Cleavage of Bacteriophage fl DNA by the Restriction Enzyme

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 69, No. 11, pp , November 1972 Cleavage of Bacteriophage fl DNA by the Restriction Enzyme of Escherichia coli B (DNA endonuclease/mutant DNA/sites of cleavage) KENSUKE HORUCH AND NORTON D. ZNDER The Rockefeller University, New York, N.Y Contributed by Norton D. Zinder, August 30, 1972 ABSTRACT We studied the cleavage of the replicativeform DNA (RF ) of bacteriophage fl and its SB mutants by purified restriction endonuclease of E. coli B. The results indicate that: (i) Circular replicative forms are broken once to yield full-length linear molecules (RF ). Such linear molecules are less susceptible than RF to endonuclease R-B. (ii) The genetic sites (SB sites) that confer on the DNA susceptibility to B-restriction are not the actual sites of cleavage. The number of possible cleavage sites is larger than the number of SB sites. We conclude this because an RF molecule produced by endonuclease R-B from RF of a mutant that has only one SB site can be circularized by denaturation and renaturation. (iii) The SB site is not modified when the DNA is cleaved, since an SB site can be used repeatedly by endonuclease R-B; the RF described in ii can be cleaved by the same enzyme after denaturation and renaturation. Bacteriophage fl (1), a small filamentous, single-stranded DNA phage (2), is susceptible to host-controlled restriction and modification by Escherichia coli B and Pi-phage lysogens (3, 4). On the fl genome there are two genetic sites that confer DNA susceptibility to restriction by the B-host (5). The two sites, called SB1 and SB2, have been mapped at two points that divide the circular fi genome into about a one-totwo ratio (6, 7). One-step mutants of f 1, which have lost either of the two SB sites (SB10 or SB20), are less restricted by the B-host than wild-type phage. The double mutant (SB10 SB20), which has lost both SB sites, is not restricted at all by the B-host. Restriction of DNA is caused by a host-specific endonuclease that can distinguish modified DNA from unmodified DNA: double-strand cleavages are produced only in unmodified DNA (8-10). Since mutations at the SB sites make the DNA resistant to B-restriction, it has generally been assumed that the B-restriction endonuclease (endonuclease R-B) cleaves the DNA specifically at the SB sites (11, 12). However, the fact that endonuclease R-B recognizes the SB site does not necessarily mean that the SB site is the actual site of cleavage. n this study, using denaturation and renaturation techniques, we examined DNA fragments produced by endonuclease R-B from replicative-form (RF ) DNA of bacteriophage fi or its SB mutants. Nathans (personal communication) used similar techniques to study the effects of endonuclease R-B on simian virus40 (SV40) DNA. Our results indicate that the SB sites are not the actual sites of cleavage. Abbreviations: endonuclease R-B and endonuclease R-P1, B- and Pl-restriction endonucleases, respectively MATERALS AND METHODS Bacteria. All the strains used are derivatives of E. coli K12. K38 is rk+mk+, and was used to prepare unmodified replicative-form DNAs. K140B and K336B are rb+mb+, and were used for the preparation of endonuclease R-B and of B-modified RF. These strains have been described (6, 7). K336P1 is rk+mk+ and Pi-lysogenic, and was used for the preparation of endonuclease R-P1 and of P1-modified RF. K336 BP1 is rb+mb+ and Pi-lysogenic, and was used to prepare endonucleases R-B and R-Pl. Bacteriophages. Wild-type fl and its derivatives, R14OR (SB20) and R132R (SB10 SB20), have been described (7). Endonuclease R-B. Cells were grown to 6 X 108 cells per ml in tryptone broth, collected, and stored at -20. The frozen cells were suspended in A-buffer (50 mm Tris HCl ph mm EDTA-5 mm 