The restriction endonucleases in Bacillus amyloliquefaciens N strain. Substrate specificities

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Biochimica et Biophysica Acta, 442 (1976) 184--196 © ElsevierScientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98662 THE RESTRICTION ENDONUCLEASES IN BACILLUS A M Y L O L I Q U E F A C I E N S N STRAIN. SUBSTRATE SPECIFICITIES

TAKEHIKO SHIBATAand TADAHIKOANDO Laboratory of Microbiology, The Institute of Physical and Chemical Research, Wako-shi, Saitama, 351 (Japan) (Received February 13th 1976)

Summary Two species of restriction endonuclease were isolated by gel filtration and DEAE-cellulose chromatography from a cell-free extract of Bacillus amyloliquefaciens (B. subtilis) N strain; a lower molecular weight endonuclease (endonuclease R.BamNI) and a higher molecular-weight one (endonuclease R.BamNx). Both of them required only Mg2÷for their activities. Endonuclease R.BamNx introduced a larger number of site-specific scissions in Escherichia coil phage k DNA than endonuclease R.BamNI did. Endonuclease R2~amNx cleaved Bacillus phage ~b105C DNA at the specific sites which are classified into two groups: one type of sites is modified by B. amyloliquefaciens H strain in vivo while the other is not affected. It was also active on DNAs of E. coil phage T7, )xdvl, Simian virus 40 (SV40) and colicinogenic factor ColEI and was inactive on DNAs of Bacillus phages 429 and M2. Endonuclease R.BamNI cleaved DNA in the same nucleotide sequences as endonuclease R.BamHI isolated from H strain by Wilson and Young. This endonuclease was active on DNAs of phage k, kdvl and SV40, and was inactive on D N . ~ of phages ~b105C, ¢29, M2 and T7, and ColEI DNA.

Introduction

Microorganisms possess host-controlled modification and restriction systems which recognize foreign D N A and degrade them in the cells.The endonucleases which degrade foreign D N A are called restriction endonucleases. R~.striction endon, tcleases recognize the specific nucleotide sequences and cut the D N A strands when the sequences are not modifie~l [1--10]. Pr~-viously we reported that the host-controlled modification and restriction systems are present in Bacillus amyloliquefaciens (previously called B. subtilis) N and H strains and B. subtilis Marburg 168 strain [11] and that the restriction

~Ss endonuclease activity and the modification m e t h y l ~ activity ~re detec.t~ the cell.freeextract of N strain [12]. In the course of pur~c~t~on of th~ enzymes, we found that N strain possesses at leasttwo species of restrictione~donucleases. One of them exhibits the same specificityas restrictionendonucl~.~ R ~ a m H I isolated from H strainby Wilson and Young [13]. The nuel~t~de ~quence at the cleavage sitesby endonucle~se R ~ m H I is known to be t (5,.30)G.G.A.T.C~ o (3°.5,)d.d.~.A~.G in which the phosphodiester bonds are broken betw~en G and G (Roberts~ ~.J.~ personal communication). The second restrictionendonuclease of N ,.~tr~dn~sd~ferent from endonucleases R ~ a m H ! and R ~ u R ~sol~ted from B. subt~l~ ~t strain by Bron et al. [14,15], and is a new one from B~c~'~us cells.In this paper isolation,substratespecificityand some of the charactersof these enzymes are de-. scribed. Materials and Methods Bacterial strains and DNAs Bacillus amyloliquefaciens (previously called B. subtiiis) N and H. s;xah~s, preparation of Bacillus phage ~105C DNA and the notation of moclification type of phage were described previously [11,12]. DNAs of Bacillus phages ~29 and M2 (these were grown on B. subtilis Marburg 168 strain) were kindly supplied by Dr. Hideo Hirokawa (our Institute). Simian vlrus 40 (SV~lq)~ DNA, Escherichia eoli phage k (kCIss~) DNA, kdvl DNA, E. col/phage T7 DNA ~ d colicinogenic factor ColEI DNA were generously provided by Dr. Nobuo Yamaguchi (The Institute of Medical Science, University of Tokyo), Mr. Tat~eo Icho (Tokyo University), Dr. Ken-ichi Matsubara (Osaka University}, Dr. Kazuo Shishido and Mr. Eiji Hayase (our laboratory), respectively.

