease does not change the sedimentation pattern of the 2 DNA from E. coli C (Fig. 2), although the restriction endonuclease activity is directly proportional to the enzyme concentration (Fig. 3). MESELSON AND YUAN8 have characterized the restriction endonueleases of E. coli K I 2 and the prophage PI. These nucleases are rather unique in that they have an absolute requirement for S-adenosyl-L-methionine and ATP. It can be seen in Table I I that this is also true for the restriction endonuclease of E. coli B. Similar results were reported with a biological assay for the restriction endonuclease activity

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Fig. 3. C o n s t a n t a m o u n t s of ~ [~H]DNA from E . coli C (4.0 • IO -14 moles) a n d ~ ? H ] D N A f r o m E . coli B (1. 5 • lO -]4 moles) were t r e a t e d w i t h v a r i a b l e a m o u n t s of B r e s t r i c t i o n e n d o n u c l e a s e ( p h o s p h o c e l l u l o s e f r a c t i o n ) a t 37 ° for 5 m i d a n d t h e r e a c t i o n s t o p p e d w i t h s o d i u m d o d e c y l sulfate. T h e a m o u n t of ;t D N A f r o m E . coli C d e g r a d e d w a s d e t e r m i n e d b y zone s e d i m e n t a t i o n as d e s c r i b e d for Fig. 2 a n d p l o t t e d as a f u n c t i o n of e n z y m e c o n c e n t r a t i o n . Fig. 4- A series of r e a c t i o n s w i t h ;t [aH~DNA f r o m E . coli C (4.o • lO -14 moles) a n d ~t [s~P]DNA f r o m E. coli B (1. 5 • lO -14 moles) w a s i n c u b a t e d a t 37 ° w i t h a c o n s t a n t a m o u n t of B r e s t r i c t i o n e n d o n u c l e a s e s (approx. 35 u n i t s ) a n d s a m p l e s r e m o v e d a t v a r i o u s t i m e i n t e r v a l s . The a m o u n t of d e g r a d e d Jl D N A f r o m E . coli C w a s d e t e r m i n e d b y zone c e n t r i f u g a t i o n a n d p l o t t e d as a funct i o n of i n c u b a t i o n t i m e in m i n u t e s . B i o c h i m . B i o p h y s . Acta,

195 (1969) 219 229

224 TABLE

D. ROULLAND-DUSSOIX, H. W. B O Y E R II

REQUIREMENTS FOR RESTRICTION ENDONUCLEASE B ACTIVITY T i l e B r e s t r i c t i o n e n d o n u c l e a s e a c t i v i t y w a s a s s a y e d a s d e s c r i b e d i n MATERIALS AND METHODS.

Complete system

zoo units

-- S - a d e n o s y l - L - m e t h i o n i n e -- A T P - - M g 2+ -- S-adenosyl-L-rnethionine + N A D + + c a f f e i n e (o.i %)

< < < < <

5 5 5 5 5

of E. coli 13 (ref. 9). Like the K I 2 and P I restriction endonucleases, the B enzyme also requires Mg~+ for activity. There is an absolute requirement for 2 DNA originating in a strain other than E. coli B. The ratio of S-adenosyl-L-methionine to ATP must be held constant (i.e., i.o) for maximum activity of the restriction endonuclease. If the molar ratio of S-adenosyl-L-methionine and ATP are not held constant, the restriction endonuclease activity decreases rapidly around ATP/S-adenosyl-L-methionine or S-adenosyl-L-methionine/ATP ratios above IO and below 0.2. However, maximum activity of the endonuclease activity was observed over a concentration range of 50-2500 nmoles of A T P and S-adenosyl-L-methionine when the ratio of S-adenosyl-L-methionine to ATP was equal to unity. The optimum pH range for restriction endonuclease activity is from pH 7.2 to 7.8, which is in fair agreement with the pH range for the KI2 and P I restriction endonucleases s. The endonuclease reaction is linear over a 5-1old concentration of enzyme (Fig. 3)- With a constant amount of e n z y m e the reaction proceeded rapidly for a few minutes and then became first order (Fig. 4).