2-mercaptoethanol) and disrupted by agitation with glass beads in a Sorvall Omnimixer. The crude extract was subjected to streptomycin sulfate precipitation, ammonium sulfate precipitation, and dialysis by the procedure of Linn and Arber (9). The dialyzed protein extract thus obtained was purified first by a DEAE-cellulose column (Cellex-D from BioRad) with a M linear gradient of NaCl prepared with B-buffer (10 mm potassium phosphate ph mm EDTA-0.5 mm dithiothreitol). The active fractions were combined, concentrated by ammonium sulfate precipitation, and subjected to gel filtration through a column of Bio-Gel A (1.5 m, mesh, from Bio Rad). The enzyme was then layered on a hydroxyapatite column (Hypatite C, Clarkson Chemical Co.) and eluted with 0.2 M, after washing with 0.1 M, potassium phosphate (ph 6.5) in the presence of 0.5 mm dithiothreitol. The purified enzyme was dialyzed against 50% glycerol prepared with B-buffer, and stored at -20. The enzyme obtained was free of any detectable amount of nonspecific endonuclease activity and exonuclease activity on double-stranded DNA. However, it was still contaminated with an exonuclease that is active on single-stranded DNA. Endonuclease R-P1. The dialyzed protein extract prepared as described above was layered on a column of Cellex-D and eluted with 0.15 M, after washing with 0.1 M, NaCl dissolved in B-buffer. The enzyme was then applied to a phosphocellulose column (P-11, Whatman) and eluted with 0.3 M, after washing with 0.2 M, NaCl solution prepared in B-buffer. The enzyme obtained was dialyzed against 50% glycerol prepared with B-buffer and stored at -20. The preparation thus

2 Proc. Nat. Acad. Sci. USA 69 (1972) obtained did not contain any detectable amount of either nonspecific endonuclease activity or exonuclease activity on singlestranded DNA. t was very slightly contamined with an exonuclease that is active on double-stranded DNA. At the enzyme concentration used for the experiments described, however, the contaminating exonuclease activity was not detectable. Radioactive RF. Radioactive RF was purified by a procedure developed by Model (to be published) that includes precipitation of host-cell DNA with high salt and sedimentation in a sucrose gradient, followed by equilibrium banding in CsCl-ethidium bromide (13). Restriction of DNA. 3H-Labeled DNA was incubated with either endonuclease R-B or endonuclease R-P1 at 370 for 30 min. The reaction mixture contained 0.1 M Tris HCl (ph 7.4), 6 mm MgCl2, 3 mm ATP, and 0.1 mm S-adenosylmethionine. The reaction was stopped by chilling in ice and addition of EDTA to give 50 mm. Samples were layered on 5-20% sucrose gradients containing 1 M NaC 1 and 0.1 M potassium phosphate (ph 7.0) and centrifuged at 50,000 rpm for 4 hr in an SW56 Spinco rotor at 180. Fractions were collected from the bottom of the tubes; radioactivity was counted. Denaturation and Renaturation. To the DNA sample dialyzed against C-buffer (10 mm Tris- HCl ph mm NaCl-0.2 mm EDTA), Tris HCl (ph 7.4) and EDTA were added to give 0.1 M and 50 mm, respectively. To 100 Mul of this sample, 3 jil of 10 N NaOH was added, and after 2 min at room temperature 10,ul of 3.3 M NaH2PO4 was added for neutralization. To anneal the denatured sample, the salt concentration was raised to 0.3 M with KCl, and the sample was incubated at 650 for 2 hr. Then it was transferred to 37. The denatured-renatured DNA thus obtained was dialyzed against C-buffer before incubations with enzymes. RESULTS f the cleavage of replicative-form (RF ) DNA of bacteriophage fi by endonuclease R-B occurs at the SB sites and if the genetic map of bacteriophage fi corresponds to the physical structure of its DNA genome, then RF of wild-type fi should be cleaved by the enzyme at two sites to produce a one-third length and a two-third length linear DNA molecule. Similarly RF of