Molec. gen. Genet. 143, 13-23 (1975) © by Springer-Verlag 1975

Restriction and Modification in B. subtilis Purification and General Properties of a Restriction Endonuclease from Strain R

Sierd Bron Department of Molecular Biology, University of Edinburgh, Edinburgh, Scotland and Department of Genetics, Biological Centre, Haren (Gn), The Netherlands

Kenneth Murray Department of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland

Thomas A. Trautner Max-Planck-Institut f/ir Molekulare Genetik, D-1000 Berlin 33, Germany, Ihnestr. 63-73

Summary. All Bacillus subtilis R-type strains show-

ing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPP1, SPO2 and 0105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400fold and is essentially free from contaminating double strand-directed unspecific exo- or endonuclease activity. Only Mg 2+ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5 × 107) have an average molecular weight of about 3 x 105.

Introduction

Since the original report of the purification of a restriction endonuclease from Escherichia coli KI2 by Meselson and Yuan (1968), the list of restriction enzymes extracted from other bacterial sources, particularly from various strains of E. coli and Haemophilus, has grown rapidly (see, for instance, Smith and Nathans, 1973; Murray and Old, 1974). Restriction endonucleases degrade foreign DNA at a limited number of sites (reviews: Arber, 1974; Arber and Linn, 1969; Boyer, 1971 ; Meselson et al., 1972; Murray and Old, 1974). Kelly and Smith (1970) were the first to show that the type II restriction endonucleases (Boyer, 1971) recognise specific nucleotide sequences in which they introduce cuts between the same base pairs in the two strands of DNA (even double-strand break), or only a few base pairs apart (staggered breaks). To the type II enzymes belong several restriction endonucleases

from Haemophilus (Smith and Wilcox, 1970; Garfin and Goodman, 1974; Gromkova and Goodgal, 1972; Middleton et al., 1972 ; Sugisaki and Takanami, 1973 ; Old et al., 1975) and from E. coli carrying resistance transfer factors (Yoshimori, 1971 ; Bigger et al., 1973; Hedgpeth et al., 1972). The recognition sites for several of these enzymes are now known (see, for example, Murray and Old, 1974), and all have in common an axis of twofold rotational symmetry, so that when read with the same polarity, the nucleotide sequences in each strand of the DNA molecule are identical. Because of their specificity, restriction endonucleases have proved useful as tools in several types of research, such as studies on the mechanism of the recognition of specific nucleotide sequences by particular proteins, nucleotide sequencing of DNA, chromosome mapping, and the construction of new combinations of genetic material that cannot be achieved by conventional genetic techniques (Arber, 1974; Murray and Old, 1974; Murray and Murray, 1974; Rambach and Tiollais, 1974; Middleton et al., 1972 ; Marx, 1973 ; Lai and Nathans, 1974; Jackson et al., 1972; Thomas et al., 1974; Morrow et al., 1974). Inappropriately modified infectious phage 2 and phage fd DNA are restricted by various helper-infected E. coli cells (Dussoix and Arber, 1965; Benzinger, 1968). Also in B. subtilis, non-modified transfecting phage SPP1 DNA is inactivated in restricting hosts (Trautner et al., 1974). Restriction-like enzymes may also play a role in the inactivation of transforming bacterial DNA after uptake by competent cells, particularly in the case of transformations between closely related species (Goodgal and Gromkova, 1974; Wilson and Young, 1972 and 1974). Increasing interest in the use of restriction enzymes in transformation studies may therefore be expected. Furthermore, as discussed by Trautner etal. (1974), studies with restriction enzymes on transforming and transfecting DNA may

14 also a u g m e n t o u r u n d e r s t a n d i n g o f D N A p r o c e s s i n g in t r a n s f o r m a t i o n a n d t r a n s f e c t i o n . T h e r e c e n t d e t e c t i o n o f a classical r e s t r i c t i o n / m o d i f i c a t i o n s y s t e m in B a c i l l u s s u b t i l i s s t r a i n R ( T r a u t n e r e t al., 1974), o f f e r e d t h e p o s s i b i l i t y to p u r i f y a n d s t u d y a restriction enzyme from a transformable/transfect a b l e b a c t e r i u m n o t u s e d h i t h e r t o in r e s t r i c t i o n / m o d i f i cation work. The availability of a simple transformat i o n / t r a n s f e c t i o n s y s t e m , w h i c h e n a b l e d us to s t u d y t h e b i o l o g i c a l c o n s e q u e n c e s o f in v i t r o t r e a t m e n t s o f b i o l o g i c a l l y a c t i v e D N A s , p r o v e d to be o f g r e a t h e l p in t h e s e studies, The present paper describes the purification and general c h a r a c t e r i z a t i o n of the B. subtilis restriction endonuclease. In the accompanying paper (Bron and M u r r a y , 1975) t h e n u c l e o t i d e s e q u e n c e r e c o g n i s e d b y t h e e n z y m e is d e c r i b e d .

M a t e r i a l and M e t h o d s t Bacterial Strains

S. Bron et al. : Restriction Endonuclease from B. subtilis Table 1. B. subtilis strains used in this study

Strain

Genotype

a) Non-restricting~non-modifying strains (r m ) 168

trpC2

parental strain for all r - m - strains

1G-20

trp C2

highly transformable/ transfectable derivative of strain 168 (Bron and Venema, 1972a)

OG-1

prototrophic

derived from 1G-20

8G-5

trp tyr adehis-nic-urarib m e t -

highly transformable derivative of 168 (Bron and Venema, 1972a)

MCB

trp-

Spatz and Trautner (1971) Trautner et al. (1974)

168(0105cts23)

thy trp(0105cts23)

temperature inducible, 0105 lysogenic strain obtained from L. Rutberg

b) Restricting~modifying strains (r +m +)

R

prototrophic

non-transformable/nontransfectable (Trautner et al., 1974). The strain has been referred to as X5 by Murray and Old (1974)

6G-R

trp tyr-nicura-rib met

transformable/transfectable derivative of 8G-5 in which the r +m + markers from strain R were introduced (Trautner et al., 1974)

OG-3R

prototrophic

obtained by transformation of 6G-R with R-DNA

4G-R(OlO5cts23)

trp-nic ura met (O105cts23)

tyr +rib + transformant of 6G-R with R-DNA; 0105 lysogenic strain obtained from L. Rutberg

The Bacillus subtilis strains used are listed in Table 1. All strains except R are known derivatives of 168 trpC2 . Bacteriophage

Stocks of phage • were prepared as described by Murray (1973). Stocks of wild type SPP1 were prepared on strains 1G-20 or MCB (yielding nonmodified SPP1.0), or strain R (yielding modified SPP 1.R) as described by Trautner et al., 1974). Non-modified 0105.0 and modified 0105.R were obtained by heat induction (15 min, 45 °) of strains 168(0105cts23) and 4G-R(0105cts23), respectively, which were grown at 32° in NY-medium to a density of approximately 5 x I0 v cells per ml. Stocks of non-modified and modified SP02.v61 (a virulent mutant of SPO2) were made by infecting exponentially growing 1G-20 and OG-3R cultures in NY-medium (A4s 0 at 1 cm lightpath is 0.4) with phage at a moi of 1 to 2. Stocks of phages 029, SPO1 and SP50 were prepared on 168 (r m ) cells exponentially growing in complete TY-medium as described by Biswal et al. (1967) and Hirokawa (1972).