Assay of restrictionendonuclease activity Unless otherwise stated, the reaction was carried out R s 37°C for 50 rain in 30 /~I reaction mixture containing 50 m M Tris- HCI buffer (pH 7.5), ,5 m M MgCl2, 0.2 m M E D T A , 5 m M 2-mercaptoethanol, 9.1/~g D N A and enzy.~e preparation. The reaction was terminated by adding 10 ~I of 40 ml~ F_~TA, 3 0 % sucrose and 0.05% Bromophenol Blue. 30/~l of scruples were elect~,~ze~d through 0.5 or 0.7% agarose gel slabs (14 × 12 × 0.6 c m with sm0nple wells (width 3 ram) at the top of the slab) in the presence of 0.5 l~glml etb~dium bzo. mide at 110 V for 1.5--2 h at room temperature as described by Sharp et al. [16] and photographed under an ultraviolet lamp. Under these conditiw_s, among the covalently closed circular D N A (cccDNA), open circular D N A (ocDNA) and linear D N A , c c c D N A migrab~ the fastestand o c D N A the slowest. Activity of the enzyme preparation was expressed by the m i n i m u m amount of protein (#g, determined by the method of Lowry etal. [17])which wasrequired for complete digestion of 0.1 ~g o~ phage ), DNA in 30 ~l of reacti,~n mixture for 50 rain at 37°C and this amount was termed the activity index. The smaller the activity index, the more active the enzyme preparation.

186

In vitro modification of DNA with the cell-free extract of H strain In vitro modification was carried out for 90 rain at 37°C in the presence of 0.2 mM S-adenosyl-L-methionine ~md the cell-free extract of H strain as described previously, using non-radioactive DNA (final concentration, 5 pg/ml) [12]. DNA treated under these conditions and untreated DNA could not be distinguished by gel electrophoretical analysis (data not shown).

Preparation of the restriction endonucleases of N strain All procedures were carried out at 4°C. (1) Ammonium sulfate fraction. Cell-free extract (streptomycin-supernatant) was prepared as described previously ~12], but 4 g of protoplasts were suspended in 15 ml of Buffer A (20 mM T~is • HCI (pH 7.5)/0.1 mM EDTA/2 mM MgCl:/2 mM 2-mercaptoethanol). The enzymes were precipitated by (NH4)2SO4 be:;ween 40 and 60% saturation from the cell..free extract, dissolved in 3 ml of Bufl'er A and dialyzed against 100 vol. of Buffer A containing 0.07 M NaCl, three times at 4°C. (2) UItrogel AcA44gelfiltration. A 3 × 40 cm Ultrogel AcA44 (LKB) column was equilibrated in Buffer A containing 0.07 M NaC1 and a 6.5 ml sample of dialyzed ammonium sulfate fraction (about 320 mg of protein) was applied to the column. Two clearly different restriction endonuclease activities on k DNA were observed in the fractions a~ayed by gel electrophoresis; the higher molec u l ~ welght active fractions (Fractions I) introduced larger number of doublestranded scissions in k DNA than the lower molecular weight active fractions did (Fractions II) (Fig. 1). (3) Purification of the lower molecular weight endonuclease (designated as endonuclease R.BamNI). The dialyzed Fractions II (about 40 mg of protein) were applied to a 1 × 15 cm DEAE-cellulose (Whatman DE52) column previously equilibrated in Buffer A. Endonuclease R.BamNI was eluted between 0.05 and 0.1 M NaC1 in Buffer A. The fully active fractions (which were defined to be able to cut completely k DNA under standard assay conditions) were pooled and used as endonuclease R.BamNL The activity index of the pooled fractions was about 3. Endonuclease R.BamNx activity was not detected in this preparation at all. (4) Purification of tire higher molecular weight endonuclease (designated as endonuclease R~BamNx, tentatively). The dialyzed Fractions I of the gel filtration (about 150 mg of protein) were applied to a 1.5 X 24 cm DEAE-cellulose column previously equilibrated in Buffer A. Endonuclease RJ~amNx was eluted soon after endonuclease RJ3amNI activity by an NaCl linear gradient in Buffer A. The fractions between 0.12 and 0.25 M NaCI were pooled. These fractions still contained significant endonuclease R~BamNI activity. Pooled DEAE-cellulose fractions (about 5.4 mg of protein) were diluted 2.5 fold with Buffer A and applied to a 1 × 5 cm DEAE-cellulose colunm equilibrated in Buffer A containing 0.07 M NaCl. Endonuclease R.BamNx was eluted between 0.1 and 0.2 M NaC1 in Buffer A (Fig. 2). The fully active (on k DNA) fractions were pooled. These fractions contained little endonuclease RJ~amNI activity. The activity index was about 4. These endonuclease preparations could be stored in liquid nitrogen without loss of activity for more than three months.

