Ge~e, 1 (1977) 291--303 291 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

SPECIFICITY OF CLEAVAGE BY A RESTRICTION NUCLEASE FROM Bacillus subtU~ (Sequence c ~ restriction sites; Bsu and HaeIII endonucleases; cleavage of dngle-stnm,~ed DNA; B. subtiUs phage SPP 1.0 and SPP 1.R; 5'-termini of DNA fragments; enzyme subsites) KURT HEININGER, WOLFRAM HSRZ and HANS G. ZACHAU

lnstitut fdr Physiologische Uhemie, Physikalische Biochemie und Zellbiologie der Univer~ sit~t Mi~nchen (Federal Republic of Germany) (Received December 3rd, 1976) (Accepted January 31st, 1977) SUMMARY

The restriction nuclease from/3, subtilis (Bsu) which cleaves in the middle of the tetra-nucieotide sequence 5'--GGCC--3' has been found to decrease its 3'--CCGCr-5' substrate specificity at high nuclease concentrations. There are special conditions, high pH, low ionic strength, and high glycerol content, which strongly enhance splitting with decreased specificity and also lead to splitting of singlestranded DNA. By sequence analyses it is shown that the reduction in specificity of Bsu corresponds to cleavage predominantly at 5LGC--3' sequences. No comparable change in specificity has been observed 3~.CG__5 , in a restriction nuclease from Haemophilus aegyptius (HaeIII), an isoschizomer of Bsu. INTRODUCTION

Type II restriction endonucleases have become increasingly useful in several types of research since they introduce cuts into DNA only at specific nucleotide sequences (for a recent review see Nathans and Smith, 1975). The substrate specificities of many of these enzymes have been determined (Roberts, 1975). With a few exceptions, the recognition sites were found to be symmetrical and to comprise between four and six nucleotide pairs. We have employed restriction nucieases also for digestion of chromatin (Pfeiffer et al., 1975; HSrz et al., 1976) and were forced to investigate more extreme conditions of digestion because of the decreased accessibility of the DNA. This led to the observation that in control experiments additional de,i

Abbreviations: The nomenclature for restriction nuelesses follows the suggestions of Smith and Nathans (1973). A, deoxyadenylic acid; C, deoxycytidyUc acid; G, deoxyguLiylic acid; T, deoxythymidylic acid; N, any of the four standard deoxyribomononucleotides.

292

fined fragments appeared in limit digests of phage DNAs with the restriction nuclease Bsu. Since this finding is of general importance for the interpretation of experiments with Bsu, we investigated digestion with Bsu in greater detaiL These studies were instigated on the one hand by an interest in the mechanism of recognition between a nucleic acid sequence and a nuclease. On the other hand, they had the practical aspect of evaluating more extreme conditions required for various applications of restriction nucleases. MATERIAIB AND METHOD8

Materials Ion exchange papers were the Whatman Chromedia DE 81 and AE 81 products, the latter kindly supplied by K. Murray. 2043 b Mgl paper was from Schleicher and Schiill, Dassel, the 5'-deoxymononucleotides from Boehringer Mannheim, [7-32P] ATP from the Radiochemical Centre, Amersha,m, iodoacetamide from Sigma Chemical Co., St. Louis, Miss., USA. DEAEcellulose w ~ the Whatman DE 52 product, and hydroxyapatite was prepared according to Bernardi (1971). Bacterial alkaline phosphatase and polynucleotide kinase were from Boehringer Mannheim, proteinase K from E. Merck, Darmstadt, DNAase I and micrococcal nuclease from Worthington Biochem. Corp., Freehold, N.J., U.S.A. Snake venom phosphodiesterase was prepared by M. Petrova according to Frischauf and Eckstein (1973) as described in Petrova et al. (1975) and generously donated.

