Random cleavage of superhelical SV40 DNA by S1 nuclease

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Biochimica et Biophysica Acta, 425 (1976) 157--167

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98527 RANDOM CLEAVAGE OF SUPERHELICAL SV40 DNA BY S, NUCLEASE

WALDEMARWALDECK, KAMAL CHOWDHURY,PETER GRUSS and GERHARD SAUER Institut fiir Virusforschung, Deutsches Krebsforschungszentrum, 69 Heidelberg (G.F.R.) (Received October 31st, 1975}

Summary SV40 DNA FO I is randomly cleaved by S, nuclease both at moderate (50 raM) and higher salt concentrations (250 mM NaC1). Full length linear $1 cleavage products of SV40 DNA when digested with various restriction endonucleases revealed fragments that were electrophoretically indistinguishable from the products found after digestion of superhelical SV40 DNA FO I with the corresponding enzyme. Concordingly, when the linear $1 generated duplexes were melted and renatured, circular duplexes were formed in addition to complex larger structures. This indicated that cleavage must have occurred at different sites. The double-strand-cleaving activity present in $1 nuclease preparations requires circular DNA as a substrate, as linear SV40 DNA is not cleaved. With regard to these properties $1 nuclease resembles some of the complex type I restriction nucleases from Escherichia coli which also cleave SV40 DNA only once, and, completely at random.

Introduction Single-strand-specific S, nuclease (EC 3.1.4.--) isolated from the eukaryotic organism Aspergillus oryzae cleaves superhelical SV40 and polyoma DNA form I (FO I) to linear duplex structures (FO III) of unit length [1,2,3]. Beard et ai. [1] have suggested, based upon results obtained by heteroduplex mapping, that cleavage occurs preferentially, depending on the salt concentrations, at either one of the regions 0.15 to 0.25 and 0.45 to 0.55 fractional length from the Eco R I restriction endonuclease cleavage site. Recently Germond et al. [3] have investigated by neutral sucrose gradient centrifugation the products obtained after cleaving polyoma DNA FO I with S, nuclease. It was proposed that the enzyme attacks the polyoma DNA in one of two or three discrete regions. Further, the suggestion was made that weakly base-paired regions brought about by a topological constraint in the superhelical molecules

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might be the site of attack for the single-strand-specific S~ nuclease [1,2,3], hence one might be able to discern and map such regions within a superhelical molecule. On the other hand, the ability of the enzyme to convert circular DNA FO II to FO III DNA structures does not depend on the superhelicity of the DNA as postulated, as we have shown that relaxed circular SV40 DNA F O I I bearing several single-strand nicks is also being converted to FO III [4]. This observation is difficult to reconcile with the above-mentioned hypothesis. Therefore, we have examined S~ nuclease-generated SV40 DNA FO III after redigestion with other restriction endonucleases. We will present evidence in this paper for random cleavage of SV40 DNA by $1 nuclease. We could not confirm, using high resolution agarose gels, that S~ nuclease attacks preferentially certain regions within the SV40 DNA. Rather the enzyme appears to bind to the DNA and then to cut only once, and, completely at random. Once the DNA is linearized, it is not cleaved for a second time. The ability of the S~ nuclease to introduce a double-strand cut clearly depends on circular DNA as a substrate, which, also in contrast to previous reports [1,3], does not have to be supercoiled [4]. Materials and Methods