Media and plates

Complete TY-medium and TY-plates have been described by Biswal et al. (1967) and Rottlfinder and Trautner (1970). Media used in transformation were described by Bron and Venema (1972a). NYmedium was described by Okubo and Romig (1966).

Buffers DNA-diluent: 10mM tris-HC1 (pH7.4), 1 mM dithiothreitol, 10 mM MgClz, 0.15 M NaC1, and 0.5% (w/v) gelatine.

Abbreviations used : A = absorbance; ATP = adenosine triphosphate; cpm=counts per minute; 2 ME=2-mercaptoethanol; moi= multiplicity of infection ; pfu = plaque-forming units; trp = tryptophan-requiring; r+m+=restricting/modifying phenotype; r - m = non-restricting/non-modifying phenotype.

Reference and comments

Enzyme Buffer: 10mM tris-HC1 (pH 7.4), and 5raM 2-ME. PM-buffer: 20 mM Na-K-phosphate (pH 7.4), and 5 mM 2-

ME. Electrophoresis Buffer. 40 mM tris-acetate, 20 mM sodium acetate, and 2 mM EDTA, pH 8.3. Phage Buffer." 20mM Na-K-phosphate (pH 7.0); 100raM NaCI; 1 mM MgSO4; 0.1 mM CaC12; 0.001% (w/v)gelatine. Chemicals: DEAE-cellulose was the Whatman DE52 product. Polyethylene glycol 6,000 and streptomycin sulphate were obtained from Merck; pancreatic DNAase from Worthington Biochem. Inc. ; agarose from BDH and ethidium bromide from Calbiochem. Unless otherwise mentioned, all other chemicals used were from BDH (AR grade). Preparation of DNA

Bacterial transforming DNA was prepared as described by Bron and Venema (1972a). The preparation of [3H]-thymidine-labeled B. subtilis DNA was described by Bron and, Venema (1972b). Phage

S. Bron et al. : Restriction Endonuclease from B. subtilis

T7 D N A was kindly supplied by Dr. G o r d o n Peters. Phage 2 D N A was prepared as described by Murray (1973). For the isolation and purification of SPP1, 0105 and SPO2 D N A the following m e t h o d was used. Lysed cultures were clarified from debris by centrifugation for 10 min at 6,000 x g. To precipitate the phage, polyethylene glycol 6,000 ( Y a m a m o t o et al., 1970) was added to 10% (w/v) and NaC1 to 0.5 M. The suspension was put at 4 ° for at least 3 hrs and the phage collected by centrifugation (15 rain at 10,000xg). The pellet, containing more than 95% of the total input phage infective centers, was resuspended in small volumes of phage buffer. 1-ml portions of the resuspended phage were layered on preformed density gradients consisting of 3 layers of CsCI in phage buffer, with the respective densities of 1,700; 1.500 and 1.300 g . cm 3. Centrifugation was carried out in a MSE SS 65, 3 x 5 rotor, for 3 hrs at 30,000 R P M and 20 °. Phage bands were subsequently collected by puncturing the bottoms of the tubes. Phage suspensions thus obtained usually showed titers of approximately 1013 pfu per ml. After removing the CsC1 by dialysis against SSC (0.15 M N a C I + 0 . 0 1 5 M trisodiumcitrate), the phages were diluted 1 : 3 with SSC. D N A was subsequently extracted twice with tetraborate-washed phenol and dialysed during at least 24 hrs with 4 1-1 volumes of SSC. D N A concentrations were determined spectrophotometrically from the A26o. The purification of SP50, SPO1 and 029 D N A has been described by Biswal et al. (1967) and Hirokawa (1972).

Transfection and Transformation Competent cultures of 8G-5 and 6G-R were either prepared according to Bron and Venema (1972a), or to Rottlfinder and Trautner (1970). Competent cultures prepared according to the first method were stored in 10 % glycerol (v/v) at - 8 0 ° and thawed shortly before use Competent cultures were exposed to 1.0 gg of transfecting D N A per ml for 30 min at 34 °, or to 2.0 pg of transforming D N A per ml for 45 min at 34 °. Further uptake of D N A was then prevented by the addition of pancreatic D N A a s e to a final concentration of 8 lag per mI. Transfectants were scored on TY-agar; transformants on appropriately supplemented minimal agar.

Gel Electrophoresis of DNA Fragments Agarose gel electrophoresis was carried out with 17 x 12 x 0.3 cm gels in EC 470 vertical gel boxes, or between vertically placed parallei glass plates (40 x 20 x 0.3 cm gels). Slab gels containing 1% (w/v) agarose and 0.5 pg/ml ethidium bromide (Sharp et al., 1973) in electrophoresis (E) buffer were made. D N A samples for electrophoresis were prepared as follows. 1 to 5 gg of D N A in 50 ~tl DNA-diluent (without gelatin) was incubated with 5 gl restriction enzyme or 5 pl enzyme buffer for 30 to 60 rain at 37 °, Reactions were stopped with I0 pl 0.2 M EDTA. After concentrating the reaction mixtures to approximately 10 pl in a dry-seal desiccator, 10 ~tl of E-buffer containing 10% (w/v) sucrose and 0.02% (w/v) bromphenoi blue tracking dye was added to each. The gel box was filled with E-buffer containing ethidium bromide (0.5 gg/ml), and 20-~1 samples were applied to wells in the gel. Electrophoresis took place for 6 to 20 hrs at a constant current of 25 to 100 mA. The gels were then photographed under ultraviolet light on Ilford FP4 films with a 4 x red filter.