Preparation of DNAs SPP 1.0 and SPP 1.R DNA were prepared from SPP I phage infected cultures of B. subtiUs strains MCB and R, respectively, by the procedtuv of Bron et al. (1975), except that CsCI gradient centrifugation of the phages was carried out in a Spinco rotor SW27 for 15 h at 22 500 rpm and 20 ° C. ;~dvl and PM2 DNA were prepared by R.E. Streeck as described (Streeck and Hobom, 1975; Streeck et al., 1974), M 13 DNA also by R.E. Streeck according to Wirtz and Hofschne~c~ (1970). These DNAs ware generously donated.

Digestion of DNA, enzyme assays, and gel electrophoresis Standard conditions for digestion with Bsu were 10 mM Tris--HCl, pH 7.4, 10 mM MgCI2, 5 mM #-mercaptoethanol, 100 mM NaCI. High pH conditions were 25 mM Tris-HCl, pH 8.5, 10 mM MgCI=, 5 mM ~-mercaptoethanol, 2 5 ~ glycerol. The buffer used for HaeIII is similar to that of Roberts et al. (1975)~ 10 mM Tris--HCl, pH 7.4, 10 mM MgCI2,5 mM ~-mercaptoethanol. For enzyme assays digestions were carried out in standard buffer aS 37°C for I h in a total volume of 50 #1 containing 3 , g of),dvl DN.~ I unit o f

293 activity was taken to be the amount of enzyme required to yield a limit digest under these conditions. Reacticns were terminated by the addition of EDTA to a final concentration of 15 raM. After the addition of one fourth volume 50% sucrose, 0.004% bromophenol blue the mixtures were analyzed by polyacrylamide gel electrophoresis as described (Philippsen and Zachau, 1972). The diethylbarbituric acid Tris--HCl system was used. The running gel contained 4% acrylamide, 0.2% bisacrylamide, 7 M urea, and the spacer gel 3% acrylamide, 0.15% bisacrylamide, 7 M urea. The gels were stained as described by Philippsen and Zachau (1972) and photographed. Restriction nucleases Bsu was prepared from the B. subtilis strain R (Trautner et al., 1974; Bron

et al., 1975) by a procedure briefly outlined before (H6rz and Zachau, 1975). This procedure is similar to the one of Bron et al. (1975) up to the DEAEcellulose chromatography step, which is the final step in the procedure of Bron et al. (1975) whereas our purification includes an additional hydroxyapatite chromatography step. 150 g cells were suspended in 300 ml buffer A (50 mM Tris--HCl, pH 7.4, 10 mM MgC12,5 mM ~-mercaptoethanol) and disrupted by sonication. Cellular debris was removed by centrifugation at 100 000 g for 60 min and the supernatant diluted with buffer A to give 150 A2~0 units/ml. A 20% (w/v) solution of streptomycin sulfate was slowly added to !~.5% final concentration. After standing at 4°C for 30 rain the precipitate was collected by centrifugation (30 min, 10 000 g) and dissolved in bur.. fer B (10 mM sodium phosphate buffer, pH 7.4, 5 mM ~-mercaptoethanol). The fraction precipitated by ammonium sulfate between 40 and 70% saturation wa~ taken, dissolved in buffer B and desalted by passage through a Sephadex G-25 column equilibrated with buffer B. The material was then chromatographed on a DEAE-cellulose column (5 × 50 cm) previously equilibrated in buffer B. After washing the column with buffer B, adsorbed material was eluted with a linear gradient from 0--0.6 M NaCI in buffer B (41 each). Bsu activity eluted at 0.4 M NaCI bound to nucleic acids. The complex (6000 A260 units) was adsorbed directly to a hydroxyapatit ~ column (6 × 20 cm) equilibrated in buffer B. The column was washed with buffer C (50 mM sodium phosphate buffer, pH 7.4, 5 mM ~-mercaptoethanol) and the nuclease eluted with 2.5 M NaCI in buffer C, i.e. conditions under which the nucleic acid material is quantitatively retained by t h e hydroxyapatite. The nuclease was concentratea by pressure dialysis (Amicon, membrane PM 10), and then dialyzed against 15 mM Tris--HCl, pH 7.4, 10 mM NaCI, 0.1 mM EDTA, 5 mM ~-mercaptoethanol, 50% glycerol and stored at --20 ° C without noticeable loss of activity for more than a year. Alternative storage was at - - 8 0 ° C in the same buffer lacking glycerol, however. Activity decreased after storage for several months under those conditions. A preparation starting from 150 g B. subtiUs cells yielded 1.5 A2s0 units protein with 40 000 units Bsu. SDS gel electrophoresis showed the presence