Cells and virus. CV-1 cells were infected at low multiplicities (0.5 plaque forming units/cell) of plaque purified SV40 strain Rh911. The infected cells were incubated either with 5 pCi/ml (15 Ci/mmol) [3H]thymidine or with [32p] orthophosphate (50 pCi/ml) in phosphate-free medium between 24 and 48 h after infection. Purification of SV40 DNA. Superhelical SV40 DNA was isolated by extraction of the NaCl-dodecyl sulfate supernatant of the infected cells with phenol and chloroform/isoamylalcohol (24 : 1, v/v), subsequent RNAase treatment, followed b y another extraction with chloroform/isoamylalcohol. Then the DNA was further purified in a CsCl-ethidium bromide density gradient as described [ 5 ] . The superhelical DNA was sedimented through a neutral sucrose gradient (5 to 20% sucrose in 1 M NaC1, 0.01 M Tris, pH 7.2, 0.001 M EDTA). The gradients were centrifuged in a Beckman SW 50.1 rotor at 46 000 rev./min for 2 h at 20°C. The FO I DNA sedimenting at 21 S was isolated and used in all experiments. S~ nuclease preparation and assay. S, nuclease was prepared as described by Vogt [6] using 20 g of s-amylase (Sigma Chemical Company, U.S.A., lot no. 103c-1550), up to and including the sulfo-sephadex chromatography. The reaction buffer (30 mM sodium acetate, 1.0 mM ZnSO4, 50 mM NaC1, 5% glycerol, pH 4.6) was the same as described by Vogt [6] except that denatured DNA was omitted from the reaction mixture. The reaction was carried o u t for 30 min at 37°C. Under this reaction condition 1 ttl of $1 enzyme rendered 4 pg of denatured SV40 F O I I DNA in 100 ttl acid soluble, while the same a m o u n t of native DNA remained completely acid precipitable. The purity of the S~ enzyme was tested b y treating 3H SV40 FO I DNA (0.5 #g) with 2 pl of $1 for 30 min and analysing the linear FO III p r o d u c t in alkaline sucrose gradients. Only the enzyme which was purified through sulfo-sephadex was free of nick-

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ing activities, as the reaction product was linear single-stranded DNA sedimenting at 16 S (see also Fig. 2). Smaller fragments appeared, in addition, when $1 nuclease purified only up to the DEAE step was used. Agarose slab gel electrophoresis. Electrophoresis was performed in vertical 1.4% agarose slab gels (Seakem agarose, mci biomedical, Rockland, Maine) according to the method described by Tegtmeyer and Macasaet [7]. The size of the analytical gels was 13.5 X 25 X 0.3 cm. 50- to 100-pl samples were applied to the gels. Electrophoresis was carried out in the cold room at 4°C at 70 V per gel for 18 h. After electrophoresis, the gel was frozen at--20°C and cut with a set of 200 razor blades (distance 1 mm). The gel pieces were transferred to plastic scintillation vials containing a mixture of 4 g/1 2,5-diphenyloxazol (PPO) in 1 part ethanol and 4 parts toluene. The samples were stored over night at 4°C until the gel slices became transparent, and they were then counted in a nuclear chicago mark II liquid scintillation counter. Preparation of restriction endonucleases and assay conditions. Eco R, was a generous gift of Drs. Philippsen and Zachau, Institut fiir Physiologische Chemie und Physikalische Biochemie der Universit~'t Mfinchen. The Eco RI reaction buffer was as follows: 12 mM MgC12, 90 mM Tris, pH 7.9, 0.1 M NaC1. Restriction endonuclease II and III from Haemophilus influenzae strain d: Restriction endonuclease from H. influenzae was prepared as described [8] up to the agarose chromatography step. From then on the following modifications were introduced. The activity-containing fractions were dialysed against 0.02 M KH2PO4 (pH 7.4), 0.1 mM EDTA, 0.01 M mercaptoethanol, 10% glycerol. The dialysate was applied to a 2.5 X 30 cm DE-52 (Whatman) cellulose column previously equilibrated with dialysis buffer and eluted with a linear KC1 gradient (0 to 0.3 M) in dialysis buffer. 6-ml fractions were collected. The activity was tested with 0.5 #g SV40 DNA in 40 ~1 0.1 X standard saline citrate *, 5/~1 10 × reaction buffer (0.5 M NaC1, 0.06 M Tris, pH 7.4, 0.06 M MgC12, 0.06 M mercaptoethanol). From each of the 6-ml fractions 5 /~1 were added to the reaction mixture. Incubation took place at 37°C for 60 min. The analysis of the cleavage pattern of the DNA was performed in 1.4% agarose gels. A broad activity spectrum ranged from fractions 30 to 130. Fractions 30 to 72 generated a Hind III restriction pattern [9], whereas fractions 74--125 contained mixed Hind II, Hind III activities. These fractions were pooled and separately applied to a 1.0 X 10 cm hydroxyapatite (Biogel HT, Bio-Rad) column and eluted with a linear 10 to 400 mM KH2PO4, pH 6.8, gradient. It was possible to separate by chromatography on hydroxyapatite Hind III completely from exonucleases. Also Hind II which eluted before Hind III was separated from contaminating nucleases. The fractions, which contained either the Hind III or the Hind II activities were dialysed against reaction buffer containing 20% glycerol and stored at 4 ° C. Restriction endonuclease from Haemophilus parainfluenzae (Hpa I and II): restriction endonuclease from H. parainfluenzae was isolated as described [10], with some modifications: The bacterial cell lysate was adjusted to 30% saturation by addition of solid ammonium sulfate, stirred for 30 min and centrifuged