Enzyme assays

15

D N A fragments produced during incubation with restriction enzyme as described in the corresponding section of materials and methods. A more convenient and very sensitive method, particularly when m a n y fractions had to be assayed, involved transfection with sensitive D N A s , for which we have chosen SPPI. Non-modified SPP1.0 and modified SPP1.R D N A were diluted to 10 pg per ml in D N A diluent. To 50-pl portions of the diluted D N A s in small test tubes 5 pl of enzyme fraction was added at 0 °. If necessary, enzyme fractions were diluted in enzyme buffer The tubes were then transferred to 34 ° for 15 rain for the restriction reaction. Controls consisted of D N A which was put through the same regime without the addition of enzyme. In those assays meant to detect the presence or absence of restriction activity it was not necessary to stop the restriction reaction after this period of incubation. In some assays, for example in the determination of optimal p H and concentration of Mg 2+, the reaction was stopped by heating the mixtures for 10 min at 68 °. This treatment inactivates the B. subtilis restriction endonuclease completely. Subsequently, 0.5 ml of competent 8G-5 (r m ) was added to each tube for 30 min at 34 ° Further uptake of D N A was then stopped with D N A a s e (8 pg/ml, final concentration) for 5 min at 34 °. Transfectants were scored on TY-plates using 1G-20 (r m ) as indicator strain. b) Quantitative Assay of Enzyme Activity. Transfection also provided a convenient assay for quantitative determinations of enzyme activity. Kinetic experiments were used in which the inactivation of transfecting activity of SPP1 D N A by a fixed a m o u n t of enzyme was followed as a function of time of incubation. Both modified and non-modified SPP1 D N A were exposed to the enzyme fractions under investigation. This allowed us to correct for the effects of eventual non-specific nuclease impurities in the preparations, which can be quantitized from the inactivation kinetics of the modified SPP1.R D N A . The method allows the detection and estimation of the restriction activity even in crude extracts of R-type strains. One unit of restriction activity was defined as that a m o u n t that is needed to inactivate in 5 rain under standard conditions (see below) 1 gg of SPP1.0 D N A to 37% residual transfecfing actavity, in the absence of an effect on SPP1 .R D N A . The following standard conditions were used. SPP1.0 D N A and SPP1 .R D N A were diluted with DNA-diluent to 10 gg per ml. Portions of 0.50 ml of each of the diluted D N A s in small test tubes were transferred to 34 ° for 5 rain. 50 pl of diluted enzyme fraction was then added to each D N A and the mixtures were incubated at 34 ° Samples of 50 pl were withdrawn after 0, 1, 2, 4, 8, 16 and 32 rain and immediately put at 68 ° for I0 min. To make sure that no inactivation occurred in the zero time control samples, these were prepared as follows. 50%tl portions of both D N A s were heated for 5 min at 68 ° before 5-pl portions of enzyme were added for another 10 min at 68 °, All samples were then chilled in ice for 5 min and subsequently used to transfect 0.5 ml of competent 8G-5 (r m ) cells for 30 rain. Further uptake of D N A was then prevented by the addition of D N A a s e to 8 gg per ml for 5 min at 34 °. Transfectants were scored on TY-plates using 1G-20 ( r - m - ) as indicator strain.

Determination of protein Protein concentrations were determined according to Lowry et al. (1951).

Purification of Restriction Endonuclease Ji'om B. subtiffs R

a) Qualitative Assays. To demonstrate the presence or absence of restriction activity in fractions obtained during the purification of the B. subtilis restriction endonuclease, two different assays were used. The first m e t h o d involved agarose gel electrophoresis of SPP1

Overnight cultures in T Y - m e d i u m were diluted 50-fold into fresh T Y - m e d i u m and the cells were grown in 50-1itre batches to late log-phase (A4so approx. 1.4). Cells (approx. 70g) were harvested

S. Bron et al. : Restriction EndonucIease from B. subtilis

16

by centrifugation and stored at - 2 0 ° until use. Batches of 25g were resuspended in 25 ml cold 50 m M tris-HC1 (pH 7.4), 10 m M 2-ME and i0 m M MgC12. Cells were disrupted in a French- or X-press, or homogenized (5 x 1 min) in the Sorvall Omnimixer, using 0.7 1.5 m m diameter glass beads. All subsequent operations were carried out at 0 ° to 4 °, To reduce the viscosity, the lysate was sonicated 3 x 10 sec at maximal output in a MSE disintegrator. Cellular debris was removed by low-speed ( 1 0 m i n ; 10,000xg) and high-speed ( 4 5 m i n ; 100,000 x g) centrifugation. The supernatant (approx. 50 ml) was diluted to 100 ml with the tris-HC1 buffer mentioned above, and stirred in ice. Streptomycin sulphate (in a 10% w/v solution) was then slowly added to a final concentration of 1.6% (w/v) over a 30-rain period. After standing at 0 ° for 45 min the precipitate, containing nearly all activity, was collected by centrifugation (10 min; 10,000 x g) and dissolved in 300 ml PM-buffer plus 1 m M MgCI2. Further purification was achieved by a m m o n i u m sulphate precipitation at the following saturation levels: 0-40%, 40 50% and 50-70%. The 50-70% precipitate, containing nearly all the restriction activity, was dissolved in 30 ml PM-buffer and extensively diaiysed against this buffer. The dialysed 50-70% fraction (20 ml with approx. 800 m g total protein) was diluted to 200 ml wlth PM-buffer and fractionated on a column (12 cm x 3 cm diameter) of DEAE-cellulose (DE52), which was washed before use with 1 1 PM-buffer at a flow-rate of 80 ml per hr. The column was loaded with the diluted 50 70% fraction at a flow-rate of 40 ml per hr and then washed with 400 ml of PM-buffer. Protein was eluted with a 700-ml continuous gradient of 0 to 0.6 M KC1 in PM-buffer. Fractions of 12 ml were collected at a flow-rate of 4 0 m l per hr. The bulk of the activity eluted in a rather broad peak (1/6 of the gradient volume) between 0.25 and 0.35 M KC1. The most active fractions were pooled (120 ml) and the enzyme was precipitated with a m m o n i u m sulphate (75% saturation). The precipitate was collected by centrifugation (20 min; 30,000 x g), dissolved in approximately 2 ml PM-buffer and dialysed against the same buffer plus 1 m M MgCI2. Stored at - 2 0 ° or - 8 0 ° in 50% (v/v) glycerol, most preparations were stable. At least one preparation has been kept at - 2 0 ° without detectable losses of activity for 1 year. Some preparations decayed rather rapidly, however. Although the cause for this inactivation is not known, we have the impression that the presence of MgC12 throughout most of the purification steps m a y be important, Occasionally DEAE-cellulose columns were run in the presence of 1 m M MgC12. In all cases stable preparations were obtained, which, however, usually had the disadvantage of being slightly contaminated with bacterial D N A . Such preparations are satisfactory in the transfection and transformation assays, but the contaminating D N A disturbs ethidium bro-

mide gel electrophoresis assays and aiso such experiments as the sequencing of the recognition site for the restriction enzyme (see accompanying paper).