294 of two prominent bands, one in the 25 000 dalton range, the other ~n excess of 40 000 da]tons. Our purification procedure cannot be compared directly in terms of purity and yield to that of Bron et al. (1975) since Bron et al. (1975) employ a t~ansfection assay for enzyme activity measurements. Some preparatio~ of Bsu of lower specific activity contained protein contaminants interfering with the gel analysis of DNA digests. A characteristic trailing of bands was observed at high nuclease concentrations which was abolished by phenol extraction or treatment with proteinase K of the digest prior to electrophoresis. This was ini~ally taken to reject the binding of Bsu to the DNA fragments in the digests. Preparations of high specific activity, however, showed hardly any binding detectable by band trailing. We interpret this to mean that the observed phenomenon was due to some other protein(s) enriched in Bsu prenarations of lower specific activiW. HaelII was prepared from Haemophilus aegyptius, obtained from H. Schaller, according to the method of Roberts et al. (1975).

Analysis o f nucleotides Determination of the 5'-terminal and penultimate nucleotides of DNA fragments was carried out according to Murray (1973) and Bron and Murray (1975) with two slight modifications. Excess [3zp] ATP after the phosphorylation step was removed by extensive dialysis only; the Sephadex chromatography step was omitted. The separation of the mononucleotides was not attempted on AE 61 paper but instead on 2043 b Mgl paper by high voltage electrophore~ at pH 3.5 (Melchers and Zachau, 1964). RESULTS

Bsu cleavage patterns of phage DNAs are affected by digestion conditions Limit digests of phage and plasmid DNAs with restriction nucleases have become a standard way of characterizing and identifying the DNA as well as the restriction nuclease. The digestion patterns should be independent of incubation time and mlclease concentration. We have noted some exception when we digested phagt, DNAs with the high concentrations of Bsu which are required for cleavage of chromatin (Pfeiffer et al., 1975) This is shown in Fig.l: track (a) shows the Bsu limit digest of),dv I DNA prepared in standard buffer except that NaCI was omitted. The pattern consists of 17 fragments (Streeck and Hobom, 1975) all present in equimolar amounts. Two of them migrate with the bromophenol blue in this gel system. When the digestion was done with two times that Ben concentration, a novel band appeared migrating just ahead of the second band in the limit digest (Fig. lb). This band became very prominent when Bsu was allowed to act upon the DNA in the presence of 12% glycerol (Fig. lc). Digestion of the DNA with even more Bsu in the presence of 12% glycerol leads to further degradation especially of large fragments. Many novel bands appear together with a background

295

a

b

c

A

d

e

f

g

h

i

i

/B

Fig.1. Effect of Bsu concentration on phage DNA digest. A. 3 #g ~.dv I DNA were incubated in 10 mM Tr~s'-HCI, pH 7.4, 10 mM MgCI~, 5 mM ~-mercaptoethanol (standard conditions min~:s NaC~) in a total volume o f 20 ~1 with Bsu (a) 3 U, (b) 6 U, (c) 6 U plus 12% glycerol, (~.) 20 U plus 12% glycerol, (e) 40 U plus 12% glycerol. 1.5 UBsu are required to give a limit d i g ~ t under these conditions. B. 3 #g PM2 DNA were incubated exactly as in A. Tracks f---j correspond to a--e. The running gel is shown without the spacer gel.