the SB10 or SB20 mutant should be cleaved at a specific site to yield full-length linear molecules with identical ends. To test whether such fragments are obtained, we prepared 3H-labeled RF DNA from fi phage-infected cells, treated it with endonuclease R-B, and analyzed it by sucrose gradient centrifugation. The only discrete products obtained were RF (full-length linear molecules without any single-strand nicks) and RF (circular molecules with a nick on one of the strands). The higher the concentration of the enzyme, the more RF and the less RF were produced. Apparently, RF is an intermediate in RF production. At an enzyme concentration that converts 70% of the RF added into RF and leaves less than 10O of the RF intact, no fragment of DNA smaller than RF was produced. A similar result was reported by Roulland- Dussoix and Boyer (10) and was interpreted to mean that the two SB sites are located close to each other on the phage genome. However, since it was not clear whether the postulated second double-strand cleavage on the RF molecule :' f) CL E 01 Cleavage of fi DNA by Restriction Enzyme 3221 ]Jm ll um FG. 1. Effect of endonuclease R-B and endonuclease R-P1 on RF. 2.7 ug (98,000 ) of [3H]RF of R14OR (SB20) was incubated with enzymes in 100-,ul reaction mixture containing 0.1 M Tris-HCl (ph 7.4), 6 mm MgCl2, 3 mm ATP, and 0.1 mm S-adenosylmethionine, unless otherwise noted. 2,ug of endonuclease R-B or 15 pg of endonuclease R-P1, or both, were used per reaction. After incubation at 370 for 30 min, the reaction was stopped by chilling to 00 and addition of 50 mm EDTA. The entire reaction mixture was layered on 3.6 ml of 5-20% sucrose gradient prepared with 1 M NaCl-0.1 M potassium phosphate (ph 7.0), and centrifuged at 50,000 rpm for 4 hr in a SW56 Spinco rotor at 18. Fractions of 5 drops were collected, and radioactivity in 7.5-,ul aliquots of each fraction was measured. Arrows marked with,, and represent the positions of RF, RF, and RF, respectively, determined by 14C-labeled markers. (A) 0-., endonuclease R-B; O-- -O, endonuclease R-B without S-adenosylmethionine. (B) 0-0, endonuclease R-P1; -- -0, endonuclease R-B plus endonuclease R-P1. The control without any enzyme and the control with endonuclease R-P1 but without S-adenosylmethionine and ATP showed exactly the same pattern as the ----O curve in (A). occurs with the same efficiency as the first cleavage on the RF molecule, we studied the effect on RF molecules produced both by endonuclease R-B and the restriction enzyme specified by phage P1 (endonuclease R-P1). B-restriction and P1-restriction are independent phenomena. Thus, B-modified RF is susceptible to endonuclease R-P1, but not to endonuclease R-B, while P1-modified RF is susceptible to endonuclease R-B, but not to endoquclease R-P1. Fig. 1 shows a sucrose gradient profile of replicativeform DNA after treatment with endonuclease R-B or with endonuclease R-P1. Under the conditions used, endonuclease R-B converted 50% of the RF added into RF and 35% into RF, leaving 15% as intact RF ; while endonuclease R-P1 converted more than 70% of the substrate into RF leaving less than 10% as intact RF. n the absence of the required cofactors, neither enzyme caused any detectable change in the sedimentation profile of RF. Surprisingly, when a mixture of endonuclease R-B and endonuclease R-P1 was used to cleave RF, we could not detect production of any significant amount of DNA smaller than RF. For further analysis of this finding, RF molecules produced by endonuclease R-P1 were purified by sucrose gradient centrifugation and used as substrate for endonuclease R-B. t