Assay for Exonuclease Activity Exonuclease activity in the enzyme preparations obtained was detected by measuring the formation of acid-soluble products when radioactive D N A was incubated with enzyme under the conditions described for the qualitative enzyme assay. 1 gg of [3H]-labeled B. subtilis 168 D N A (spec. act. 140,000 cpm per ixg of D N A ) in 100 p.l DNA-diluent without gelatine was incubated with approximately40 units of DEAE-cellulose, or 50 70% a m m o n i u m sulphate fraction for 30 min at 34 °. This a m o u n t of enzyme was sufficient to inactivate in a simultaneously carried out transfection experiment SPP1.0 D N A to less than 10-4% survival. As a control, D N A was similarly incubated in the absence of enzyme fraction. Reactions were stopped by the addition of non-labeled B. subtilis carrier D N A (0.6 ml with 100 ~tg of D N A ) and 0.2 ml of 6% perchloric acid. After leaving the mixtures for 15 rain at 0 °, D N A precipitates were removed by centrifugation (15 min at 30,000 x g and 2°). Radioactivity in the supernatants was counted after mixing 0.3-ml samples with 5.0 ml scintillation liquid (77% v/v toluene; 23% v/v triton X-100; 0.5% w/v PPO and 0.005% w/v POPOP) in the Mark II Nuclear Chicago liqmd scintillation counter. The counts were corrected for the radioactivity in the supernatant from the incubation mixture without added enzyme fraction. Input radioactivity was measured in a control from which the non-labeled carrier D N A was omitted and which was not centrifuged after the addition of perchloric acid.

Results 1. Restriction Nuclease in Strain R

The presence of restriction nuclease activity in the restricting/modifying B. subtilis strain R was studied using a simple biological assay system, in which the sensitivity of modified and non-modified transfecting SPP1 D N A against various cell fractions was studied. Similar assay systems have been used successfully by Takano et al. (1968) and Linn and Arber (1968) to demonstrate restriction activity in various E. coli strains carrying particular host-specificities. Table 2

Table 2. Effect of partially purified enzyme fractions on transfecting SPP1 D N A Fraction

SPP1.0 D N A SPP1.R D N A

control (no enzyme)

crude extract

100 100

0.1 10

strp. sulph, precipitate

strp. sulph, supernatant

a m m o n i u m sulphate 040%

40-50% 50-70%

> 70%

DEAEcellulose column

0.1 50

15 25

70 70

i0 40

80 90

0.1 95 100

0.1 4040

SPP1.0 and SPP1.R D N A were incubated with enzyme fractions obtained from strain R as described in materials and methods. Transfecting activities of the treated D N A s are expressed relative to those of non-treated controls (Set at 100%). The most active enzyme fractions were diluted such as to give 0.1% residual transfecting activity with SPP1.0 D N A . Streptomycin sulphate supernatant was diluted to the same extent as the streptomycin sulphate precipitate. The a m m o n i u m sulphate fractions were diluted to different degrees. Compared to the volume of the crude extract, the 0 4 0 % fraction was tested undiluted; the 40-50% fraction 2-fold; the 50 70% fraction 40-fold and the 70% supernatant 4-fold diluted.

S. Bron et al. : Restriction Endonuclease from B. subtilis

17

shows the effects of various enzyme fractions from

B. subtilis strain R on the transfecting activity of modified and non-modified SPP1 DNA. The results show that a restriction activity can be extracted from strain R which specifically inactivates the non-modified SPP1.0 DNA. This activity is detectable in the crude extract, which inactivates the SPP1.0 DNA to a considerably higher extent that the SPP1.R DNA. During the successive purification steps most of the activity is recovered in the streptomycin sulphate precipitate, and subsequently in the 50 70% ammonium sulphate fraction. The results also show that non-specific nucleases in the crude extract, responsible for the inactivation of the SPP1.R DNA, are gradually removed in the course of the purification. In the active fraction obtained from the DEAE-cellulose column non-specific nuclease activity is hardly detectable. Since both single- and double-stranded breaks in the DNA inactivate the transfection event, we conclude that the DEAE-cellulose fraction is virtually free from contaminating non-specific endonucleases acting on doublestranded DNA

2. Correlation between Restrictb~g PbenoLvpe of Cells and the Presence of Restriction Enzyme The experiments described above were carried out with enzyme fractions extracted from the original R (r +m +) strain, which was non-transformable and non-transfectable. Potentially competent r+m + strains of the 168 type were obtained by introduction (transformation) of the r+m + markers from strain R into the 168 genetic background of the strain 8G-5 (Trautner et al., 1974). This possibility of transfer allowed us to investigate whether enzyme occurs in all R strains with the r +m ÷ phenotype. To this aim, we analysed strains 1G-20 ( r - m ) , MCB (r-m-), 6G-R (r+m+),

and R (r*m +) for presence of enzyme. The results (Table 3) show that none of the ammonium sulphate fractions from strains 1G-20 or MCB exhibits specific activity against nonmodified DNA : all fractions inactivate SPP1,0 and SPP1.R DNA to the same extent. However, the 50-70% ammonium sulphate fractions from the r + m + strains R and 6G-R contain specific activity against SPP1.0 DNA. It can be concluded that restriction enzyme can only be extracted from those strains that are phenotypically r+m +. Therefore, a complete correlation exists between the restricting phenotype of a cell as measured by phage infection or transfection and the presence of the restriction enzyme.

3. Purity and Activity of the Enzyme Preparations a) Levels of Enzyme Activity; Degree of Purification. To quantitate the activities and the degrees of purity of the various enzyme fractions, we followed the kinetics of inactivation of transfecting SPP1 DNA by the various enzyme fractions. Fig. 1A shows that the crude extract inactivated both the SPP1.0 and SPP1.R DNA, although the first type considerably more than the second. With the DEAE-cellulose fraction (Fig. 1B) inactivation was observed only with the SPP1.0 DNA. These data confirm two conclusions drawn in the foregoing sections; 1) from strain R an activity specific against non-R-modified DNA can be extracted, and 2) the DEAE-cellulose preparation obtained is essentially free from contaminating nonspecific nuclease activity. The curves presented in Figs. 1A and IB show that inactivations of DNA follow single-hit kinetics, indicating that one event, probably one cut of the molecule, results in loss of activity. This type of inactivation kinetics is the basis of the definition of the unit of enzyme activity (see materials and methods).