* S S C , s t a n d a r d saline c i t r a t e : 0 . 1 5 M N a C 1 / 0 . 0 1 5 M s o d i u m citrate.

160 at 15 000 rev./min in a Heraeus Christ Zeta 20 centrifuge at 4°C for 30 min. The supernatant was adjusted to 70% saturation by addition of solid ammonium sulfate. The precipitated protein was collected by centrifugation and resuspended in 10 ml of buffer solution (0.01 M KH~PO4, pH 7.4, 0.001 M mercaptoethanol) and desalted on Sephadex G25 (3 cm X 30 cm column). The final volume of 60 ml was adjusted to 1 M NaC1 by addition of solid NaC1 and applied to an agarose (A 0.5 m, Biogel, Bio Rad) column equilibrated with 1 M NaC1, 0.02 M Tris, pH 7.4, 0.01 M mercaptoethanol as described [10]. The enzyme activity was tested using SV40 DNA FO I as a substrate as mentioned above, except that the following 10 X concentrated reaction buffer was used: 0.1 M Tris, pH 7.4, 0.1 M MgC12, 0.06 M KC1, 0.01 M mercaptoethanol. Incubation was performed at 30°C for 60 min. The separated Hpa I and Hpa II were layered on h y d r o x y a p a t i t e columns and eluted with a linear potassium phosphate gradient as during isolation of the Hind enzymes. The fractions which contained either Hpa I or Hpa II activity were pooled, dialysed against reaction buffer containing 50% glycerol and stored at 4°C. For cleaving either FO I or FO III with Hind or Hpa restriction endonucleases the reaction mixtures contained 5 pl of 10 X concentrated Hind or Hpa reaction buffer as described above, 0.01 pg to 0.1 gg DNA and 5 to 10 pl of Hind or Hpa restriction endonucleases. The total volume was adjusted to 50 pl by addition of distilled water. The reaction was stopped by addition of EDTA to a final concentration of 10 mM. Before being applied to the gels one fifth of the volume of a solution containing 8 M urea, 50% sucrose and 0.05% bromophenolblue was added to the samples. Conversion o f SV40 DNA FO I to FO III with $1 nuclease. To convert 3Hor 32P-labeled SV40 DNA FO I to FO III, 10 #g DNA were incubated in S~ buffer with 30 pl of $1 nuclease (total vol.: 250 #l) at 37°C for 30 min. The reaction was stopped by addition of one-fifth the volume of a solution containing 8 M urea, 0.1 M EDTA, 0.05% bromophenolblue and 50% sucrose. Then the mixture was applied to a preparative 1.4% agarose gel for electrophoretic separation of the $1 generated FO III ($1 FO III) from residual F O I I . The size of the gels was 9 X 10 X 0.3 cm and the actual distance, after electrophoresis (2 h, 100 V) between FO III and F O I I was approximately 2 cm. From the gels, a small (1 cm) reference section was separated and stained (10 min in distilled water containing 5 #g/ml ethidium bromide). The agarose containing the $1 FO III was excised using the stained gel section under ultraviolet light (254 nm) as a reference. Then the agarose was transferred into a small glass homogenizer, and the DNA was recovered, after addition of 5 ml distilled water by homogenization (10 strokes). The agarose was pelleted by centrifugation for 10 min at 15 000 rev./min in a Heraeus Christ Zeta 20 centrifuge. The supernatant containing the S~ FO III input was adjusted to 1 M NaC1 and mixed with 2 X the volume of ice cold ethanol. After 2 h at --20°C the precipitated DNA was pelleted in a Heraeus Christ Minifuge (15 min, 6000 rev./min). The pellet, which still contained some agarose, was suspended in 0.5 ml of 0.05 X SSC. Usually more than 80% of the FO I DNA was converted to FO III and was recovered as such by this method. Further purification of S~ generated FO III for secondary digestion with restriction nucleases. The S~ FO III obtained by this procedure is {probably