3 3222 Biochemistry: Horiuchi and Zinder FG r m b Resistance of RF produced by endonuclease R-P1 to endonuclease R-B. [PH]RF was prepared by incubation of 13 ug (420,000 ) [PH]RF of R140R (SB20) with 75 ug of endonuclease R-P1 at 370 for 30 min in a 300-ul reaction mixture. The reaction was stopped, centrifuged in a neutral sucrose gradient. Fractions were collected, of which 7.5-jul aliquots were counted, as described in Fig. 1. Peak fractions of RF were pooled and dialyzed against C-buffer ug of the [2HJRF thus obtained was incubated with 5.6 pg of endonuclease R-B in a 100-l reaction mixture with or without S-adenosylmethionine. After incubation at 370 for 30 min, the reaction was stopped, and the whole content was layered on a sucrose gradient and centrifuged as described in Fig. 1. Fractions of 4 drops were collected and counted. * *, with 0.1 mm S-adenoyslmethionine; , without S-adenosylmethionine. When 0.17 pug of ['HJRF of R14OR was incubated with 5.6 pg of endonuclease R-B under the same conditions, about 50% of the RF was converted into RF. There was no detectable degradation of RF by the enzyme (Fig. 2). We also prepared RF produced by endonuclease R-B from RF of wild-type fi and from its SB20 mutant, R14OR, and used them as substrates for endonuclease R-B. n neither instance did we detect formation of any DNA that sediments slower than RF. These results strongly suggest that RF is less susceptible than RF to endonuclease R-B. Another possible explanation for the above observation is that both endonuclease R-P1 and endonuclease R-B cleave fi RF DNA at a very small number of specific sites that are so close to each other that the large product of the second cleavage cannot be distinguished from RF by sucrose gradient centrifugation. However, this explanation is unlikely because of the results shown in Fig. 3 that indicate that the RF molecules produced by endonuclease R-P1 had been cleaved at various sites. 1 r 000O Proc. Nat. Acad. Sci. USA 69 (1972) Um 1 A anm -v a ~ ~ ~ cpam m - %A- 0%O FG. 3 Sensitivity of RF produced by endonuclease R-P1 to endonuclease R-B after denaturation and renaturation. ['H]RF of R14OR (SB20) produced by endonuclease R-P1 was prepared by pooling RF peak fractions of sucrose gradient (solid line in Fig. 1B). After dialysis against C-buffer, Tris -HCl (ph 7.4) and EDTA was added to give 0.1 M and 50 mm, respectively. An aliquot (0.15 pg) of RF thus obtained was centrifuged in a sucrose gradient (A....A). The rest of the RF (1.5 pg/ml) was subjected to denaturation and renaturation. An aliquot (0.12 ug) of this renatured material was centrifuged in a sucrose gradient (O- -0). Aliquots (0.16 ug) of the renatured DNA were dialyzed against C-buffer and incubated with 4 pg of endonuclease R-B at 370 for 30 min in a 200-ul reaction mixture and centrifuged in a sucrose gradient (0-*) as described in Fig. 1. Fractions of 4 drops were collected and counted. When 0.16 pg of [2HJRF of R14OR was incubated with 4 ug of the endonuclease R-B under the same conditions, about 75% of the RF was converted into RF. 0 2C C Co A9 FG. 4. Denaturation and renaturation of RF produced by endonuclease R-B from RF of R14OR (SB20). [3H]RF was prepared by incubating 13 ug (420,000 ) of [3HJRF of R14OR with 21 ug of endonuclease R-B at 370 for 30 min in 600 pl of reaction mixture, and centrifuging in sucrose gradients as described in Fig. 1. The peak fractions of RF were combined and dialyzed against C-buffer. (A) An aliquot (0.33 ug) of RF thus obtained was centrifuged in a sucrose gradient ( ). The rest of the RF (2.5 pg/ml) was subjected to denaturation and renaturation ug of the renatured material was centrifuged ijn a sucrose gradient (0-0). (B) 0.16 ug of the renatured DNA was incubated with 4.5 pg of endonuclease R-B at 370 for 30 min in 150-pl reaction mixture in the presence (0-*) or absence ( ) of 0.1 mm S-adenosylmethionine. The contents of the reaction mixtures were layered on sucrose gradients and centrifuged as described in Fig. 1. Fractions of 4 drops were collected and counted.