Table 3. Restriction enzyme in various B. subtilis strains Donor strain

1G-20 ( r - m - )

M C B (r m - )

D N A assayed

SPP1 0

SPP1 .R

SPP1.0

a m m o n , sulph. : 0-40% 40 50% 50-70% > 70%

17.5 11.3 20.4 27.8

18.0 11.7 21.6 24.6

30.4 13.2 35.6 14.8

6G-R (r+m +)

R (r+m +)

SPP1 .R

SPP1.0

SPP1 .R

SPP1.0

SPP1.R

26.4 14.4 31.9 15.7

42.0 15.5 0. l 20.8

58.0 45.0 55.0 31.1

53.2 8.1 0.1 38.0

68.4 26.2 60.3 43.9

B. subtilis strains 1G-20 ( r - m - ) , MCB (r m - ) , 6 G - R (r+m+), and R (r+m +) were grown in 4-1 batches TY-broth to late log-phase. After harvesting, cells were disrupted and enzyme fractions were made as described in materials and methods. The various a m m o n i u m sulphate fractions abtained were assayed for their ability to inactivate modified and non-modified SPP1 D N A using the qualitative transfection assay of enzyme activity. Values in the table are entered as transfecting activities of the enzyme-treated D N A s relative to the activities of controls (no enzyme), which were set at 100%. Compared to the volume of the original crude extracts, the 0 4 0 % fractions were tested undiluted; the 40-50% fractions 2-fold; the 50 70% fractions 40-fold; and the 70% supernatants 4-fold diluted.

S, Bron et al. : Restriction Endonuclease from B. subtilis

18 1024

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32

Fig. 1 A and B. Kinetics of inactivation of SPP1.0 and SPP1.R DNA by crude extract (A), and DEAE-cellulose fraction (B). SPP 1.0 and SPP1.R DNA were exposed to 100-fold diluted crude extract or to 500-fold diluted DEAE-cellulose fraction for various periods of time and assayed for transfecting activity. Relative transfecting activities are expressed as the number of transfectants obtained with enzyme-treated samples relative to that number in control samples. The results are averaged from three experiments. The plaque numbers actually counted in the control samples (0 rain) totalled: (A) SPP1.0 2828; SPP1.R 1355. (B) SPP1.0 3119; SPP1.R 1497. o - - - - o SPP1.0 DNA, o ...... o SPP1.R DNA

Since, as can be c o n c l u d e d f r o m Fig. 1 B, the restriction e n z y m e does n o t affect the activity of modified D N A , one can d e t e r m i n e the level of restriction enzyme activity even in i m p u r e enzyme extracts, like the one s h o w n in Fig. 1A, by correcting the effect o n n o n - m o d i f i e d SPP1.0 D N A for that o n m o d i f i e d S P P 1 . R D N A . T a b l e 4 gives results o f an e x p e r i m e n t in which the levels of restriction activity in the v a r i o u s enzyme fractions o b t a i n e d d u r i n g p u r i f i c a t i o n were q u a n t i f i e d as just described. T h e increase in specific activity shows that in terms o f total p r o t e i n the D E A E - c e l l u l o s e fract i o n was purified a p p r o x i m a t e l y 400-fold f r o m the crude extract. T h e results also show that in this experim e n t , in which the assays were d o n e s o o n after the

Table 4. Restriction activity in fractions obtained during purification

crude extract strep, sulph, pellet amm. sulph. 50-70% DEAE-cellulose column

10

o

100

A

10-

t3~ c

\o, 100

1

10

total activity (units)

total protein (mg)

specif, activ. (units/rag)

4.50 x 105 3.95 x l0 s 3.78 x l0 s 3.21 x l0 s

749 124 18.8 1.3

0.6 x l0 s 3.2 x 103 20.1 x 103 247.0 x 103

Restriction activities were determined within one day after the fractmns were available as described in the text. Entries for total activity and total protein are calculated assuming that all of the crude extract was purified through each of the next steps.

-2 A 10

i

;

16

-2 B 10

I

4 ; time of incubation(rain)

I

16

Fig. 2 A and B. Kinetics of inactivation of DNA from B. subtilis phages. DNAs were exposed to DEAE-cellulose fraction (approx. 3U per ~tg of DNA) for various periods of time and assayed for transfecting activity. Relative transfecting activities are expressed as in Fig. 1. (A) o - - e 0105.0 DNA, o ..... o 0105.R DNA, m--m SPO2.0 DNA, ~ ..... [] SPO2.R DNA. (B) o - - o SPP1.0 DNA, o ..... o SPP1.R DNA, m--m SP50.0 DNA, • . . . . . . . • SPO1.0 DNA, o - - o 029.0 DNA

fractions were available, a p p r o x i m a t e l y 70% of the activity in the crude extract was recovered in the D E A E - c e l l u l o s e fraction. Severe losses of activity were observed, however, w h e n enzyme fractions were kept for extended periods of time before being concentrated.

b) Contaminating Non-spec~c Nuclease Activity. The foregoing results have d e m o n s t r a t e d that the D E A E - c e l l u l o s e p r e p a r a t i o n s were essentially free f r o m c o n t a m i n a t i n g non-specific endonucleases acting o n d o u b l e - s t r a n d e d D N A . E x p e r i m e n t s n o t s h o w n here indicated, however, that this enzyme fraction has some slight unspecific activity against single-stranded D N A . T o investigate whether u n d e r the c o n d i t i o n s used exonuclease activity was present, we m e a s u r e d the prod u c t i o n of a c i d - s o l u b l e r a d i o a c t i v i t y from tritiated B. subtilis 1 6 8 D N A after i n c u b a t i o n with 5 0 - 7 0 % a m m o n i u m sulphate a n d D E A E - c e l l u l o s e enzyme fraction. F r o m an i n p u t radioactivity of 82,000 cpm, 0.34% was m a d e acid-soluble by the a m m o n i u m sulphate fraction, d e m o n s t r a t i n g the presence of some exonuclease activity. W i t h the D E A E - c e l l u l o s e fraction less t h a n 0.01% of the i n p u t radioactivity was m a d e acid-soluble, i n d i c a t i n g that this e n z y m e was essentially free f r o m exonuclease activity.