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owing to strongly binding proteins contained in the S~ nuclease preparation) not always susceptible to further digestion with restriction endonucleases. This is evidenced by treatment of the isolated S, FO III with Hind or Hpa restriction endonucleases. As revealed by electrophoresis, such doubly digested S, FO III preparations remained frequently unaltered as FO III. We attempted, therefore, to remove putative binding proteins by treatment of the S, FO III with an equal volume of 8 M urea, 0.1 M EDTA for 30 min at 37°C. Subsequently the DNA solution was absorbed at 60°C to a hydroxyapatite column to prevent, on the one hand, re-binding of polypeptides to the DNA, and, on the other hand, to effectively remove contaminating agarose which also might occasionally impair successful redigestion with restriction endonucleases. The column (a 5 ml plastic syringe) containing at the bottom a Whatman glass fiber filter pad (GF/C) of 1 cm diameter was loaded with 2 ml of hydroxyapatite suspended in 1 mM potassium phosphate buffer. After adsorbing the DNA, the column was washed with 5 ml of 0,14 M sodium phosphate buffer. Elution of the doublestranded DNA took place with 5 ml (0.5 ml portions) of 0.4 M potassium phosphate buffer. The distribution of radioactivity was determined using 10 #1 aliquots which were dried on filters and counted in a liquid scintillation counter. The peak fractions containing the S, FO III were pooled and dialysed over night against 3 liters of 0.05 × SSC with two changes in between. This method reveals a "reactive" S, FO III (see Fig. 2 for product analysis) which is susceptible to digestion with various restriction endonucleases. The same procedure can be used to remove binding proteins from the DNA substrate that may occasionally be contained in restriction endonuclease treated DNA preparations. Alkaline velocity sedimentation. The DNA was sedimented through 5--20% sucrose dissolved in 0.9 M NaC1, 0.1 M NaOH, 0.01 M Tris, 0.001 M EDTA, 0.05% sarcosyl. Before adding the samples, 20 pg of denatured calf thymus DNA were layered on the gradient to prevent loss of single-stranded DNA. The centrifugation took place in a Beckman SW 41 rotor at 30 000 rev./min for 18 h at 20°C. Eight drop fractions were collected on Whatman paper filter pads (3 MM, 23 mm diameter), dried and counted in toluene containing 4 g/1 2,5-diphenyloxazole in a scintillation counter. Denaturation and renaturation of DNA. SH S, FO III DNA (10 pl) in distilled water was denatured in an Eppendorf plastic vial (7 min in a boiling water bath) and then quenched in ice. 5/~I of 5 M NaC1 were then added, and the DNA was allowed to reassociate at 65°C for 18 h. The concentration of the DNA was 3 pg/ml. Results

Isolation and analysis of S, nuclease-generated SV40 DNA FO III To obtain S, generated linear SV40 FO III (S, FO III) superhelical SV40 DNA FO I was reacted with S, nuclease and the reaction product subjected to agarose gel electrophoresis. In gels stained with ethidium bromide and examined under ultraviolet light the FO III can be readily distinguished from both F O I I and FO I (Fig. 1). The section of the gel containing FO III was isolated, and the DNA was extracted by homogenization of the gel as described in

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Fig. 1. A g a r o s e slab gel e l e c t r o p h o r e s i s of v a r i o u s f o r m s o f S V 4 0 D N A . (a) C o n v e r s i o n of S V 4 0 FO I D N A t o F O I n a n d F O I I b y t r e a t m e n t w i t h S l n u e l e a s e . (b) S V 4 0 FO I D N A . (c) F O I I D N A g e n e r a t e d b y D N A a s e - t r e a t m e n t as d e s c r i b e d [ 4 ] . T h e gel was s t a i n e d w i t h e t h i d i u m b r o m i d e a n d p h o t o g r a p h e d u n d e r u l t r a v i o l e t light u s i n g a p o l a r o i d c a m e r a MP4.