of heterogeneous cleavage products (Fig. 1 d,e). This change in cleavage pattern is detected equally well with DNA from the phages PM2 (Fig.If--j), T7 or SPP 1.0 (not shown). The additional fragments are ~:ever produced in stoichiometric amounts; with further increases of enzyme concentrations more and more fragments of smaller size are produced, indicating that there is never a plateau value obtained with the characteristics of a new limit digest. This degradation of fragments in |imit digests is only observed when high

296

o

b

¢

A

d~

e

C

Fig.2. Effect of digestion conditions on Bsu digestion patterns of phage DNAs. A. 3 pg ~dv 1 DNA were incubated in 50 mM Tris--HCl, 10 mM MgCIs , 5 mM ~-mercaptoethanol within a total volume of 20/~1 with 8 U Bsu. The pH of the Tris buffer was 6.5 (a), 7.0 (b), 7.5 (c), 8.0 (d), 8.5 (e). B. 3 pg SPP 1.0 DNA (f) and SPP 1.R DNA (g) were digested with 2 U Bsu in 30 pl, at standard conditions. In h--j digestion of SPP 1.R DNA was done at high pH conditions with 2 U (h), 4 U (i), and 8 U (j) Bsa C. 3/Jg M 13 DNA were digested with 3 U ~cu at standard (k) and high pH conditions (!). Undegraded M 13 DNA remained in the spacer gel which is omitted in the photo.

c o n c e n t r a t i o n s of Bsu are used. Just h o w m u c h Bsu is required, however, to show t h e first sign o f f u r t h e r degradation ~ strongly d e p e n d e n t o n t h e react i o n m e d i u m . As s h o w n in F i g . l , glycerol stimulates t h e f o r m a t i o n o f additional b a n d s in Bsu digests. NaCI, on t h e o t h e r h a n d , is i n h i b i t o r y , even t h o u g h it stimulates the a c t i v i t y o f Bsu, m a x i m a l activity being b~.~ween 0.1 and 0.2 M NaCI (HSrz ~,~d Zachau, 1975; Bron e t al., 1975). T h e p H o f t h e

297

reaction medium was found to exert a strong influence: while the pH optimum for Bsu is around 7.4, the formation of the additional bands increases with increasing pH up to pH 8.5 (Fig.2). At even higher pH values all degradation is strongly suppressed. Taken together, conditions most conducive, to the formation of additional bands, which will be referred to as high pH conditions, are 25 mM Tris--HC1, pH 8.5, 10 mM MgClz, 5 mM ~-mercapto, ethanol and 25% glycerol. At these conditions as little as a twofold excess of Bsu suffices to generate additional bands, while at Bsu standard conditions cleavage with reduced specificity is first observed at a 20--40-fold exc~s of Bsu. The additional activity of Bsu can be assayed conveniently with DNA from the phage SPP 1 grown in amodifyingB, subtilis host (Trautner et ai., 1974). This SPP 1.1t DNA is completely resistant to digestion with Bsu at standard conditions since all Bsu cleavage sites are methylated, in contrast to SPP 1.0 DNA which is not modified (Gi2~nthert et al., 1975). Fig. 2f and g shows digests of the two DNAs obtained at standard conditions. At the high pH conditions, SPP 1.R DNA can be degraded to heterogeneous cleavage products with some indication of discrete bands (Fig.2h--j). It is remarkable that single-stranded M 13 DNA gives a pattern of sharp bands after degradation w_ith Bsu at high pH conditions, while there is very little if any breakdown at standard conditions (Fig.2k--l). Characteristically again, no limit digest is obtained at the high pH conditions. We will show in the following that the additional acti~Sty in our Bsu preparations is not due to a contaminating enzyme but a property of Bsu itself. Nucleotide sequences cleaved by Bsu at different digestion conditions