4. Specificity of the Restriction Enzyme a) Effects on Phage DNAs. B. subtilis strain R restricts phages SPP1, SPO2 a n d 13105, b u t several other B.

s. Bron et al. : Restriction Endonuclease from B. subtilis subtilis p h a g e s are resistant ( T r a u t n e r et al., 1974). It was t h e r e f o r e o f i n t e r e s t to c o m p a r e the sensitivity o f these p h a g e s to r e s t r i c t i o n in vivo with the sensitivity o f their D N A s to r e s t r i c t i o n e n z y m e in vitro. Fig. 2 A shows the effect o f D E A E - c e l l u l o s e e n z y m e f r a c t i o n on t r a n s f e c t i n g 0105 a n d S P O 2 D N A . I n a c t i v a t i o n occurs with the n o n - m o d i f i e d D N A s only. T h e r e f o r e , D N A s f r o m p h a g e s SPP1, 0105 a n d S P O 2 all b e h a v e s i m i l a r l y on t r e a t m e n t with the r e s t r i c t i o n e n z y m e ; even the relative sensitivities o f the three D N A s are a l m o s t identical. Fig. 2B shows t h a t the D E A E - c e l l u lose f r a c t i o n does n o t i n a c t i v a t e D N A f r o m p h a g e s 029, SPO1 a n d SP50 ( g r o w n on 168 r - m - ) , which are u n a f f e c t e d b y r e s t r i c t i o n in vivo ( T r a u t n e r et al., 1974). These results d e m o n s t r a t e t h a t a c o m p l e t e correl a t i o n exists b e t w e e n the sensitivity o f a p h a g e to rest r i c t i o n b y R ( r + m ÷) cells, a n d the sensitivity o f its D N A to r e s t r i c t i o n e n z y m e f r o m strain R. A l t h o u g h n o b i o l o g i c a l test was done, p h y s i c a l e x p e r i m e n t s to be d e s c r i b e d in section 6 d e m o n s t r a t e t h a t also D N A s f r o m E. coli p h a g e s 2 a n d T7 r e p r e s e n t s u b s t r a t e s for the enzyme. b) Effects on Bacterial Transforming D N A . I n a d d i t i o n to n o n - m o d i f i e d p h a g e D N A , n o n - m o d i f i e d transf o r m i n g B. subtilis D N A (trpC2 m a r k e r ) is also specifically i n a c t i v a t e d b y the e n z y m e in vitro, a l t h o u g h at a m u c h l o w e r r a t e (Fig. 3). In a d d i t i o n to the trpC2 m a r k e r , 5 o t h e r m a r k e r s were investigated, w h i c h were all f o u n d to be s i m i l a r l y inactivated. T h e r e f o r e , like transfection, t r a n s f o r m a t i o n can be used to a s s a y the r e s t r i c t i o n enzyme. T h a t transfecting D N A is m o r e sensitive to r e s t r i c t i o n in vitro t h a n t r a n s f o r m i n g D N A is n o t surprising. In t r a n s f e c t i o n i n t a c t p h a g e c h r o m o somes are r e q u i r e d to p r o d u c e p h a g e p r o g e n y , whereas in t r a n s f o r m a t i o n relatively short f r a g m e n t s o f D N A are still effective, albeit with a r e d u c e d efficiency.

5. Conditions f o r in vitro Restriction a) Cofactor Requirements. T h e B. subtilis restriction e n z y m e requires o n l y M g 2+ ions as cofactor. A T P a n d S - a d e n o s y l m e t h i o n i n e d o n o t affect the e n z y m e activity. Fig. 4 shows the effect o f M g 2 + ions on the r e s t r i c t i o n reaction. M a x i m a l i n a c t i v a t i o n o f transfecting SPP1.0 D N A o c c u r r e d at c o n c e n t r a t i o n s a r o u n d 10 m M MgC12. Since t r a n s f e c t i o n with S P P 1 . R D N A was i n d e p e n d e n t o f the c o n c e n t r a t i o n o f M g 2 +, indic a t i n g t h a t the efficiency o f t r a n s f e c t i o n was n o t affected b y the a l t e r a t i o n s in the c o n c e n t r a t i o n o f M g 2 +, we c o n c l u d e t h a t the r e s t r i c t i o n e n z y m e is optim a l l y active at a b o u t 10 m M M g 2 +. A t c o n c e n t r a t i o n s b e l o w 0.5 m M MgC12 restriction activity was h a r d l y detectable. W i t h o u t M g 2+ no r e s t r i c t i o n at all occurred. N e i t h e r C a 2 + n o r M n 2 + can r e p l a c e M g 2 +

19

1024

101- l 10 °

-1

8 116 214 312 time of incubotion(min)

Fig. 3. Sensitivities oF transfecting and transforming DNA against restriction enzyme. DNAs fi-om B. subtilis OG-1 ( r - m ) and R (r+m+), and phage SPP1.0 and SPPI.R were diluted to 10 pg per ml in DNA-diluent and incubated at 34° with DEAE-cellulose fraction (approx. 3U per gg of DNA) for various periods of time. Relative transfecting activities of the SPP1 DNAs, and relative transforming activities (trpC2 marker) of the bacterial DNAs were then determined, e - - e SPP1.0 DNA, w - - m B. subtilis OG-I ( r - m ) DNA, ©..... o SPP1.R DNA, cJ. . . . . . . ~ B. subtilis R (r+m +) DNA

102-, > ID 101,g "5

g ~6

I

I

0.1 1 10 I[)0 concenlration Mg2+(mMl

Fig. 4. Dependence of restriction activity on Mg 2+ ions SPP1.0 DNA was diluted to 10 gg per ml in DNA-diluent from which MgC1z was initially omitted. 0.1 ml of 500-fold diluted DEAE-cellulose fractaon (approx. 20U) was mixed at 0° with 0.9 ml of DNA. Samples of 50 gl were then transferred to small test tubes containing 5 gl MgC12 solutions at various concentrations. After mixing, the samples were incubated at 34° for 15 min. Controls without enzyme were included. To stop the reactions~ the samples were heated For 10 rain at 68°. Transfecting activities at the various concentrations of Mg 2 + were subsequently determined as described for the qualitative enzyme assay

in the reaction. In an e x p e r i m e n t in which p h a g e 2 D N A was i n c u b a t e d with D E A E - c e l l u l o s e f r a c t i o n in the presence o f 5, 10, o r 50 m M CaC12, or MnC12, n o r e d u c t i o n in m o l e c u l a r weight was o b s e r v e d u p o n a g a r o s e gel electrophoresis. In the c o n t r o l e x p e r i m e n t where MgC12 was present, a s h a r p r e d u c t i o n in m o l e c u l a r weight o f the 2 D N A was o b s e r v e d ; the f r a g m e n t s

20

S. Bron et al. : Restriction Endonuclease from B. subtilis

Table 5. Effect of NaC1 on restriction activity

1

2

3

4

5

DNA NaCl-concentration

SPP1.0

SPPI.R

0.00 0.05 0.08 0.10 0.15 0.20 0.30 0.50

22.8 13.5 7.9 5.3 4.9 5.0 10.0 20.8

95 114 109 102 98 105 97 i01

M M M M M M M M

control (no enzyme; 0.15 M NaC1)