Materials and Methods. It is essential for further analysis that tightly b o u n d proteins contained in $1 nuclease preparations are carefully separated from the DNA, as failure to do so, will often render $I FO III inaccessible to further digestion with restriction endonucleases. We have found treatment of the $1 FO III with 8 M urea, 0.1 M EDTA followed by chromatography on hydroxyapatite (see Methods) a satisfactory m e t h o d to produce "reactive" FO III which can be subjected to further digestion with restriction endonucleases. A typical $1 FO III preparation obtained by this m e t h o d is shown in Fig. 2, where an aliquot of the "reactive" $1 FO III was re-electrophoresed in agarose and where another aliquot (see insert) was analysed together with a 16 S sedimentation marker in an alkaline velocity gradient. It may be seen that the DNA runs as a homogeneous peak in the gel and that it contains very few, if any, singlestrand nicks, as revealed by the sharp sedimentation profile in the alkaline gra-

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Fig. 2. A n a l y s i s of t h e S l g e n e r a t e d F O I I I b y e l e c t r o p h o r e s i s a n d alkaline v e l o c i t y s e d i m e n t a t i o n . 3H S! F O I I I (spec. a c t . 2.4 • 105 c p m / # g ) w a s i s o l a t e d as d e s c r i b e d f r o m a gel a n d a n a l i q u o t was re-electxop h o r e s e d in a n a g a r o s e slab gel ( 1 3 . 5 X 2 5 X 0.3 e m ) . F r a c t i o n s 1 t o 4 0 w h i c h c o n t a i n e d n o radioactiVity w e r e o m i t t e d f r o m t h e F i g u r e . A l k a l i n e v e l o c i t y s e d i m e n t a t i o n (insert) o f a n a l i q u o t o f t h e s a m e 3 H Sl F O I I I . T h e a r r o w s i n d i c a t e t h e p o s i t i o n o f E c o R I g e n e r a t e d 3 2 p S V 4 0 FO I I I m a s k e r D N A .

163 dient. S~ nuclease preparations which were only purified up to the DEAEcellulose step [6], and that were not further subjected to chromatography on sulfo-sephadex (as was the enzyme used in this work) were contaminated with nucleases and introduced, as a consequence, single-strand nicks into the S~ FO III.

Cleavage o f $1 FO III DNA with various restriction endonucleases To re-investigate whether $1 nuclease cleaves SV40 DNA in a specific region, S1 FO III was digested with various restriction endonucleases, and the products were electrophoresed in agarose gels. If, for example at moderate salt concentrations (50 mM NaC1) cleavage would occur, as reported [1] preferentially in one of two regions, one would expect to find, after redigestion of the S~ FO III with restriction endonucleases, characteristic alterations of the cleavage pattern. If, on the other hand, $1 nuclease would attack the DNA in a random fashion the fragments produced should be indistinguishable from those obtained after digestion of circular DNA. This pattern, however, should be superimposed on an increased background. The last possibility is borne out by the data shown in Fig. 3a where cleavage of S~ FO III with Hpa I is depicted. The

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Fig. 3. C l e a v a g e o f 3 2 p S V 4 0 S 1 F O III D N A a n d 3 H S V 4 0 F O I D N A w i t h H p a I a n d H i n d III r e s t ~ c t i o n e n d o n u e l e a s e s . T h e H p a I (a) a n d H i n d I n (b) c l e a v a g e p r o d u c t s o b t a i n e d f r o m a m i x t u r e o f S1 F O III a n d F O I w e r e a n a l y s e d b y e l e c t r o p h o r e s l s i n a g a r o s e slab gels. T h e S1 F O III u s e d in the inserts w a s generated in presence of 2 5 0 m M NaCI. F r a c t i o n s 1 t o 7 0 (a) a n d 1 t o 5 0 (b) w h i c h d i d n o t c o n t a i n a n y r a d i o a c t i v i t y w e r e o m i t t e d f r o m t h e figures.