In order to find out which sequences are cleaved by the additional Bsu activity, digests were prepared with Bsu from PM2, SPP 1.R, and M 13 DNAs at high pH conditions. Gel patterns were obtained as shown in Fig. lj, 2j and 21, respectively. The 5'-terminal nucleotides of the fragments in these digests were determined.. The results are listed in Table I and are compared to those obtained with a Bsu digest of PM 2 DNA at standard conditions. It is clear that the additional activity also generates fragments which bave predominantlyC at the 5'-terminal position even though there is a small lbut significant proportion of 5'-termin~ other than C. This is best seen in the digest of SPP 1.1t DNA since the standard cleavage sites can no longer be cleaved because of the modification. In PM2 DNA cleaved at high pH conditions, the 5'-termini reflect both activities of Bsu and, consequently, a higher percentage of C is obtained. The highest value is found, as expected, when digestion with Bsu is performed under standard conditions. We then turned to the analysis of the penultimate nucleotides and compared Bsu digests of SPP 1.0 DNA at standard conditions and SPP 1.R DNA at high pH conditions. The fragments were labeled at the 5'-terminal positions and digested with pancreatic nuclease. The resulting oligonucleotides

298

TABLE I 5'-TERMINAL NUCLEOTIDES OF Bsu FRAGMENTS PRODUCED UNDER DIFFERENT CONDITIONS Phage DNAs were digested with Bsu under the conditions indicated and gave digestion patterns as those shown in Fig. I f , ij, 2j, ~nd 21, respectively. 5e-terminal nucleotides o f the fragments in the digests were det=~mined as described in METHODS. The relative amounts (in percent) o f the four nucleotides are listed. DNA PM2 PM2 SPP 1.R M 1~

Standard conditions High pH conditions High pH conditions High pit conditions

T

G

A

C

1.5

0.1

1.1

97.3

1.4

1.4

1.0

96.2

2.9

1.8

3.3

92.0

6.3

3.5

1.9

88.3

CCIAC)

©

CCG CCT

~

CCA

d25 CG AE 61 (pH 3.5)

CCC r..~ A

OBlue

marker

cc 0

Fig.3. Fractionation of oligonucleotides in a pancreatic DNAase digest o f terminally labeled Bsu fragments. SPP 1.R DNA was digested with Bsu at high pH conditions as shown in Fig.2B track (j). The fragments were labeled at the 5J-terminal position and digested with pancreatic DNAase. The digest was ionophoresed on AE 81 paper (pH 3.5) until the blue marker had migrated approximately 40 era, and t h e n in the second dimension on DE 81 paper (pH 2.0) as described by Murray (1973). Radioactive nucleotides were detected by autoradiography. The identities of the oligonueleotides were established from their electrophoretic mobilities o n t h e two ion, exchange papers and from the mobilities of the products formed on partial digestion with snake venom phusphodiesterase (Murray, 1973). The analogous procedure With SIP 1.0 DNA digested with Bsu at standard conditions gave a similar electropherogram, lacking however among others the radioactive spots which are shaded.

299

were resolved by twodlmensional electrophoresis according to Murray (1973) as shown for SPP 1.R DNA in Fig.3. The: analogous procedure with SPP 1.0 DNA gave a similar pattern, lacking however a number of radioactive spots. Some of those which are clearly resolved and are only present in the SPP 1.R digest are shaded in Fig.3. It is apparent that, in contrast to the standard Bsu activity (Bron and Murray, 1975), the additional ac~vity yields fragments which have all four possible nucleotides in the 5'-penultimate position. The relative ~mounts of the dinucleotides CA, CG, CT and CC in these experiments were quite similar in the autoradiograms. The Bsu activities in different preparations and their stability in inactivation studies

In order to show that Bsu itself and not a contaminating nuclease was responsible for the cleavage with reduced specificity we first compared relative amounts of both activities in a number of Bsu preparations differing in purity. Each preparation was assayed for Bsu activity as described in METHODS. In parallel the additional activity was assayed by measuring the relative intensity of the novel bands (see Fig.l) in ~dv 1 DNA digests. It turned out that in all preparations the ratio of Bsu activity to additional activity was identical. We then proceeded to investigate heat inactivation and inhibition by iodoacetamide for the two activities (Fig.4). In these experiments the additional activity was assayed by digestion of SPP 1.R DNA under high pH conditions. Residual activity after various degrees of inactivation was determined by comparing the respective digestion pattern to patterns of the type shown in Fig.2g--j, which had been produced by a series of known enzyme concentrations. It is evident that within experimental scatter of the points, inactivation curves for both activities are identical. Haelll does not show an analogous reduction in specificity

