Site-Specific Cleavage of Single-Stranded DNA by A

Proc. Nat. Acad. Sci. USA VoL 72, No. 7, pp. 2555-2558, July 1975 Biochemistry Site-specific cleavage of single-stranded DNA by a Hemophilus' restriction endonuclease (fl phage DNA/endo R-HaeIII/fragments of single strands) KENSUKE HORIUCHI AND NORTON D. ZINDER The Rockefeller University, New York, N.Y. 10021 Contributed by Norton D. Zinder, April 15,1975

ABSTRACT Single-stranded viral DNA of bacteriophage E. coli strain used for the preparation of RFI, was very gen- fI is cleaved into specific fragments by endo R-HaeIII, a re- erously provided by Dr. S. Hattman (23). Preparation of striction endonuclease isolated from Hemophilus aegyptius. 32P-labeled DNA has been described (12). The sites of the single strand cleavage correspond to those of the double strand cleavage. A single-stranded DNA fragment Endonucleases. Endonucleases R.HaeIII (6, 12), R-HaeII containing only one HaeIII site is also cleaved by this en- (11, 12), and R-Hind (4) were isolated as described previous- zyme. This observation suggests that the reaction of single- ly (12). Endo R-EcoRII, isolated by the method described by stranded DNA cleavage does not require the formation of a Yoshimori (5), was supplied by Dr. G. F. Vovis. symmetrical double-stranded structure that would result Denaturation and Renaturation. Alkali denaturation and from the intramolecular base-pairing between two different subsequent renaturation of DNA were carried out as de- HaeIII sites. Other restriction endonucleases may also cleave scribed previously (24) except that acetic acid was used in- single-stranded DNA. stead of NaH2PO4 to neutralize the alkali. Various restriction endonucleases have been isolated which Gel Electrophoresis. Procedures for slab gel electropho- recognize specific base sequences in double-stranded DNA resis, radioautography, and extraction of DNA from the gel (1-3). Those that cleave DNA at specific sites (4-12) have have been described previously (12). A gel containing a been particularly useful for the physical dissection and sub- 2.5-7.5% gradient of polyacrylamide (25) was used for the sequent analyses of DNA genomes. The recognition sites for analysis of DNA fragments, while a 1.4% agarose gel was these enzymes have been shown to have a 2-fold rotational used for the preparation of DNA fragments. symmetry (13-19) and, hence, the enzymes. are believed to act only on double-stranded DNA. RESULTS AND DISCUSSION Bacteriophage- fi contains a covalently closed, circular Endo R.HaeIII, a restriction endonuclease isolated from H. single-stranded DNA of 1.8 X 106 daltons (20). Upon infec- aegyptius (6), cleaves the double-stranded replicative form tion of sensitive cells of Escherichia coli, replication of the DNA (RF) of bacteriophage fi at nine specific sites to yield phage DNA takes place through the formation of double- nine fragments, which have been named HaeIII-A through stranded, circular replicative form DNA (RF). The RF, con- I. These fragments have been ordered so as to construct a sisting of viral and complementary strands, can be isolated circular cleavage map of the fI genome (12) (See Fig. 1). from the fl-infected cells and used as a substrate for en- Upon polyacrylamide gel electrophoresis, the endo R*HaeIII donucleolytic reactions. digest of fI RFI gives the pattern shown in Fig. 2a. Endo R-HaeIII*, a restriction endonuclease isolated from Hemophilus aegyptius (6), has been shown to cleave the RF of bacteriophage fI to produce nine specific fragments which have been ordered to construct a circular map of the fi genome (12). The present paper reports that endo R- HaeIII cleaves single-stranded viral DNA of fi at specific sites and that the sites of the single strand cleavage apparently correspond to those of the double strand cleavage. MATERIALS AND METHODS DNA. Single-stranded viral DNA of phage fI was prepared as described previously (21). Covalently closed, circular replicative form- DNA (RFI) of fi was prepared by the modified procedure (12) of the method described by Model and Zinder (22). K415 (F+ mec- endo I- thi- rK-mK+), the