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restriction endonuclease Hpa I cleaves SV40 to three large fragments, 43, 38 and 19%, respectively, of the length of SV40 DNA [10]. A comparison between SV40 DNA FO I and $1 FO III cleaved by Hpa I does not reveal, apart from an increased background, a difference in the resulting cleavage patterns. In each case three products were generated. There is no evidence for the appearance of a new product or, for the preferential attack of a particular region \ of the SV40 DNA molecule by $1 nuclease which would have altered recognizably the cleavage pattern. To investigate whether cleavage of SV40 FO III by S1 nuclease under conditions of high salt (250 mM NaCl) t o o k place in a particular region [1], $1 FO III was isolated as described and cleaved with Hpa I restriction endonuclease. A typical Hpa I cleavage pattern was revealed (see insert in Fig. 3a) which did not differ from the control (FO I cleaved with Hpa I nuclease). We conclude, therefore, that at high salt SV40 FO I DNA was also cleaved by $1 nuclease at random. The restriction endonuclease Hind III has recognition sequences that are different from those recognized by Hpa I [9]. Six products are formed, the largest of which contains the region 0.15 to 0.25 fractional length from the Eco R1 recognition site which is suspected of being one of the preferred sites of attack by $1 nuclease [1]. Hence, cleavage of $1 FO III by Hind III should considerably change the electrophoretic mobility of the largest fragment, as approximately one third of the sequences should be removed after double digestion. In constrast to this prediction, it is clear from the data shown in Fig. 3b that the mobility of all the $1 FO III-Hind III fragments remained quite unaffected. Rather, the same cleavage pattern was again generated as in the case of Hind III-cleaved FO I. It may also be seen in Fig. 3b, that the size of the peaks of the large fragments is relatively decreased as compared with smaller fragments. Furthermore, there is, similar to the Hpa I-cleavage of S~ FO III, an elevated background (Fig. 3a). The insert in Fig. 3b shows the Hind III cleavage pattern of an $1 FO III which had been generated in S~ buffer containing 250 mM NaC1. Again a typical Hind III cleavage pattern was obtained. These results confirmed the above-mentioned observation with Hpa I, namely that cleavage of SV40 DNA FO I by $1 nuclease occurs completely at random both in high and in low salt. Denaturation and renaturation o f $1 FO III That cleavage of FO I takes place at random and not at a specific site can also be shown by denaturation and renaturation of $1 FO III and analysis of the reassociation products in agarose gels. If cleavage t o o k place at a specific site, FO IH molecules of unit length would be expected to occur as reassociation products. The result depicted in Fig. 4 does not comply with this expectation. The vast majority of the reassociation products consisted of large, probably tandemly arranged structures, from randomly cleaved and partially basepaired strands. Approximately 10 to 15% of the reassociation products formed ring structures which co-electrophoresed with DNAase-generated SV40 DNA F O I I . Formation of circular molecules can be explained by assuming random cleavage of the DNA by $1 nuclease. Single-strands with different starting points and different termini can form partially base-paired duplexes with over-