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Fig. 1. Ftactic, n a t i o n o f t h e lowel" a~cl M g h e t m o l e c u ~ we~jh~ ~ e ~ e ~ e ~ l < ~ e~,~v~tJ~-ge~ N s t r a i n b y U l t r o g e l A c A 4 4 ( L K B ) gel f t l ~ a ~ o n . A 3 × 4 0 e m column, w ~ e ~ u ~ v ~ ' ~ ~u ~ f f e ~ ~ ¢ ~ a ~ i n g 0 , 0 7 M NaCL A s a m p l e o f 6 , 5 me o f d~ l y~ecl a m m e m u m Sulfate f m e ~ (~ ~ ~ ~ " ~ ~-~ pried t o t h e c o l u m n a nd t h e c o l u m n w a s elutecl w i t h B u f f e t A e c ~ u ~ 0 . 0 7 M NaC~ ~ ~ m l i~w ~h, 5.5~ m l f r a c t i o n s w e r e c o l l e c t e d arid 1 0 ~Jl-~mples ~ e t e a ~ y e c l tot" e v ~ o ~ c ~ e a ~ ~c~v~t~e$ ~ ~ * ~k D N A unde~ s t a n d a r d c o n d i t i o n s . Ttea~ecl D N A s ( 3 0 ~l} w e r e elec~l~h~vt_~m~ ~b~u$~h (~.'~% ~ t e ge~ ~ ~ t h e pre~cnce o f O.5 ~g~ml e t h i d i u m b r o m k l e a~ 1 1 0 V fe~r 2 h, ~ - - f f i ~ - ~ , t ~ e £ n ~ : ~ ¢ e ~ t ~ b , ~ - ~--~- , t h e h i g h e r m o l e c u l a r w e i g h t a c t i v e fl'aelior~; I IS !, ~ e lowe~" m o l e ~ we~t ~,~ ~¢t~w: ~t~* g r a p h , profiles o f gel e l e e l ~ ophot e s e s o f She t ~ e ~ e d D N A s ,

Preparation o f restriction endonuclease o f H strait~ Ammonium sulfate fraction prepared from tile celts of H s ~ i ~ was dk~'t~y applied to a DEAE-cellulose column previo~sly equilibrated in B~:ffer ~\o 2~ endonuclease was eluted from the column between 0,05 a~.d 0,2 ~ b:aC1 a ~ i~ probably the same as the endonuclease R,Baml is¢)lated bF Wilson ~ h ' o u ~ [13], We will call the endonuclease of H strain endonuclea~ R ~ m i l ] ia d~is paper. Results

Restriction endonucleases in B. am'yloliquefaciens N stn~in Previously we demonstrated restriction endonuclease activity on ~ c ~ a $ phage ¢105C DNA in the cell-free extract prepared from B. amy~k)I~quefi~c~s (B. subtilis) N strain [12]. As described in Materi~s and Methgds, ~.wo s,'~ie.~ of the endonucleases active on E. coli phage X DNA were isolated from the cellfree extract of N strain by Ultrogel AcA44 gel filtration and by DEAE-celiulose chromatography. We designate the lower molecular weight endonuclease as ca. donuclease R.BamNI and the higher molecular weight one as er,donuc:ea~ R.BamNx, tentatively, according to the nomen".iature of Smitt~ and Nat ham [18]. When the E. coil phage k DNA treated wi~h endonuclease Rk~amNI wa.~

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analyzed by gel electrophoresis, five discrete bands were observed (Fig. 3). On the other hand, endonuclease R.BamNx cleaved phuge ~ DNA into more than fifteen fragments having discrete molecular weights (Fig. 3). When endonuclease R.BamNx was assayed with phage ¢105C DNA, the results were dependent on the host strain on which the phage was grown. Phage ~105C • N (grOwn on N strain} DNA carrying the N strain-specific modification was not attacked by this endonuclease {Fig. 3). This shows that this enzyme is a typical restriction enzyme. Phage ~105C • 168 (grown on B. subtilis 168 strain) DNA was cleaved by this endonuclease into about seventeen discrete fragments, while phage ¢105C- H (grown on H strains) DNA produced only eight fragments {Fig. 3). These results indicate that the sites cleaved by endonuclease P~.BamNx are classified into two groups; one type of site is modified by the H strain while the other is not affected.