Molecular size estimate The elution of the B restriction endonuclease close to the void volume of a Sephadex G-2oo column suggested that the molecular weight of the enzyme to be 200 ooo daltons or greater. Indeed, zone sedimentation analysis with catalase as a marker protein (tool. wt. ---- 244 ooo) showed that the restriction endonuclease sedimented faster than catalase with a molecular weight estimate of about 300 ooo daltons. We therefore estimate a molecular weight in the range of 200 000-300 ooo daltons. Speci]icity o/the B restriction endonuclease. Experiments in vivo predict that the 13 restriction endonuclease would attack unmodified bacterial and episomal DNA as well as unmodified 2 DNA or other phage DNA. We used ~H-labeled DNA extracted from a mutant bacterial strain of E. coli B which no longer modified 2 DNA as a substrate in the restriction assay. The results are presented in Fig. 5, and it can be seen that the ]3 restriction endonuclease brings about a degradation of the unmodified bacterial DNA. This degradation was complete, since treatment of the same amount of unmodified bacterial DNA with 12o units of the 13 restriction endonuclease did not change the sedimentation pattern of the degraded DNA. Zonal sedimentation of the E. coli chromosomal DNA leads to skewed l~iochim. Biophys. Acta, 195 (1969) 2 1 9 - 2 2 9

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Fig, 5. SH-labeled bacterial D N A (4/zg) e x t r a c t e d f r o m an r - m - m u t a n t of E. coli 13 and ~ [3H]D N A f r o m E. coli B were t r e a t e d w i t h 4 ° u n i t s of the B restriction endonuclease in the absence (A) and presence (B) of S-adenosyl-L-methionine for 3 ° rain at 37 °. The reactions were s t o p p e d w i t h s o d i u m dodecyl sulfate and subjected to zone centrifugation (80 m i n at 6o ooo rev./min, S W 65 rotor, io °) in 5-2o % sucrose gradients and fractionated. The ~ [32P]DNA f r o m E. coli B peak (27 • io" daltons) was used as a m a r k e r to e s t i m a t e the molecular weights of the E. coli DNA. 0-0, 3H; O - - - O , 3~p.

distribution with a broad peak at 6o-lO s daltons. Treatment with the B restriction endonuclease and subsequent zonal sedimentation leads to a more homogenous distribution of smaller molecular weight D N A with a very broad peak at about 23" lO s daltons. The degradation was dependent on the presence of S-adenosyl-L-methionine. The male specific phage, fd, is restricted in vivo by E. coli B but not E. coli KI2 (ref. 18). We have examined the activity in vitro of the B restriction endonuclease with modified and unmodified fd replicative form D N A . In neutral sucrose gradients, the fd replicative form preparations usually display two species of D N A , one with an S value of about 26 (replicative from I) and one with an S value of about 17 (replicative form II) (Fig. 6). The relative amounts of I and II were variable from

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Fig. 6. Zone centrifugation of 9.o • IO-z4 moles of 3H-labeled fd replicative f o r m f r o m E. coli and 8.1 • lO -14 moles of 3zP-labeled replicative f o r m f r o m E. coli B. The D N A (in IOO/zl) was ered on a i3.5-ml 5-20 % sucrose gradient (0.05 M Tris (pH 7.5), o. ~ mM E D T A , o.i M NaC1) centrifuged for 9 h at 38 ooo rev./min, io °. The e s t i m a t e d S values for the t w o p e a k s of D N A determined b y a p r e v i o u s e x p e r i m e n t w i t h 3H-labeled replicative f o r m D N A f r o m E. coli K I 2 )t [8zP]DNA frorp E. coli B (34 S). O - Q , 3H; O - - - O , 3 z p .

KI2 layand was and

Fig. 7. A m i x t u r e of 3H-labeled fd replicative form D N A from E. coli K I 2 (6.0. lO T M moles) and 3~P-labeled fd replicative f o r m D N A from E. coli B (2. 7. io -1~ moles) w a s i n c u b a t e d at 37 ° for 3 ° rain w i t h 2o u n i t s of the B restriction endonuclease and t h e n subjected to zone centrifugation as described in Fig. 6. 0 - 0 , SH; O - - - O , 32p. Biochim. Biophys. Acta, 195 (I969) 219-229