4 Proc. Nat. Acad. Sci. USA 69 (1972) n this experiment, a preparation of RF, which was produced from RF by endonuclease R-P1 and purified by sucrose gradient centrifugation, was denatured with sodium hydroxide and annealed by heating at 650 for 2 hr. The sucrose gradient profile of this renatured sample shows that more than 60% of the DNA now sediments at the position of the circular form, RF (Fig. 3). Electron micrographs showed that these structures were actually circular. About 30% of the DNA sedimented faster than RF without forming any discrete peaks. This DNA represents polymers produced by annealing together of more than two molecules of single strands. Only 10% or less of the DNA sedimented at the original position of RF. Renaturation of the RF preparation, without previous alkaline denaturation, did not cause any change in the RF profile in sucrose gradient centrifugation. This excludes the possibility that the circularization and polymer formation were due to singular "sticky" ends that had somehow been separated during the cleavage reaction. Since it appears that a collection of circularly permuted molecules had been produced, these results indicate that the cleavage of RF does not occur at a fixed point. Note also (Fig. 3) that the structures obtained by denaturation and renaturation of RF (P1) are now susceptible to cleavage by endonuclease R-B and once more produce RF. Therefore, the inability of RF to serve as a substrate for endonuclease R-B must be due to its linearity and not to other irreversible changes caused by the restriction endonucleases. n order to test whether the cleavage of RF by B-restriction endonuclease specifically occurs at the SB sites, we performed the experiment shown in Fig. 4A. 3H-Labeled RF of R140R (SB20 mutant phage) was incubated with B- restriction endonuclease, and the resulting RF was purified by sucrose gradient centrifugation. n an alkaline sucrose gradient this RF preparation gave a single peak, the position of which corresponds to that of the full-length, linear single strands of fi DNA. Since R14OR genome has lost the SB2 site by mutation and retains only one SB site (SB1), the RF samples obtained should be a homogeneous population of linear molecules with identical ends if RF had been cleaved at the SB1 site. The result shown in Fig. 4A indicates that this is not the case. Upon alkaline denaturation followed by renaturation at 650, this RF from R140R became either circularized to give RF or polymerized to give larger molecules. Less than 5% of the total DNA was at the original RF region. Renaturation without previous denaturation did not cause any change in the sedimentation profile of the RF produced by endonuclease R-B. Therefore, although the SB site is indispensable for cleavage by endonuclease R-B, it cannot be the site of actual cleavage; again, we appear to have a circularly permuted set of molecules. The susceptibility of this denatured-renatured DNA of R14OR to the second attack by endonuclease R-B was also examined. The result shown in Fig. 4B indicates that when this material was incubated with the enzyme in the presence of required cofactors, RF and the polymers are cleaved once more to give RF molecules. Since there was only one SB site per RF molecule of R140R, that SB site must have survived the original treatment. Sedimentation profiles of alkaline sucrose gradients of this denatured-renatured DNA before and after the second incu- Cleavage of fi DNA by Restriction Enzyme 3223 il 1 Y2 1 FG. 5. Alkaline sucrose gradient centrifugation of replicative form DNA of R14OR (SB20) that was repeatedly treated with endonuclease R-B. The same samples as seen in Fig. 4 were layered on 3.6 ml 5-20% sucrose gradients prepared with 0.4 N NaOH-0.6 M NaCl and centrifuged at 50,000 rpm for 6 hr at 180 in SW56 rotor of Spinco. Fractions of 4 drops were collected and counted. The arrow marked 1 represents the position of a fulllength linear strand of fi phage DNA. The arrow marked 1/2 represents the calculated position of a half-length strand on the basis of Studier's equation (14). RF produced by endonuclease R-B from RF of R14OR was subjected to denaturation and renaturation, and an aliquot was layered (A- - -A). The renatured sample was incubated with endonuclease R-B in the presence (0-0) or absence ( ) of 0.1 mm S-adenosylmethionine. bation with endonuclease R-B, in the presence or absence of S-adenosylmethionine, are shown in Fig. 5. DNA subjected to the second cleavage did not show any discrete peaks, but gave an almost linear distribution ranging from the position of full-length linear strands toward the position of small fragments. The absence of S-adenosylmethionine in the second incubation with the enzyme did not change the sedimentation profile: no single-strand nicks had been introduced by the manipulations themselves. Therefore, we may conclude that the second treatment produced, in a specific manner, an RF that could be resolved upon denaturation to fragments of various sizes, about equal in number. Given an original set of circularly permuted double-stranded molecules, this is the expectation regardless of whether the second break was at a fixed or random point. DSCUSSON Among the known systems of DNA restriction and modification, the one manifested by E. coli B has been most extensively studied. n this system, modification represents methylation of bases caused by a specific enzyme, DNA methylase M-B (15). Measurement of the number of bases methylated in vivo (16) and in vitro (17) in fd phage DNA has indicated that DNA is modified by methylation of two bases per available SB site. This result strongly suggests that the SB sites represent the modification sites. On the other hand, the B-specific restriction enzyme, endonuclease R-B, does not