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Fig. 3. Cleavage of DNAs of phages k and O105C with the restriction .'ndonucle~es of N st~a~. DNAs ~[ phages ~., O105C • 168 and 0105C • H (0.3/Jg each) were treated with e." donuelease R.~a~Nt (9 ~g ¢~ Dr~)~ tein) or endonuelease R.BamNx (13 ~ugof protein) in 90 ~1 of reaction mixture under stand~d ¢~ n~li¢~ons but the concentration of MgCI2 was 10 rnM. Treated DNAs were ev :ratted wilh phenol, p~e¢ipi¢&'t¢4 b~ addition of ethanol and dissolved in 20 ~1 of 50 rnM Tris - HCI buffer (pH T.5) et~nt~iIl~,g 0.2 : ~ [ ED~'A, 8% sucrose and 0.01% Brornophenol Blue. 10 al of the samples were analyzed, 0,1 ~zgofol05C- N [)NA was treated with endonuclease R.BamNx (4 tzg of protein) in 30 ~1 of ~eactio~ rnixtulx- and ~n&ly~, Treated DNAs were electrophoresed through O.T% agarose gel at tlO ~" for 1.5 h. ~. FcoRI g¢agl~le~s ~ ~, DNA; 2. untreated 3. DNA; 3. 3. DNA treated with endonueleas0 R ;~amN~;4. X DNA pre~iou~y ~cdifled with the cell-free e×trac: of H strain and then treated with endonnelease R.8~mNL 5, unCteatefl ~)~05C • 168 DNA; 6. 0105C • N DNA t~eated with endonuclease Rj3amNx; T OIOSC - H DNA ¢,~&~ed ~-ilh ~-~donuclease R.BamNx; B. 0105C. 168 DNA treated with endonuelease Rjga,~Nx: 9, ~-~coRl fragn~en¢,=:~)~ X DNA; 10. untreated ,\ DNA: 11. ,k DNA treated with endonuclease R,3amNx; 12. X DNA pre~,io~s~y ~¢~odified with the cell-free ~'xtract of H strain and then treated with endonuelease RJ~amNx.

~• h e f r a g m e n t s o f p h a g e s k a n d 0 1 0 5 C D N A s p r o d u c e d o f t h e N strain will be d e s c r i b e d in d e t a i l later.

by the endonuc]eases

Substrate speci[tcity o f endonuclease R . B a m N x D N A s o f Bacillus ( v i r u l e n t } p h a g e s 0 2 9 a n d M 2 d i d n o t s e e m t o t ~ atta::ked by endonuclease R~B~unNx (Fig. 4). Colicinogenic factor ColEl DNA appeared to be cleaved by this enzyme to produce three fra~m~ents. When SV40 DNA was treated

with

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the sum of roughly estimated molecular weights of these bands was about half o f t h a t o f w h o l e S V 4 0 D N A ( F i g . 4 ) . I n t h e c a s e o f E. coli p h a g e T 7 D N A , n : o r e than fifteen discrete fragments were separated by gel electrophoresis (Fig. 4). When phage 0105C DNAs were treated with this enzyme, twenty-one bonds w e r e o b s e r v e d f o r ¢ 1 0 5 C • 1 6 8 D N A a n d e l e v e n b a n d s f o r ~ 1 0 5 C • H D N ~ , in the profile of gel electrophoresis (Fig. 3). Four bands (Band - - i v ) i n fl~e

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Fig. 4, Cleavage of D N A s wi~h endcnuclease R.BamNx. D N A s of phages M 2 and ~29 (0.1 pgeach) were t r e a t e d w i t h t h i s e n z y m e ( 4 U~ o f p r o t e i n ) i n 3 0 sal-reaction m i x t u r e u n d e r t h e s t a n d a r d c o l t d i t i o n s . P h a g e T 7 D N A , C o l E l D N A , S V 4 0 D N A a n d h d v l D N A ( 0 . 3 p g e a c h ) w e r e t r e a t e d w i t h t h i s c ~ y m e ( 1 3 ~ug o f p r o t e i n ) in 9 0 p l r e a c t i o n m i x t u r e a n d t h e n p u r i f i e d as d e s c r i b e d i n F i g . 3. T r e a t e d D N A s w e r e e l e c t r o p h o r e s e d t h r o u g h 0 . 7 % a g a r o s e gel a t 1 1 0 V f o r 1 . 5 h. 1. u n t r e a t e d M 2 D N A ; 2. t r e a t e d h i 2 D N A ; 3. u n t r e a t ed 0 2 9 D N A ; 4. t r e a t e d 0 2 9 D N A ; 5. u n t r e a t e d T 7 D N A ; 6. t r e a t e d T 7 D N A ; 7, Re~;i:¢.l f r a g m e n t s o f h D N A ; 8. u n t r e a t e d C o l E I D N A ; 9. t r e a t e d C o l E I D N A ; 10. u n t r e a t e d S V 4 0 D N A ; 11, t ' e a t e d S V 4 0 D N A ; 12. E c o R I f r a g m e n t s o f h D N ~ ; 13. E c o R I f r a g m e n t s o f h D N A ~ 14. u n t r e a t e d k d v l D N A ; 1 5 . t r e a t e d h,d v l D N A .