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Fig. 4 . D e n a t u r a t i o n a n d r e n a t u r a t i o n o f S 1 F O III S V 4 0 D N A . S 1 g e n e r a t e d F O III w a s d e n a t u r e d and r e n a t u r e d as d e s c r i b e d in m a t e r i a l s a n d m e t h o d s . T h e r e a s s o c i a t e d D N A w a s e l e c t r o p h o r e s e d in an agarose gel. T h e a r r o w s i n d i c a t e t h e p o s i t i o n o f 3 H F O II m a r k e r D N A w h i c h w a s p r o d u c e d b y D N A a s e - t r e a t m e n t o f F O I as d e s c r i b e d [ 4 ] , and t h e p o s i t i o n o f E c o R I 3 2 p F O III S V 4 0 D N A w h i c h w e r e e l e c t r o p h o r e s e d in a p a r a l l e l t r a c k . Fig. 5. Treatment of Eco R I generated F O IIl S V 4 0 D N A with S 1 nuclesee. 32p S V 4 0 D N A F O I was converted with 2 N1 of Eco R I restriction endonuclease to form III. T h e reaction was stopped after 1 h by addition of 0.I M E D T A to a final concentration of 10 raM. T h e n the reaction mixture was subjected to treatment with urea and subjected to chromatography on hydroxyapatite as described in materials and methods. T h e 32p F O III D N A was mixed with 3 H F O I D N A and incubation with 10/~I of S I nuclease for 30 rain. T h e reaction was stopped by addition of one-fifth of the reaction v o l u m e of 8 M urea, 0.1 M E D T A , 5 0 % sucrose, 0.05% bromophenolblue. Electrophoresis was performed in an agarosc gel. T h e arrow indicates the position of Eco R I F O III which was eleetrophoresed in a paralleltrack of the s a m e gel.

lapping ends that are then circularized [15]. Only 1.5 to 2 % of the strands formed after reassociation F O III molecules of unit length. This reaction product could either be due to a small amount of molecules that had been cleaved by $I nuclease at a specific site,or, within a specific small region. Attempts to cleave linear SV40 DNA with $1 nuclease. S1 nuclease cleaves, besides SV40, various circular D N A s such as p o l y o m a [3] and the k dv-120 plasmid D N A [1] to unit length linear molecules. We also find that the E. coli plasmid COLE1 D N A is converted by $1 nuclease to open linear FO III D N A (unpublished). These results, together with the observation that cleavage occurs in S V 4 0 D N A at random sites, suggest, that only circular D N A molecules can be cleaved, which once they are linearized, cannot be cleaved further. To test this hypothesis, we have used as a substrate Eco R1 generated linear SV40 D N A as a substrate for $1 nuclease. It may be seen that the linear D N A retained unit length size after treatment with the S~ nuclease (Fig. 5). It should be pointed out that the Eco R I FO III was "reactive", as it proved to be susceptible to attack by other restriction endonucleases such as Hind III. To test the activity of the S~ nuclease 3H S V 4 0 D N A FO I was added to the reaction mixture. Almost all FO I D N A was converted by the S~ nuclease to FO III, the rest being converted to FO II. A similar result was obtained, when SV40 D N A FO I was cleaved first with

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Hind III restriction endonuclease and then re-digested with $1 nuclease. No alteration of the cleavage pattern as compared to Hind III treated FO I could be noticed. It is also of interest that $1 FO III which is susceptible to digestion with various restriction nucleases cannot be cleaved any further by a second treatment with S~ nuclease. These lines of evidence suggest that only circular DNA serves as a substrate for the cleaving activity of $1 nuclease. Discussion The results presented in this paper have shown that superhelical SV40 DNA FO I is opened b y S1 nuclease to form linear duplex rods of full length. Cleavage occurs, in contrast to other reports [1,3] completely at random and not within one or two regions regardless of the salt concentrations used (either 50 mM or 250 mM NaC1). The different observations made b y us may be explained b y the fact that we have employed in our experiments $1 generated FO III DNA which had been subjected to protein-removing-procedures which rendered the DNA susceptible to further digestion with restriction endonucleases. It is conceivable that failure to remove either DNA binding proteins or, presence of other nucleases in $1 nuclease preparations may account for the results obtained b y others. It is equally possible that failure b y other groups to cleave non-superhelical circular SV40 DNA b y $1 nuclease to FO III may be accounted for b y similar problems. We have shown that SV40 DNA F O I I bearing on average four single-strand nicks per molecule can be readily converted b y $1 nuclease to unit length FO III without the appearance of smaller fragments [4]. This observation together with the fact that non-superhelical circular closed fd RF DNA can be converted to linear double-stranded rods of unit length by S~ (manuscript in preparation) shows, that neither the presence of single-strand nicks nor superhelicity with ensuing weakly base-paired regions (1,2,3) are necessary prerequisites for the double-strand activity of S~ nuclease. Rather, we would like to suggest an entirely different mode of action which does not invoke the single-strand specificity of the enzyme to explain its action on double stranded circular DNA. There is a striking similarity between restriction endonucleases t y p e I isolated from E. coli (Eco B [ 1 1 , 1 2 ] , Eco P1 [13, 14] ) and S, nuclease: the enzymes introduce only one double strand cut per molecule of SV40 DNA and, cleavage occurs at random [ 1 5 , 1 6 ] . The difference being that S~ nuclease reacts in the absence of ATP and S-adenosylmethionine which is required b y the prokaryotic t y p e I restriction endonucleases [ 1 1 ] . It is not yet known whether S~ nuclease recognizes a specific binding site at the DNA from which it travels along the substrate to make only one double-strand cut like Eco B and Eco P1 restriction endonucleases. We are currently investigating the ability of various restricted SV40 DNA fragments to bind S~ nuclease. Also, we have attempted, as y e t unsuccessfully, to dissociate the singlestrand activity from the double-strand-cleaving activity b y chromatography of $1 nuclease both on sulfo-sephadex and on hydroxyapatite columns. We find, however, that the double-strand-cleaving activity requires the same buffer and pH as the single-strand activity in order to be active (unpublished data). Fur-