226

D. ROULLAND-DUSSOIX, H. W. BOYER

one preparation to another, and replicative form I I could be eliminated b y repeating the alkaline denaturation and neutralization step (see MATERIALS AND METHODS) and filtration through a nitrocellulose membrane filter. Therefore, replicative form I corresponds to the twisted circular replicative form DNA and replicative form I I corresponds to replicative form DNA with one or more single strand breaks per molecule 19. Treatment of a mixture of 3H-labeled replicative form from E. coli K I 2 and a2p_ labeled replicative form from E. coli B with the restriction endonuclease but without S-adenosyl-L-methionine did not change the normal sedimentation pattern of the DNA. However, in the presence of the enzyme plus S-adenosyl-L-methionine, most of the unmodified replicative form DNA sediments as a third species (replicative form I I I) and usually a small amount of replicative form II, while the modified DNA sediments as Species I and I I in the same relative amounts as in the control (Fig. 7)- The S value for Species I n is i5 relative to polio virus replicative form RNA (Fig. 8). Polio replicative form RNA is a linear molecule with a molecular weight of about 4" lO6 daltons and the hydrodynamic properties of this molecule are similar to linear DNA molecules 2°. The relative molecular weight estimate for the fd replicative form I n is 2.8.Io 6 daltons. This is in fair agreement with molecular weight estimates of virion fd DNA 21. The ratio of replicative form I I to replicative form I n s values is i . i and corresponds to the ratio expected for non-twisted circular DNA and linear DNA. Since we cannot detect a fourth species of replicative form DNA after exhaustive treatment with the restriction endonuclease, we conclude that there is either one double strand scission per unmodified fd replicative form molecule or several double strand scissions close to one another. ARBER AND Kt~HNLEIN22 recovered a m u t a n t of the fd phage (fd/~1), which was restricted Ioo-fold less than the wild type phage by E. coli B, and from this

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Fig. 8. A m i x t u r e of 3H-labeled fd r e p l i c a t i v e form D N A f r o m E. coli K I 2 (I.2. l o 13 moles) a n d polio v i r u s 32p-labeled r e p l i c a t i v e form R N A (1.o.io -13 moles) w a s t r e a t e d w i t h 5o u n i t s of B r e s t r i c t i o n e n d o n u c l e a s e for 3 ° m i n a t 37 ° a n d s u b j e c t e d to zone s e d i m e n t a t i o n as d e s c r i b e d in Fig. 6. Po lio v i r u s r e p l i c a t i v e f o r m R N A is a l i n e a r m o l e c u l e w i t h a n S v a l u e of 17 a n d m o l e c u l a r w e i g h t of 4" lO6 daltons21. 0 - 0 , 8H; (2)- - - O , 32p. Fig. 9. A m i x t u r e of a l l - l a b e l e d fd r e p l i c a t i v e f o r m D N A from E. coli K I 2 (1.2. i o -la moles) a n d 8q°-labeled fd /'1 r e p l i c a t i v e f o r m D N A from E. coli K I 2 ( i . o . lO -18 mole) w a s t r e a t e d w i t h 5 ° u n i t s of B r e s t r i c t i o n e n d o n u c l e a s e for 3 ° m i n a t 37 ° a n d s u b j e c t e d to zone s e d i m e n t a t i o n as d e s c r i b e d in Fig. 6. Q - O , 8H; O - - - O , 82p.

Biochim. Biophys. Acta, 195 (1969) 219 229

227

E. coli g RESTRICTION ENDONUCLEASE

mutant they isolated a double mutant (fd/~1#~) which was not restricted at all. These mutants were interpreted by assuming that there are two sites on the unmodified wild type fd replicative form susceptible to the B restriction endonuclease, one on the unmodified fd #1 replicative form and none on the unmodified fd/~/~2 replicative form. Zonal centrifugation of 8~P-labeled/z 1 replicative form from E. coli KI2 and all-labeled replicative form from E. coli KI2 treated with the B restriction endonuclease results in identical sedimentation patterns for the/~1 replicative form and wild-type replicative form D N A (Fig. 9). On the basis of this result, the two sites postulated for the fd replicative form would have to be less than 5 ° nucleotides apart. This is based on the assumptions that the fd replicative form D N A contains about 5000 nucleotide base pairs 21 and that a 1-2 fraction difference in sedimentation patterns would be discernible. We therefore propose an alternative explanation for these data, that is, the wild type fd replicative form has one site (a sequence of nucleotide base pairs) susceptible to the B restriction endonuclease and the/'1 replicative form has a mutated site for which the enzyme has a lowered affinity. A second mutation in this site could then lead to the restriction-insensitive fd #1#2 mutant.