0105C • 168 DNA profiies and three bands (Bands i--iii) in the 0105C • H DNA profiles gradually faded away as the reaction progressed and this indicates that these four and three bands are not ~;he final products of the reaction. When phage h DNA wa,~ treated with endonuclease R.BamNx, twenty bands were observed, but three of them (Bands i--iii) could not be detected when phage ~ DNA was previously modified in vitro with the cell-free extract of B. amyloliquefaciens H strain under conditions described in Materials and Methods (Fig. 3). In these modifying conditions, phage ~ DNA became resistant t~, endonuclease R ~ a m N I activity (Fig. 3), but the DNA would not aquire the modification to resist endonuclease R.BamNx activity since the susceptibility of phage ~105C • 168 DNA to endonuc!~ase R.BamNx was not affected by the tre a t men t (data not shown}. For this reas,.)n, Bands i--iii of phage )~ DNA fragments probably would not be produced by endonuclease R.BamNx but by contaminating endonuclease R.BamNI in endonuclease R 2 a m N x preparations. Xdvl DNA treated with endonuclease R.BamNx exhibited four bands in the profile of gel electrophoresis (Fig. 4).

Endonucleases R.BamNI and R.BamHI Wilson and Young reported that restriction endonuclease (R.BcmHt) was is,:)o lated from B. amyloliquefaeiens H strain [13]. Since the H and N st~b~s were closely related, we compared the specfficities of the restriction er~om~e]e~a of the N strain and endonuclease R.BamHI, and it was revealed t~at eadvnucleases R.BamNI and R.BamHI cleaved DNA in the same nucle~fifie ~q~e~ce~. This conclusion was drawn from two experimental resuits; {1) As ~hown ~ Fig. 5, when k DNAs treated with endonuclease R ~ a m N L endonuc~ea~ R ~ m H ~ and both of them together were analyzed by agarose gel slab e~ectrophore~i~ a~l of the treated DNAs exhibited the same pattern. (2) The celPfree extract of H strain conferred the BamNI-type modification on ~ DNA as descrfi.~d b~ the previous section {Fig. 3). R.J. Roberts {Cold Spring Harbour Labomt<~W) ~d~, obtained the same conclusion {personal commurdcationL Endonuclease RJ3amNI did not seem to attack DNAs of p h ~ e s 029. ~I2~ 0105C • 168 and T7, and ColEl DNA (Fig. 6). Ti¢is ~mdonuclease intr,_~h~ce(~ olae cut in SV40 DNA or kdvl DNA (per monomer DNA) (Fig. 6L

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Fig. 5. C o m p a r a t i v e s t u d i e s o n e n d o n u c l e a s e s R . B a m N I a n d R , B a m H L P h a g e X D N A w ~ ~ e ~ I ~ u n d e r t h e s t a n d a r d c o n d i t i o n a n d e l e c t r o p h o r e s e d t h r o u g h 0 , 7 % a g a r o s e gel a t 1 1 0 V f ~ r 2 h . 1, u n ~ = ~ D N A ; 2. h D N A t r e a t e d with endonuclease R . B a m Y l for 50 r a i n ; 3 , ,\D N A ~ r e ~ e f l f~.r~ ~'i~h ~donue~ease R , B a m N I f o r 2 0 r a i n a n d f o l l o w e d b y t r e a t m e n t w i t h bo~.h endonucIease,~; R . ~ a m H ] a n d R . B a m N ~ f ~ r 3 0 m i r a 4. k D N A t r e a t e d f ir s t w i t h e n d o n u c l e a s e R B a m H l for 2 0 r a i n a n ~ f o l I o w e d b y t h e ~ e a l m e m w i t h b o t h e,'tdonucleases R.l?.amNI a n d R . B a m H I f o r 3 0 m i n ; 5, ,k D N A treate~t wi~h e~ d o n u e l e ~ s e R , ~ m H I f o r 5 0 m;.n.