100.0

100.0

50-glsamples of SPP1.0 and SPP1.R DNA (at 10gg per ml) in DNA-diluent to which variable amounts of NaC1 were added, were incubated with approx. 2 U of DEAE-cellulose fraction for 15 min at 34°. Reactions were stopped by heating at 68 ° for 10 min. Competent 8G-5 (r m-) cells, 0.5ml, were then added for 30rain at 34°. Transfecting activities were determined as described in materials and methods (qualitative enzyme assay). Values entered are expressed (in %) as the number of transfectants obtained in enzyme-treated samples relative to that number in control samples (no enzyme). produced showing the characteristic, discontinuous l e n g t h d i s t r i b u t i o n w h i c h will b e d i s c u s s e d in c o n n e c t i o n w i t h Fig. 6. b) Effect o f N a C l on Restriction in vitro. T a b l e 5 s h o w s t h e effect o f v a r i o u s c o n c e n t r a t i o n s o f N a C 1 o n r e s t r i c t i o n in vitro, as a s s a y e d b y t r a n s f e c t i o n . T h e various NaC1 concentrations do not change the transf e c t i n g a c t i v i t y o f SPP1 . R D N A , i n d i c a t i n g t h a t t r a n s 10 Fig. 6. Agarose gel electrophoresis of DNAs restricted in vitro. 2to 5-gg samples of SPPI.0, SPP1.R, T7.B and 2.C DNA were incubated with approximately 500 U of DEAE-cellulose fraction for 1 hr at 37 °, and prepared for agarose gel electrophoresis (5 hrs at 50 mA in the EC 470 gel box), Sample nr. 1 : Control T7.B DNA (no enzyme added). 2: T7.B. 3: 2.C DNA. 4: SPP1.R DNA. 5: SPP1.0 DNA

b

>

101

o o'1 c"

-5

100

c

~5

4 10

Gio

,

,

,

,

71o

,

,

,

i

10

,

pH Fig. 5. pH optimum for in vitro restriction. SPP 1.0 D NA was diluted to 10 ~tg per ml in various tubes with DNA-diluent in which the tris-HC1 (pH 7.4) was replaced by 20 mM Na-K-phosphate of pH varying from 5.8 to 8.2.50-gl samples were incubated with approx. 5 U of DEAE-cellulose fraction for 15 min at 34°. Restriction was then stopped by heating for 10 min at 68 °. Transfecting activities of the samples were determined as described for the qualitative enzyme assay. Transfecting activities are given relative to the nonenzyme-treated controls

f e c t i o n itself w a s n o t a l t e r e d b y t h e v a r i o u s c o n c e n t r a t i o n s o f salt. T r a n s f e c t i n g activities o f e n z y m e t r e a t e d S P P 1 . 0 D N A s are, h o w e v e r , a f f e c t e d b y t h e salt a n d s e e m to be m o s t r e d u c e d at 0.10 to 0.20 M NaC1. T h i s i n d i c a t e s t h a t t h e r e s t r i c t i o n e n z y m e is o p t i m a l l y s t i m u l a t e d b y t h e s e c o n c e n t r a t i o n s o f salt. c) p H Optimum. T h e t r a n s f e c t i o n a s s a y f o r e n z y m e a c t i v i t y w a s also u s e d to d e t e r m i n e t h e p H o p t i m u m o f t h e r e s t r i c t i o n e n z y m e . Fig. 5 s h o w s t h a t t h e e n z y m e is o p t i m a l l y a c t i v e at p H 7.4.

6. Products o f in vitro Restriction A g a r o s e gel e l e c t r o p h o r e s i s was u s e d to c h a r a c t e r i z e the molecular weight distribution of DNA fragments

S. Bron et al. : Restriction Endonuclease from B. subtilis 1

2

3

4

5

21

6

section 2 that the restriction enzyme can only be extracted from phenotypically r +m + strains. Some 10 to 25 different fragments seem to be formed from SPP 1, SPO2, 13105, 2, and T7 DNA (Figs. 6 and 7). That this is an underestimate of the number of different fragments actually formed (since in the ethidium bromide-agarose gels used here low-molecular weight DNA fragments are not well resolved and not well stained) is shown in paragraph 6 (Fig. 5) of the accompanying paper (Bron and Murray, 1975). By terminally labeling the cleavage products with 32p, the numbers of fragments in limit digests of SPP1 and 2 DNA were estimated to be about 80 and 200, respectively. Discussion

In an earlier paper (Trautner et al., 1974) we described B. subtilis strain R, which restricts and modifies phages

Fig. 7. Agarose gel electrophoresis of restricted 0105, SPO2 and SPPI DNA. 2-gg samples of 0105.0; 0105.R; SPO2.0; SPO2.R; SPP1.0 and SPP1 .R D N A were incubated for 1 hr at 37 ° with 500 U of DEAE-cellulose fraction. Electrophoresis took place between glass plates (140x 20 x 0.3 cm) for 20 hrs at 25 mA. Sample nr. 1: SPP1.0 DNA. 2:SPP1 .R DNA. 3:SPO2.0 DNA. 4: SPO2.R DNA. 5:0105.0 D N A 6: 0105.R D N A

produced by restriction in vitro. Figs. 6 and 7 show limit digests of various phage DNAs. Modified SPP1.R, SPO2.R and 0105.R DNA are not affected by the enzyme, but the non-modified types of DNA (SPP1.0, SPO2.0 and 0105.0) are fragmented heavily; each DNA yielding a fragmentation pattern different from that of the others. Fig. 6 also shows that DNAs from the E. coli phages 2 and T7 are fragmented. The characteristic fragmentation patterns for SPP1.0 DNA shown in Fig. 6 could only be obtained with extracts from R-type (r+m --) cells. SPP1.0 and SPP1.R DNA incubated with extracts from MCB (r m ) or 168 ( r - m - ) cells yielded fragmentation patterns identical to each other in which no discrete bands were present, indicating that only non-specific nucleases were present. This confirms the conclusion from