A two-step endonuclease attack Another similarity between the K and B restriction endonuclease is the twostep nature of the double strand scissions. MESELSON AND YUAN s demonstrated that

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F i g . i o . M i x t u r e s of 3 H - l a b e l e d fd r e p l i c a t i v e f o r m DiWA f r o m E. coli K I 2 ( 5 . 0 - lO -14 m o l e s ) a n d 32P-labeled p o l i o v i r u s r e p l i c a t i v e f o r m R N A ( i . o - lO -13 m o l e s ) w e r e t r e a t e d w i t h IO u n i t s o f B r e s t r i c t i o n e n d o n u c l e a s e for (A) 5 rain a t 37 ° w i t h o u t S - a d e n o s y l - L - m e t h i o n i n e , (B) 3 ° sec a t 37 ° w i t h S - a d e n o s y l - L - m e t h i o n i n e , (C) 6 o sec a t 37 ° w i t h S - a d e n o s y l - L - m e t h i o n i n e a n d (D) 1 2 o sec a t 37 ° w i t h S - a d e n o s y l - L - m e t h i o n i n e a n d s u b j e c t e d to z o n e s e d i m e n t a t i o n as d e s c r i b e d in F i g . 6. 0 - 0 , ~H; C)- - - O , ~*P. F i g . i i . T h e a m o u n t s of 3 H - l a b e l e d fd r e p l i c a t i v e f o r m (IRF) D N A s e d i m e n t i n g w i t h 26, 18 a n d 15 S v a l u e s w e r e e s t i m a t e d b y i n t e g r a t i n g t h e a r e a s u n d e r t h e c u r v e s (see F i g . I o ) a n d p l o t t e d as a f u n c t i o n of e x p o s u r e to t h e ]3 r e s t r i c t i o n e n d o n u c l e a s e .

Biochim. Biophys. Acta, 195 (1969) 2 1 9 - 2 2 9

228

D. ROULLAND-DUSSOIX, H. W. BOYER

the K restriction endonuclease first converted unmodified 2 DNA twisted circles to non-twisted circles and then to linear fragments. We have observed the same effect with the unmodified twisted fd replicative form DNA (replicative form I) and the B restriction endonuclease. The first change in sedimentation pattern is the conversion of replicative form I to replicative form I I (Fig. loB) and eventually the appearance and accumulation of replicative form I I I (Figs. IoC and IoD). By integrating the areas under the curves, an estimate of the amounts of replicative forms I, I I and I I I was obtained and plotted as a function of exposure of the replicative form I DNA substrate to the B restriction endonuclease (Fig. I I ) . The kinetics of the reaction are typical for a two-step reaction with the replicative form I I DNA representing the intermediate substrate 23. DISCUSSION

I t is now clear that the restriction or exclusion of non-modified DNA b y a bacterial cell is brought about b y a specific endonuclease, introducing double strand scissions b y a two-step process at a limited number of sites along the molecule. The restriction endonuclease of E. coli B reported here and elsewhere 9, and the restriction endonucleases of E. coli K I 2 and the prophage P I (ref. 8) share a number of interesting similarities. All three enzymes require Mg 2+, S-adenosyl-L-methionine and ATP for activity, and they all have molecular weights on the order of magnitude of 300 ooo daltons. They differ in their substrate specificities, requiring in each case a unique site on a DNA molecule, defined as a sequence of nucleotide base pairs. The number of sites per molecule can only be estimated at the present time, (with the assumption that there are not a number of contiguous sites) at about 3-5 per ~vir DNA and ,~+ + DNA, one per fd replicative form molecule, and about IOO per bacterial chromosomal molecule. The finding that unmodified bacterial DNA is a substrate for the B restriction endonuclease confirms the role of the restriction and modification systems in interstrain transfer of DNA by conjugation *,5. The role of S-adenosyl-L-methionine and ATP in the enzymatic activity of the restriction endonucleases is not known. It has been reported that the restriction activity in vitro controlled b y RTF-2 does not require S-adenosyl-L-methionine and A T P in crude extracts m, and this has been verified with a more purified preparation of the R restriction endonuclease (D. ROULLAND-DussoIX, unpublished data). Thus the requirement of S-adenosyl-L-methionine and ATP m a y be peculiar to one class of restriction endonucleases. I t has been proposed that the E. coli KI2, B and P I restriction endonucleases are members of a family of restriction endonucleases differing primarily in substrate specificity zS. Indeed, the K and B restriction and modification systems are controlled b y isoalleles 4 and genetic complementation occurs between the K I 2 and B r - m + and r - m - mutants 25. The large molecular weight of the restriction endonucleases are compatible with their apparent multimeric nature 25. Mutants of the P I system do not complement the K or B mutants ~5, but it is evident that the PI, K and B restriction endonucleases are very similar. It will be interesting to compare further the R restriction endonuclease to the other restriction endonucleases. Although we cannot eliminate here the possibility of several adjacent sites on the fd replicative form susceptible to the B restriction endonuclease, the data Biochim. Biophys. Acta, 195 (1969) 219-229