FIG. 1. Cleavage map of bacteriophage fl, which shows the Abbreviations: RF, replicative form DNA; RFI, a covalently closed, location of the endo R-HaeIII fragments (A,B,...... 1), the cleavage circular double-stranded DNA molecule. sites of endo R-HindII (the solid arrow labeled HindI on the out- *The nomenclature proposed by Smith and Nathans (32) for re- side of the circle), endo R-HaeII (the three solid arrows on the in- striction-modification enzymes has been used, i.e., endo refers to side of the circle), and endo R-EcoRII (the two dashed arrows on endonuclease, R to restriction, Eco to E. coli, Hin to H. influen- the outside of the circle) (ref. 12; Vovis, Horiuchi, and Zinder, sub- zae, Hae to H. aegyptius, Hpa to H. parainfluenzae, etc. mitted for publication). 2555 Downloaded by guest on September 28, 2021 2556 Biochemistry: Horiuchi and Zinder Proc. Nat. Acad. Sci. USA 72 (1975)

a b c HaeIII fragments. The result shown in Fig. 2b reveals nine single-stranded fragments, the positions of which are 0 -- 0 marked on the figure by Arabic numerals 1-9. An additional A - _ 2 top band is due to the radioactivity of a portion of the complementary strand hybridized to the full-length viral strand. B Additional faint bands due to the self-annealing of a small -3.. portion of the HaeIII fragments are also seen in the figure. c---- _ The striking resemblance between the pattern of the viral strand from the double strand digest (Fig. 2b) and the pattern of the viral strand digest (Fig. 2c) is obvious: among the q'sppw x Iftwoft vel..*, 4 eight bands shown in Fig. 2c seven had a mobility identical 5 to that of the bands shown in Fig. 2b. This strongly suggests ,4 that the sites of the single strand cleavage correspond to EF --~ those of the double strand cleavage. Apparently one of the nine cleavage sites was not cleaved when single-stranded DNA was used as the substrate, yielding a fragment that is 7 labeled X in Fig. 2. By use of a higher concentration of endo G _- R-HaeIII and a longer incubation time, we have observed H conversion of a portion of fragment X into fragments 5 and 9 7 (data not shown). Therefore, the resistance of this site in the single-stranded form to the enzyme is quantitative rather than qualitative. Quantitative differences in the sensitivity among other HaeIII sites to the single strand cleavage FIG. 2. Gel electrophoresis of phage fl RFI and single-strand- have also been observed by using lower enzyme concentra- ed DNA digested with endo R-HaeIII. To prepare the samples (a) tions (data not shown). The results described above strongly and (b), we incubated 1.4 gg of 32P-labeled fl RFI (34,000 cpm) with 4 Ml of an endo R-HaeIII preparation at 370 for 2 hr in an 80 suggest that the cleavage of the single-stranded DNA is cata- Ml reaction mixture containing 7 mM Tris-HCl, pH 7.4, and 7 mM lyzed by endo R.HaeIII, not by a contaminating enzyme in MgCl2. (a) A 20 Ml aliquot of this reaction mixture was analyzed by the preparation. gel electrophoresis as described previously (12) on a slab contain- In order to correlate the viral strand fragments (Fig. 2c) to ing a 2.5-7.5% linear gradient of polyacrylamide. (b) The other ali- the double-stranded fragments (Fig. 2a), we cut out portions quot (60 gl) was mixed with 19 Mg of nonradioactive, intact, single- of the dried gel that correspond to each radioautographic stranded viral DNA of fi, and was subjected to alkali denaturation, subsequent renaturation, and ethanol precipitation. The pre- band and measured the radioactivity. The result shown in cipitate was dissolved in 20 ,ul of the reaction buffer described Table 1 indicates that the viral strand fragments 1, 2, 3, 7, 8, above and analyzed by gel electrophoresis. (c) Two and two-tenths and 9 (see Fig. 2c) correspond to the double-stranded frag- micrograms of 32P-labeled single-stranded fl DNA (8400 cpm) was ments A, B, C, G, H, and I (see Fig. 2a), respectively. Pre- incubated with 10 gl of the endo R-HaeIII preparation at 370 for 2 liminary results of experiments in which RF fragments pro- hr in the reaction buffer described above, and analyzed by gel electrophoresis. The figure is a radioautograph of a single slab gel. Double-stranded HaeIII fragments (a) are indicated by capital let- Table 1. Radioactivity found in the area of a slab gel ters on the left side of the figure. HaeIII fragments of the viral corresponding to the radioautographic bands from the endo strand (b) are indicated by Arabic numerals on the right side of R-HaeIII digests of phage fl RFI and single-stranded DNAa the figure. For a description of fragment X, see text. 0 represents the origin. The intact molecules of both fl RFI and single-stranded (a) (b) (c) fl DNA remain at the origin of the gel (data not shown). Double-stranded Viral strand Viral strand fragments of RFI fragments of fragments of viral digest RFI digest DNA digest In order to test whether this endonuclease can cleave fl single-stranded DNA, we incubated the viral DNA extracted Radio- Radio- Radio- from purified 32P-labeled fl phage with the enzyme and activity activity activity then analyzed it by gel electrophoresis. The result shown in Bandb (%) Bandb (%) Bandb ( Fig. 2c reveals the production of eight discrete fragments. This in itself indicates that the endo R.HaeIII preparation A 42 1 37 1 40 cleaved the single-stranded fl DNA at specific sites. B 26 2 27 2 26 To examine whether the sites of the single-stranded DNA C 13 3 14 3 14 cleavage correspond to the sites of the RF cleavage, we car- DEF 14 4 6 4 5 ried out the following experiment. 32P-Labeled ft RFI was 5 6 incubated in the presence of endo R.HaeIII. After the reac- 6 4 6 5 G 2.6 7 tion was stopped, a 40-fold molar excess of nonradioactive, 2.5 H 1.7 8 (2.5)c 8 1.9 viral DNA was and the mixture single-stranded fl added, I 1.3 9 1.0 9 1.2 was to alkali denaturation and renatu- subjected subsequent X 6.5 ration. By this procedure most of the complementary strand component of the HaeIII fragments was annealed to the a A slab gel was prepared in the same way as that shown in Fig. 2. full-length viral strand. The hybrid molecules thus formed The area of the gel corresponding to each radioautographic band at the upon the was cut out and the radioactivity was measured in a liquid scin- remain the top of gel electrophoresis, leaving tillation counter. viral strand component of the fragments free to migrate into b For the nomenclature of the bands, see Fig. 2. the gel. Gel electrophoretic analysis of this sample thus al- Includes the radioactivity due to self-annealing of the fragment lows one to observe only the viral strand component of the HaeHI-G. Downloaded by guest on September 28, 2021 Biochemistry: Horiuchi and Zinder Proc. Nat. Acad. Sci. USA 72 (1975) 2557