5 3224 Biochemistry: Horiuchi and Zinder cause any cleavage in either modified DNA or SB,' SB20 mutant DNA. Therefore, endonuclease R-B must recognize the same SB sites as DNA methylase M-B. t has been assumed that the SB sites also represent the specific sites of cleavage by the endonuclease (11, 18). The fact that X phage DNA, upon digestion with restriction endonuclease, yields only fairly large fragments (8) has been taken to support this notion. Moreover, the restriction endonuclease from Hemophilus influenzae (19, 20) cleaves DNA into specific fragments (21). However, there has been no clear evidence that the SB sites are the actual sites of cleavage by endonuclease R-B. The results we describe (Fig. 4) indicate that the full-length linear DNA molecules produced by endonuclease R-B from RF of the SB20 mutant form circular molecules or polymers upon denaturation and renaturation. Thus, the SB site is not the specific cleavage site and the number of potential sites of cleavage on fl replicative-form DNA is much larger than the number of SB sites. A control experiment without denaturation before reannealing excluded the possibility that the circularization or polymerization of the RF was due to the presence of sticky ends. Although the cleavage does not occur at the SB site, the presence of the SB site on the DNA molecule is an absolute requirement for cleavage by the enzyme, since RF of the SB, SB20 double mutant cannot be cleaved at all. We interpret this to mean that the SB sites are recognition sites for the enzyme and not cleavage sites. Possibly endonuclease R-B travels along the DNA molecule from the recognition site to the cleavage sites. A very large ATPase activity observed in this reaction (Horiuchi and Zinder, to be published) might be related to movement of the enzyme. The results in Fig. 4 also show that the RF produced from RF of the SB20 mutant by endonuclease R-B can be cleaved again by the same enzyme if the RF were subjected to denaturation and renaturation before the second incubation with the enzyme. The RF of this mutant contains only one available BB site (SB1), and this SB site therefore must have been used repeatedly by the enzyme. A possibility that the second cleavage is due to an artifact produced by the denaturation and renaturation procedures was excluded by a control experiment in which RF of the SB10 SB20 double mutant produced by endonuclease R-P1 was used for denaturation and renaturation and the subsequent incubation with endonuclease R-B. We observed that RF is a much poorer substrate for endonuclease R-B than is RF (Figs. 1 and 2). Since RF is as sensitive as RF, the resistance of RF to the enzyme cannot be due to the lack of superhelical structure. The possibility that the resistance is due to modification by contaminating DNA methylase M-B is excluded because RF produced by R-P1 also showed the same resistant properties. n addition, when those RF molecules were circularized by denaturation and renaturation, they could now be cleaved by endonuclease R-B. Thus, it would appear that it is the circularity of DNA that is required by this endonuclease. However, we know that linear DNA molecules of X phage (8) and T7 phage (Horiuchi and Vovis, unpublished) are ex- Proc. Nat. Acad. Sci. USA 69 (1972) cellent substrates for the enzyme, yielding fragments of average molecular weight of about 5 X 106. Since the molecular weight of replicative-form DNA of fi is 3.5 X 106, one possible explanation would be that linear DNA molecules are required to be larger than a certain size to be susceptible to endonuclease R-B, while for circular DNA the size limitation is not applicable, since long linear molecules share topological features with small circular molecules. Endonuclease R-P1 also produced only RF from RF (see Fig. 1), in spite of the fact that the RF molecule has more than one possible site of cleavage by this enzyme (Fig. 3). Preliminary results suggest that this phenomenon is at least in part due to specific modification of DNA, rather than to the linearity of the DNA. n the experiments presented in this report, endonuclease R-P1 was used in the presence of S-adenosylmethionine. t should be noted, however, that cleavage of DNA by endonuclease R-P1 does not require S-adenosylmethionine, although it is stimulated by its presence (Horiuchi and Zinder, unpublished). t seems clear, especially for endonuclease R-B, that the simple hypothesis relating the specificity of cleavage to sites of methylation and potential sites of mutation to resistance to cleavage must be reevaluated. Most likely these sites are recognition sites for the enzyme and not the sites of cleavage. Whether there is any specificity for the cleavage sites remains to be shown. We thank Drs. Gerald F. Vovis and Peter Model for helpful discussions and for aid with some of the experiments. 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