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Fig. 6. Cleavage of DNAs with endonuclease R.BamNI. DNAs of phages M2, 029, 0105C and T7 (0.1 ~g each) were treated with endonucleau~e ~.BemNI (6 ~g of protein) and DNAs of ColEI, SV40 and kdvl were treated with this enzyme (3 Ug of protein) under the standard conditions. Treated DNAs were electrophoresed through 0.5% (A)or 0.7"% (B) agarose gels at 110 V for 1.5 h. 1, untreated r,i2 DNA; 2. treated M2 DNA: 3. untreated 029 DNA; 4. treated 029 DNA; 5. untreated 0105C. 168 DNA; 6. treated 0105C. 168 DNA; 7. untreated T7 DNA; 8. treated T7 DNA;9. untreated ColEl DNA; 13. treated ColEI DNA; 11. untreated SV40 DNA: 52. treated SV40 DNA: 13. b;cc~RIfragment.; of ~, DNA;14. untreated Xdv~ DNA : 15. treated ,\dvl I)NA.

TJ:e requirements o f cofactors for the restriction endonuclease activities According to the classification of restriction endonucleases by Boyer [2], t y p e I e n d o n u c l e a s e a c t i v i t i e s r e q u i r e , i n a d d i t i o n t o M g 2., A T P a n d S - a d e n o s y l L - m e t h i o n i n e o r s t i m u l a t i o n b y t h e n ) . T y p e II e n d o n u c l e a s e s r e q u i r e o n l y M g :÷ for their activities. Both of the endonucleases R.BamNI and R.BamNx belong t o t i l e t y p e I1 e n z y m e ; i e. t h e y r e q u i r e o n l y M g : * f o r t h e i r a c t i v i t i e s . T h e o p t i m u m c o n c e n t r a t i o n o f M g 2. f o r t h e a c t i v i t m s d i f f e r e d f r o m e a c h o t h e r . E n d o n u c l e a s e R . B a m N x w a s f u l l y a c t i v e b e t w e e n 1 0 a n d 2 0 m M MgCI~, w h i l e e n d o n u c l e a s e R.BamNI w a s b e t w e e n 1 a n d 1 0 m M ( F i g . 7).

Other characteristics o f the restriction endonucleases Endonuclease R.BamNx was eluted from a phosphocellulose

column between about 0.3 and 0.45 M NaCI in Buffer A and from a hydroxyapatite column between about 0.2 and 0.35 M potassium phosphate buffer (pH 7.0), while endonuclease R.BamNI was eluted from a phosphocellulose column in a

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A

B

Fig. 7. MgC12 dependel.'Cy o f t h e r e s t r i c t i o n e n d o n u e l e a s e a c t i v i t i e s o f t h e ~; s t r a ~ , ( A ) 0 , 1 ,~g o f p h a s e X D N A w a s t r e a t e d w i t h e n d o n u c l e a s e R.BamNI ( 1 . 5 ~ g o f p r o t e i n ) u n d e r t h ~ s ~a n ~a r d c ~ n d i t i ~ s b u t ~he c o n c e n t r a t i o n s of Mg C I : w e r e 0 ( 2 ) , 0 . 3 ( 3 ) , 1 ( 4 ) , 2 ( 5 ) , 5 ( 6 ) , 10 ( 7 ) a n d 2 0 m ~ (AL T ~ e a t e d DNA_~ ~ ¢ ~ e e l e c t r o p h o r e s e d t h r o u g h 0 . 5 % a g a r o s e gel a t 1 1 0 V f o r 1 . 5 h, ( | ~ u n t r e a t e d :' D N A . (B~ O,~ * ~ o f p h a g e D N A w a s t r e a t e d w i t h e n d o n u c l e a s e R,BatnN× ( 3 g g o f p r o t e i n ) u~lder t h e :ta:~dard eo~
broad range of NaC1 concentrations (0,1--0.5 M) in Buffer A ~data not shown), When endonuclease R.BamNx was purified by phosphoceihfio~ chromatography from the fractions of the first DEAE-cellulose chromat c~aphy descriL~d in Materials and Methods, the activity index was reduced to ~bout 9.6, whEe that of the fractions of DEAE-cellulose rechromato~mphy from the same material was around 4. However, by means of phosphocellulose cnromato~¢phy, endonuclease R.BamNI activity could not be removed from the endonuctease R.BamNx preparation. Neither endonucleases R.BamNI nor R~BamNx were stimulated by NaCL but were inhibited above a concentration of 100 mM (Fig. 8k Discussion We isolated at least two species of restriction eadonucleases from ceils of B.

amy loliquefaciens N strain; endonucleases R.Bam NI and R.Bam N x. Endomm~ease R.BamNx cleaved phage 0105C DNA at tile specific sites whicE are clarified into two groups: one type of site is modified in vivo by B. amy~o~iquefi~ci-