We investigated if a loss of specificity of the type observed for Bsu could be detected also with HaeIII, a restriction nuclease which also recognizes GGCC (Roberts et al., 1975). The result was that under standard conditions described for digestion with HaeIII (Roberts et al., 1975) no digestion beyond the limit digest could be demonstrated with ),dr I DNA even at a 40-fold excess of HaeIII. The analyses are not shown since standard limit digests were obtaiined in all cases. In keeping with this result, no digestion of SPP 1.R DNA was z~bserved. High pH conditions inhibit HaeIH more strongly than Bsu, but even at high enzyme concentrations no evidence for a reduction in specificity was noticed.

300

B

!oo 8

i00

so .-~

O nf

a

0 con~ Inhib. (raM)

|

|

I

10 20 time(rain)

tO

Fig.4. Inactivation of ~.su activitie~ A. 5 ~1Bsu (10 000 U/ml) were incubated with different iodoacetamide concentrations for 30 min at 37°C in 15 mM Tris--HCl, pH 7.4, 10 mM NaCI, 0.1 mM EDTA, 50% glycerol. Bsu activity remaining after treatment with iodoacetamide was assayed as described in METHODS by subsequent digestions of ~dv 1 DNA at standard conditions in the absence of B-mercaptoethanol. The additional activity was assayed with SPP 1.R DNA at higiz pH conditions. To this end, one half of the reacted enzyme was incubated for 3 h with 3/zg SPP 1.R DNA in a total volume of 30 ~1 at high pH conditions except that no 0-mercaptoethanol was present. In parallel, analogous digestions were carried out with known m o u n t s of Bsu in the range between 2 and 24 U (as, e.g., in Fig. 2h--j). From a comparison of the digestion patterns, activity remaining in the reacted enzyme could be determined. Percent standard (a) and additional (e) Bsu activities remaining are plotted vs. concentration of iodoacetamide. B. 5 ~1 Bsu (10 000 U/ml) was incubated for various times at 50°C in 15 mM Tris--HCl, pH 7.4, 10 mM NaCi, 0.1 mM EDTA, 5 mM 0-mercaptoethanol, 50% glycerol and aiterwards cooled on ice. Remaining activity was measured with ~,dv I DNA at standard and SPP 1.R DNA at high pH conditions as described in A, but with 5 mM O-mercaptoethanol present in the assays. Percent remaining standard (v) and additional (e) Bsu activities are plotted vs. time of inactivation at 50°C. DISUCSSION We have investigated t h e finding t h a t a t certain c o n d i t i o n s additional fragm e n t s appear in Bsu l i m i t digests of phage DNAs. It b e c a m e clear t h a t these fragments are p r o d u c e d p r e d o m i n a n t l y b y cleavage at t h e d i n u c l e o t i d e sequence GC rather t h a n t h e GGCC site cleaved b y Bsu at s t a n d a r d conditions. T h a t t h e splitting at sequences o t h e r t h a n G G C C is a p r o p e r t y o f Bsu itself and n o t d u e to a c o n t a m i n a t i n g nuclease is indicated b y several lines o f evidence. First o f all it w o u l d have to be a r a t h e r specific nuclease c o n t a m i n a n t to a c c o u n t for t h e sharp additional bands in t h e gel patterns. T h e ratio o f additional activity to Bs u activiW is c o n s t a n t in Bsu p r e p a r a t i o n s at d i f f e r e n t stages o f purification; b o t h activities s h o w identical inactivation curves in h e a t d e n a t u r a t i o n or after t r e a t m e n t w i t h iodoacetamide. Finally, t h e gradual transition f r o m specific cleavage at G G C C t h r o u g h a stage o f defined additional bands in gel p a t t e r n s to cleavage a t N G C N makes t h e participation o f a n o t h e r nuclease e x t r e m e l y unlikely.