SPP1, 13105 and SPO2, both in infection and transfection. The commonly used 168-derived strains lack this restriction/modification system. In the present paper we show that an enzyme can be extracted from R (r+m +) strains, but not from 168-derived r m strains, with the property of a restriction endonuclease, i.e. it inactivates transfecting activity of DNAs from sensitive phages grown on 168 i f - m - ) strains, but is without effect on the corresponding DNAs extracted from R-grown phages. Similarly, transforming DNA extracted from 168 cells is inactivated by the enzyme, whereas R-type DNA is resistant. (The observation that 168-type transforming DNA is not inactivated by competent R cells in vivo (Trautner etal., 1974) will be discussed in a subsequent paper.) The enzyme thus specifically inactivates DNAs lacking R-specific modification. Agarose gel electrophoresis showed that several non-modified DNA substrates are fragmented into discrete segments, and since no exonuclease activity was demonstrable under the conditions used, we conclude that the fragmentation was caused by endonuclease activity. Following the nomenclature of Smith and Nathans (1973), we call the enzyme endonuclease R. Bsu R, or, when no confusion arises, shortly endo R.R. Endo R.R. requires only Mg 2+ as cofactor. Its activity is stimulated by NaC1, but ATP and S-adenosyl methionine have no effect (results not shown). From its cofactor requirement and the nature of its products, the enzyme is a type II restriction endonuclease (Boyer, 1971). In line with this is the preliminary determination of the molecular weight of endo R.R by gel filtration on Sephadex G-200, which gave values of around 70.000 (S. Bron and E. Luxen; unpublished results). Agarose gel electrophoresis showed that a considerable number of specific cleavage products is formed

22 from suitable substrates such as SPP1, SPO2, 0105, 2 and T7 D N A . The numbers of fragments in limit digests of SPP1 and 2 D N A were estimated to be about 80 and 200, respectively. This means that the average molecular weight of the SPP 1 D N A fragments is about 3 x 105 (mol. weight c h r o m o s o m e : 25 x 106), and of the 2 D N A fragments 1.5 × 105 (tool. weight c h r o m o s o m e : 3 0 x 106). The nucleotide sequence at the site of action of the enzyme upon D N A , which on the basis of the above results m a y be expected to be small, is described in the accompanying paper (Bron and Murray, 1975). An unexpected property of the enzyme was its high binding affinity also to modified D N A . This is reflected by its coprecipitation with nucleic acid in the streptomycin sulphate precipitation step in the purification procedure, and by its elution pattern on DEAE-cellulose columns, where it elutes in or just before the D N A peak. Also we found recently that the enzyme binds equally well to modified and non-modified doublestranded D N A entrapped in polyacrylamide columns. Complete correlation was found between the presence of endo R . R and the restricting phenotype of a cell (Table 3). Strain R and its r ÷m ÷ derivative 6 G - R contained the enzyme, whereas no specific activity could be extracted from the 168-derived strains 1G-20 and MCB. This is strong evidence that endo R . R is responsible for the restricting properties of R-type strains. The preparations of endo R . R obtained sofar were purified approximately 400-fold. A preliminary electrophoretic investigation on polyacrylamide gels showed that 8 different proteins are still present in the active DEAE-cellulose fraction. Encouraging results for further enzyme purification were obtained recently using affinity c h r o m a t o g r a p h y on columns with double-stranded D N A entrapped in polyacrylamide. The availability of a simple transformation/transfection system is an attractive aspect of the B. subtilis restriction system. Helper-mediated transfection has been shown before (Takano etal., 1968; Linn and Arber, 1968) to provide useful assays for the detection of restriction enzymes in E. coll. Transfection also provided us with a convenient method to detect and to quantitate restriction activity. In addition to transfection, transformation could be used as an assay for endo R.R. However, transfection is more sensitive than transformation. This is not surprising in view of different target sizes of transfecting and transforming D N A . In addition to providing qualitative and quantitative assays for endo R.R, transfection proved also useful in studies on the optimal conditions for the in vitro restriction reaction. Furthermore, with the aid of competent cells, the in vitro effects of restriction

s. Bron et al. : Restriction Endonuclease from B. subtilis enzymes on a variety of biologically active D N A s can be investigated. Studies of this type, the detailed results of which will be presented in a subsequent paper, have already shown that endo R. Bsu R does not affect single-stranded transfecting SPP1 D N A (both modified and non-modified). Heteroduplex D N A consisting of one modified and one non-modified strand is, however, inactivated by the enzyme, although not at the same high rate as non-modified homoduplex D N A . A surprising observation in the restriction/modification system of B. subtilis strain R is that, Unlike phages SPP1, SPO2 and 0105, m a n y other phages grown in r m strains are resistant to restriction in vivo (Trautner et al., 1974). The present results show that the D N A from the resistant phages is insensitive to restriction enzyme in vitro. This is remeniscent of the behaviour of phage T7 D N A towards one of the restriction enzymes of H. influenzae (Old et al., 1975). It is possible that these D N A s lack the nucleotide sequence recognised by the enzyme, but this seems very unlikely statistically. Endo R. Bsu R recognises the tetranucleotide sequence 5 ' . . . N - - G - - G - - C - C N - - . . . Y (see accompanying paper), which in, for example, the D N A s from phages SP50, SP82 and H1 (mol. weight approx. 100 x 106) would be expected to occur by chance approximately 300 times. Alternative possibilities are being investigated now. Acknowledgements. Sierd Bron would like to thank the Dutch Organization for Pure Research (ZWO) for nominating him for a fellowship under the European Science Exchange Programme, and the British Royal Society for supplying it. Sierd Bron would also like to thank the European Molecular Biology Organization for supplying him with a short-term research fellowship. The work was supported in part by the Science Research Council (U.K.). We thank Bngitte Pawlek and Erik Luxen for their expert technical assistance.

References Arber, W. : DNA modification and restriction. Prog. Nucleic Acid Res. Mol. Biol., 14, i 37, Acad. Press Inc. (1974) Arber, W., Linn, S. : DNA modification and restriction. Ann, Rev. Biochem. 38, 467 500 (1969) Benzinger, R.: Restriction of infectious bacteriophage fd DNA's and an assayfor in vitro host-controlled restriction and modification. Proc. nat. Acad. Sci. (Wash.) 59, 1294-1299 (1968) Bigger, C.H., Murray, K., Murray, N.E.: Recognition sequence of a restriction enzyme. Nature (Lond.) New Biol. 244, 7-10 (1973) Biswal, N., Kleinschmidt,A.K., Spatz, H.Ch., Trautner, T.A. : Physical properties of the DNA of bacteriophage SP50. Molec. gem Genet. 100, 39-55 (1967). Boyer, H.W.: DNA restriction and modification mechanisms in bacteria. Ann. Rev. Microbiol. 25, 153-176 (197I) Bron, S., Murray, K. : Restriction and modification in B. subtilis. Nucleotide sequence recognised by restriction endonuclease R. Bsu R from strain R. Molec. gen. Genet. 143, 25-33 (1975) Bron, S., Venema, G. : Ultraviolet inactivauon and excision-repair

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Communicated by W. Arber Received June 16, 1975