194 1 2

34

5

A

'~ 2 3 4

5~5 7

[B

Fig, 8. E f f e c t o f N a C l o n t h e r e s t ~ i c t i n n e n d o n u c l e a s e a c t i v i t i e s o f N strah~. ( A ) 0 . 1 # g o f p h a g e h D N A w a s t r e a t e d w i t h e n d o n u c l c a s e R , B a m NI ( 1 . 5 ~g o f p r o t e i n ) u n d e r t h e s t a n d a r d c o n d i t i o n s b u t i n t h e pres e n c e o f N a C h T h e c o n c e n t r a t i o n s w e r e 1 3 m M 41), 6 3 r a m 42), 0 . 1 1 M ( 3 ) , 0 . 2 1 M ( 4 ) a n d 0 . 4 1 M ( 5 ) . T r e a t e d D N A s w e r e c l e c t r o p h o r e s e d t h r o u g h 0 . 5 % a g a r o s e gel f o r 1 . 5 h. ( B ) 0.1 # g o f p h a g e h D N A w a s t r e a t e d w i t h e n d o n u c l e a s e R . B a m N x ( 3 # g o f p r o t e i n ) u n d e r t h e s t a n d a r d c o n d i t i o n s b u t in t h e p r e s e n c e o f NaCI; t h e c o n c e n t r a t i o n s w e r e 0 r a m ( 1 ) , 1 0 r a m 42), 5 0 r a M 43). 0.1 M 44), 0 . 2 M ( 5 ) a n d 0 . 4 M (6). T h e t r e a t e d D N A s w e r e e l e c t r o p h o r e s e d t h r o u g h 0 . 7 % a g a r o s e gel a t 1 1 0 V f o r 1 . 5 h.

ens H strain while the other is not affected (Fig. 3). These results may be inter-

preted in two ways. (1) Endonuclease R93amNx preparation consists of one species of enzyme. The modification system of the H strain alters some of the sites on DNA at which endonuclease R . B a m N x cleaved the DNA when these sites are unmodified. (2) Endonuclease R.BamNx preparation consists of two species of the enzymes. One activity is blocked by the H strain-specific modification on the substrate DNA and the other is not. Although in chromatographies on DEAE-cellulose, phosphocellulose and hydroxyapatite, and gel filtration, endonuclease R 2 a m N x behaved as an enzyme, at present we cannot conclude which interpretation is right. Further :ratification and the nucleotide sequence analysis at the cleavage sites will give the answer. Endonuclease R.BamNI cleaved DNA in the same nucleotide sequence of

(5'-3')G-G-.A-T-C-.C (3'-5')C.C-T-A-G.G

195 ( R o b e r t s , R.J., p e r ~ n a l c o m m u n i c a t i o n } as e n d o n u c I e a s e R . B a m H 1 i e l a t ~ : ~ f r o m H s t r a i n b y W i l s o n a n d Y o u n g [ 1 3 ] , as d e s c r i b e d in t h e R e s u l t s . R e c e n t l : , , B r o n et al. [ 1 4 , 1 5 ] r e p o r t e d a r e s t r i c t i o n e n d o n u c l e a s e ( e n d o n u c l e ~ = ~ RA%uR~ o f B. subtilis R s t r a i n w h i c h cleaved D N A s t r a n d s in t h e s e q u e n c e o f

(5'-3'}0.G.C-C (3'-5')C-C-G-G E n d o n u c l e a s e R . B a m N x is d i f f e r e n t f r o m t h i s e n z y m e , b e c a u s e e m i o a u c } e a ~