301

The possibility that the recognition sequence is reduced from the standard tetranucleotide sequence only to a trinucleotide sequence (GGC or GCC) is very improbable in view of symmetry considerations and also in view of the finding of similar ~mounts of all four nucleotides in the 5'-penultimate positions of the cleavage products. A decrease in specificity of Bsu has some practical implications and is interesting for mechanistic considerations. One practical aspect of this work was to give some quantitative estimates as to the degree of specificity of substrafe recognition for Bsu. Specificity is strongly dependent on reaction conditions, but even at conditions of maximal specificity cleavage begins to occur at sequences other than GGCC when a 20-fold excess of Bsu is employed. Concentrations of Bsu higher than this have been used in digestions with intact nuclei (Pfeiffer et al., 1975; HSrz et al., 1976). However, according to the available criteria, cleavage proceeded specifically in these experiments. A possible reason for this specificity might be that the Bsu concentration effective within the nuclei is lower than the applied concentration. At digestion conditions of minimal specificity as little as a 2-fold excess of Bsu suffices to lead to nonspecific cleavage. Reaction media approaching these conditions may be reached inadvertently, e.g. by the use of rather dilute preparations of Bsu stored in 50% glycerol. The finding that specificity is largely retained for the central dinucleotide pair of the Bsu recognition site while it is lost for the two outer nucleotides was not necessarily to be expected and is interesting as far as the mechanism of recognition between the nuclease and the nucleic acid is concerned. Those properties would fit the concept well that the catalytic sites of depolymerizing enzymes are constructed of subsites recognizing a series of adjoining residues of the polymer chain. This concept has been formulated for proteinases, amylases and nucleases (see Allen and Thoma (1976) for a number of examples). In our specific case it would entail separate subsites with different affinities for at least the four nucleotide pairs of the recognition sequence. Within the transition from cleavage at GGCC to cleavage at NGCN, certain sequences must be strongly favored. Otherwise at the onset of the loss of specificity, there would not be just a few characteristic fragments present in large amounts in the digestion patterns. A graduated relaxation of specificity affecting subsites individually would account for our findings. A comparable change in specificity has been described' for the restriction nuclease EcoRI (Polisky et al., 1975). At certain conditions cleavage occurs at the tetranucleotide sequence AATT instead of GAATTC, endowing this nuclease with a novel activity which the authors termed EcoRI ÷. The analogy with Bsu holds even with respect to the conditions of lower specificity (low ionic strength, high pH). In the case of EcoRI+, it was also observed that even though specificity is relaxed at positions 1 and 6 of the original EcoRI sequeI~ce, the nucleotides present in these positions strongly affect the kinetics of splitting by the EcoRI ÷ activity. The concept of subsites would well account for these properties as well as the finding that EcoRI splits different

302

cleavage sites on DNA of phage ), at very different rates (Thomas and Davis, 1975). In this latter case subsites for bases outside the hexanucleotide recognition site might play a role. With Bsu even more than with EcoRI it is difficult to reach a limit digest under conditions of reduced specificity. This complicates but may not preelude the use of Bsu as a GC-specifie nuclease. For a few restriction nucleases splitting of single-stranded DNA has been reported (Horiuchi and Zinder, 1975; Blakesley and Wells, 1975; Blakesley, 1976). The finding that cleavage of single stranded M 13 DNA with Bsu is facilitated at conditions of reduced specificity can be explained in different ways. It might be that relaxation of specificity makes single-stranded DNAs a more acceptable substrate, Alternatively one might follow the arguments: of Blakesley (1976) that splitting only occurs at locally base paired regions~ Obviously, there is a much greater probability of a dinucieotide sequence being present in a base-paired region than of a tetranucleotide sequence. The fact that HaeIH does not exhibit a Bsu type reduction in specificity shows that even among isosehizomers the mechanism of recognition is different. It is certainly reasonable to assume, though, that for HaeHI as well as for other restriction nucleases, conditions exist at which specificity can be manipulated. For theoretical considerations as well as practical purposes it would be interesting to know if the principle of nucleic acid recognition shared by Bsu and EcoRI is a more universal property of restriction nucleases. ACKNOWLEDGEMENT