E. coli B RESTRICTION ENDONUCLEASE

229

reported here are most easily interpreted as only one site per replicative form molecule susceptible to the B restriction endonuclease. The fd replicative form molecule, if it has one site susceptible to the B restriction endonuclease, should prove useful as a substrate for stoichiometric measurements and for examining the molecular events of the endonucleolytic attack. In addition, the/'1- and/*2-type mutants should provide excellent substrates for investigating the role of proteins interacting with specific sequences of nucleotide base pairs. ACKNOWLEDGMENTS

This investigation was supported by U.S. Public Health Service Research Grant No. 5 RoI GM 14378; by U.S. Public Health Service Training Grant No. 5 ToI AI 00299; by NSF Grant No. GB 8169; and by a Jane Coifing Childs Memorial Fund for Medical Research Award to one of the authors (D. R-D). REFERENCES W. ARBER, Ann. Rev. Microbiol., 19 (1965) 365 • D. D u s s o l x AND W. ARBER, J. Mol. Biol., 5 (1962) 36. W. ARBER AND D. DUSSOlX, J. Mol. Biol., 5 (1962) 18. H. BUYER, J. Bacteriol., 88 (1964) 1652. J. PITTARD, J. Bacteriol., 87 (1964) 1256. W. ARBER, J. Mol. Biol., i i (1965) 247. W. ARBER, Syrup. Soc. Gen. Microbiol., April ~968, Cambridge U n i v e r s i t y Press, Cambridge (I968). 8 M. MESELSON AND R. YUAN, Nature, 217 (1968) i i i o . 9 S. LINN AND W. 2~kRBER, Proc. Natl. dead. S¢i. U.S., 59 (1968) 13oo. I O R . ROBLIN, J. Mol. Biol., 31 (i968) 5i. I I C. LARK, J. Mol. Biol., 31 (1968) 4Ol. 12 H. JANSZ, P. POUWELS AND J. SCHIPHOIST, Biochim. Biophys. dcta, 123 (1967) 626. 13 H. DORWALD AND ]-I. HOFFMAN-BERLING, J. Mol. Biol., 34 (1968) 331. 14 K. SHORTMAN AND I. R. LEHMAN, J. Biol. Chem., 237 (1964) 2964. 15 L. FEINER, P. SADOWSKI, M. GOLD, J. HURWlTZ, Federation Proc., 27 (1968) 395. 16 E. BURGI AND A. HERSHEY, Biophys. J . , 3 (1963) 309 • 17 G. KELLENBERGER, M. ZICHICHI AND J. WEIGLE, Proc. Natl. Acad. Sci. U.S., 47 (1961) 869. 18 W. ARBER, J. Mol. Biol., 20 (1966) 483. 19 A. BURTON AND R. SINSHEIMER, J. Mol. Biol., 14 (1965) 327 . 2o J. M. BISHOP AND G. KOCH, J. Biol. Chem., 242 (1967) 1736. 21 D. A. MARVIN AND H. SCHALLER, J. Mol. Biol., 15 (i966) i. 22 W. ARBER AND V. KOHNLEIN, Pathol. Microbiol., 3 ° (1967) 946. 23 W. CLELAND, Biochim. Biophys. dcta, 67 (1963) lO 4. 24 T. TAKAMO, T. WATANABE AND T. FUKASAWA, Virology, 34 (1967) 29 °. 25 H. BUYER AND D. ROULLAND-DUssoIx, J. Mol. Biol., 41 (1969) 459. i 2 3 4 5 6 7

Biochim. Biophys. Acta, 195 (1969) 219-229