R.BamNx d o e s n o t i n t r o d u c e so m a n y scissions i n t o D N A as e n d o m ~ c ] ~ R , B s u R ; f o r e x a m p l e , e n d o n u c l e a s e R . B a m N x c:eaved ,~dvt D N A i n t o ~wo {or three} f r a g m e n t s p e r m o n o m e r D N A {Fig. 4 *}, w h i l e e n d o n u d e a s e R.Bs~aR produced fifteen fragments [20]. B o t h o f e n d o n u c l e a s e s R . B a m N I a n d R ~ a m N x are t y p i c a l r e s t r i c t i o n enz y m e s , b e c a u s e t h e y c a n n o t a t t a c k D N A m o d i f i e d e i t h e r in vivo o r in v i t r o }a an N strain-specific t y p e { Fig. 3), We f o u n d p r e v i o ~ s ' y t h e t y p i c a l host~c~}~rob led m o d i f i c a t i o n a n d r e s t r i c t i o n active o n p h a g e ~ 1 ~ 5 C a m o n g B. ~my~o~iq~:e[~* cience N a n d H s t r a i n s a n d B. subtilis M a r b u r g 1 6 8 s t r a i n [ 1 1 ] . E a d o n u c ] e a s e R . B a m N x s e e m s p r o b a b l y t o p a r t i c i p a t e in in vivo r e s t r i c t i o n o f ~ 1 0 5 C b y N s t r a i n , since this e n z y m e a t t a c k s D N A c,f ~ 1 0 5 C g r o w n o n H s t r a i n o r 1 6 S s t r a i n (Fig. 3). O n t h e o t h e r h a n d , n e i t h e r e n d o n u c l e a s e R . B a m N t n o r R.t3amHI s e e m s to p a r t i c i p a t e in r e s t r i c t i o n o f :phage @105C b y N o r H s t r a i n , s~nce t h e s e e n d o n u c l e a s e s d o n o t a t t a c k ,5105C D N A at all (Fig. 6). E n d o n u c l e a s e s having t h e s a m e n u c l e o t i d e s e q u e n c e spec}ficity as e n d o n u clease R . B a m N I w e r e also d e t e c t e d ( d a t a n o t s h o w n } in t h e cells o f o t h e r t ~ o s t r a i n s o f B. amyloliquefaciens, ? . s t r a i n ( t y p e s t r a i n ) a n d K s ~ i n [21~o Acknowledgements We t h a n k Miss K. H o s h i n o f o r h e r t e c h n i c a l a s s i s t a n c e d u r i n g t h e s e h~w~t~gat i o n s a n d Dr. R.J. R o b e r t s f o r his p e r s o n a l c o m m u n i c a t i o n s . Th~s s t u d y w a s s u p p o r t e d b y a g r a n t f o r s t u d i e s o n " L i f e S c i e n c e " at this ] n s t i t u t e a n d ~ par~ by a grant from the Ministry o f Education of Japan. References 1 Arbor, W. (1974) in Progress in Nucleic Acid Research am] MolecularB~olo~,.~(Cohn. "~V.t"..,ed.). Vol. 14, pp. 1--37, Academic Press, New York 2 Boyer, H.W. (1971) Annu. Roy. MicrobioL 25, 153--176 3 Meselson, M,, Yuan, R. and Heywood, J. (1972) Annu. Roy. Biochem, 41,447--~86 4 Kelly, J~., T,J. and Smith, H,O. (1970) J. Mol. Biol. 51,393--409 5 Hedgpeth, J., Goodman, H.M, and Boyer, H.W. (1972) Proc. Na~l. Acad. SOL U,S, t;9, 34:4S--3452 6 Bigger,C.H., Mu~ay, K. and Murray, N.E. (1973) Nat. New B~ol. 244, 7--10 7 Boyer~ H.W,, Chow, L.T., Dugaiezyk, A.~ Hedgpeth, J, and Goodman, H.M. (1973) Na~, New B~ol, 244, 40--43 8 SugisakLH. and Takanami, M. (1973) Nat. New Biol. 246° 13S--140 9 narfin, D.E. and Goodman, H.M. (1974) Bioehem. Biophys. Res. Con:~mun.59, 10~1--116 I0 Old, R., Murray, K, and Roizes, G. (1975) J. Mol. Biol. 92, 331----339 11 Shibata, T. and Ando, T. (1974) MoL Gen. Goner. 131,275--2S0 * Roughly estimated molecular weights of the bands of the treated ~,dvl DNA (Fig. 4) axe 4.2 - ~0~, 2.8 • 10 ~, 1.3--1.4 • 10~ and ~ 0.2 " 10~, while the molecular weight of ~he monomer DNA o~ ~.dvt is 4.3 • 106 [19].

196 12 13 14 15 16 17 18 19 20 21

Sh,lbata0 T. and Ando~ T. (1975) MoL Gen. Genet. 135. 269--279 Wilson, G.A. and Yours, F.E. (1975) J. Mol. Biol. 97,12~--125 Bron, S.. Mun~ay, K. and Trautne~, T.A. (1975) Mol. Gem Genet. 143,13--23 Bron, S. and Murray, K. (1975) Mol. Gen. Genet. 143, 25--33 Sharp° P.A., Sudden, B. and Sambrook0 J. (1978) Biochemlst~y 12, 8055--8068 Lowry, O.H., Rosebmugh, N.J., Farr, A. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Smith~ H.O. and Nathans° D. (1973) J. Mol. Biol. 81, 419--423 Hobom° G. ~nd Hogneu, D.S. (1974) J. MoL Biol. 86, 65--87 Streeck0 R.E. and Hobom, G. (1976) Eur. J. Biochem. ST, 595--606 Welker, N.E. and Campbell, L.L. (1967) J. Bacter/ol. 94,1124--1130