We thank K. Murray for a gift of Whatman AE 81 paper and T.Trautner for the B. subtilis strains and phage SPP 1 and also for communicating, prior to publication, his results on the behaviour of Bsu in streptomycin precipitation. We are grateful to R.E. Streeck for gifts of )~dvl, PM2, and M13 DN.~ REFERENCES Allen, J.D. and Thoma, J.A., Biochem. J., 159 (1976), 105--120. Bernardi, G., in Cantoni, G.L. and Davis, D.R. (Eds.), Proe. Nucl. Acids Res., Harper and Row, New York, Vol. II, 1971, pp. 455--499. Blakesley, R.W., Fed. Proe., 35 (1976) 1518. Blakesley, R.W. and Wells, P~D., Nature (London), 257 (1975) 421--423. Bron, S. and Murray, K,, MoL Ge~ Genet., 143 (1975) 25--33. Bron, S., Murray, K. and Trantner, T.A., Mol. Gen. Genet., 143 (1975) 13--23. Frischanf, A.M. and Ecksteln, F., Eur. J. BioehenL, 32 (1973) 479--485. Giinthert, U., Stutz, J. and Klotz, G., Mol. Gen. Genet., 142 (1975) 185--19~t. Horiuehi, K. and Zinder, N.D., Proc. Natl. Aead. Sei. USA, 72 (1975) 2555--2558. HSrz, W. and Zaehau, H.G., FEBS Syrup., Budapest, 33 (1975) 403--408. H~rz, W., Igo-Kemenes, T., Pfeiffer, W. and Zaehau, H.G., Nucl. Acids Res., 3 (1976) 3213--3226. Melehers, F. and Zael~u, H.G., Bioehhn. Biophys. Aeta, 91 (1964) 559--572. Murray, I~, Biochem. j., 131 (1973) 569--583. Nathans, D. and Smith, H,O., Annu. Rev.Biochem., 44 (1976) 27~--293.

303 Petrova, M., Phflippsen, P. and Zachau, H.G., Biochim. Biophys. Acta, 395 (1975) 455--467. Pfeiffer, W., H~rz, W., Igo-Kemenes, T. and Zachau, H.G., Nature (London), 258 (1975) 450--452. Philippsen, P. and Zachau, H.G., Biochim. Biophys. Acta, 277 (1972) 523--538. Polisky, B., Greene, P., Garfin, D.E., McCarthy, B.J., Goodman, H.M. and Boyer, H.W., Proc. Natl. Acad. Sci. USA, 72 (1975) 3310-3314. Roberts, R.J., in ~'asman, G.D.(Ed.), CRC Handbook of Biochemistry and Molecular Biology, Nucleic Acids, Vol. II, CRC Press, Cleveland, Ohio, 1976, pp. 532--534. Roberts, R.J., Breitmeyer, J.B., Tabachnik, N.F. and Myers, P.A., J. biol. Biol., 91 (1975) 121--123. Smith, H.O. and Nathans, D., J. Mol. Biol., 81 (1973) 419--423. Streeck, R.E. and Hobom, G., Eur. J. Biochem., 57 (1975) 595--606. Streeck, R.E., Philippsen, P. and Zaehau, H.G., Eur. J. Biochem., 45 (1974) 489--499. Trautner, T.A., Pawlek, B., Bron, S. and Anagnostopoulos, C., Mol. Gen. Genet., 109 (1974) 181--191. Wirtz, A. and Hofschneider, P.H., Eur. J. Biochem., 17 (1970) 141--150. Communicated by W. Fiers.

Specificity of cleavage by restriction nuclease from Bacillus subtilis.

Ge~e, 1 (1977) 291--303 291 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands SPECIFICITY OF CLEAVAGE BY A RESTRICTI...
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