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ther experiments will be necessary to clarify the mechanism by which SV40 circular DNA is opened by $1 nuclease to linear FO III. It has been shown that the supercoiled replicative DNA of ~ X 1 7 4 is not susceptible to the double-strand cleaving activity of S~ nuclease. Upon treatment with the enzyme only relaxed circles were generated by introduction of singlestrand nicks, but no conversion to linear DNA could be noticed [17]. This observation, namely that not all circular DNA species can be cleaved open by S~ nuclease, lends support to the suggestion that $1 enzyme requires a specific base sequence for binding and, therefore, might represent a eukaryotic restriction endonuclease which bears close similarities to the prokaryotic restriction enzymes type I. Acknowledgements One of us (K. Chowdhury) is recipient of a fellowship o f the Deutsche Akademische Austauschdienst. COLE1 superhelical DNA was kindly provided by W. Goebel. Eco RI restriction endonuclease was a generous gift of P. Philippsen and H.G. Zachau. We thank A. Fried for critical reading of the manuscript. References 1 Beard, P., Morrow, J.F. and Berg, P. (1973) J. Virol. 12, 1303--1313 2 M~chali, M,, de Rocondo, A.M. and Girard, M. (1973) Biochem. Biophys. Res. Commun. 54, 1306 m 1320. 3 Germond, J.E., Vogt, V.M. and Hirt, B. (1974) Eur. J. Biochem. 43, 591--600 4 Chowdhury, K., Gruss, P., Waldeck, W. and Sauer, G. (1975) Biochem. Btophys. Res. Commun. 64, 709--716 5 Waldeck, W., Kammer, K. and Sauer, G. (1973) Virology 54,452--464 6 Vogt, V. (1973) Eur. J. Biochem. 33, 192--200 7 Tegtmeyer, P. and Macasaet, F. (1972) J. Virol. 10, 599--604 8 Smith, H.O. and Wilcox, J. (1970) J. Mol. Biol. 51,379--391 9 Darma, K.J., Sack, G.H. and Nathans, D. (1973) J. Mol. Biol. 78, 363--376 10 Sharp, P.A,, Sugden, B. and Sambrook, J. (1973) Biochemistry 12, 3055--3063 11 Linn, S. and Arber, W. (1968) Proc. Natl. Acad. Sci. U.S. 59, 1300--1306 12 Smith, J.D., Arber, W. and K~ihrdein, U. (1972) J. Mol. Biol. 63, 1--8 13 Haberman, A., Heywood, J. and Meselson, M. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3138--3141 14 Brokes, J . P , Brown, P,R. and Murray, K.C. (1972) J. Biochem. 127, 1--10 15 Adler, S.P. and Nathans, D. (1973) Biochim. Biophys. Acta 299,177--188 16 Morrow, J.F. and Berg, P. (1972) Proe. Natl. Acad. Sci. U.S. 69, 3365--3369 17 Godson, G.N. (1973) Biochim. Biophys. Acta 308, 59--67