a b c d e f (K. Murray, cited in ref. 26). Since there are nine HaeIII- sensitive sites on the fI RF (12), it might be that the enzyme cleaves single-stranded fl DNA only when two different HaeIII sites on the molecule pair, even transiently, to form a double-stranded symmetrical structure. Alternatively, the enzyme may act directly on the single-stranded structure as a base-sequence-specific endonuclease like T4 epdonuclease IV (27-S0). To distinguish between these possibilities, we tested a single-stranded DNA fragment that contains only one HaeIII site for its sensitivity to endo R.HaeIII. Endo R. FIG. 3. Cleavage of the viral strand of fragment EcoRII-B by EcoRIT cleaves fl and fd RFI at two sites, producing two -endo R-HaellI. 32p-Labeled and 3H-labeled preparations of Eco- fragments, EcoRII-A and EcoRII-B (Vovis, Horiuchi, and Rh1-B were prepared by incubation of radioactive ft RFI preparations with endo R-EcoRII and subsequent agarose gel electropho- Zinder, submitted for publication; S. Schlagman, S. Hatt- resis as described elsewhere (Vovis, Horiuchi, and Zinder, submit- man, M. S. May, and L. Berger, submitted for publication). ted for publication). The viral strand of EcoRI-B was isolated as The fragment EcoRII-B contains only one endo R-HaeIII follows. Seventeen-hundredth pmol of 32P-labeled EcoRai-B (6000 site, the site between HaeIII-B and HaeIII-C (see Fig. 1). cpm) was mixed with 17 pmol of nonradioactive, intact single- We, therefore, cleaved 32P-labeled fi RFI with endo REco- stranded viral DNA of fi, subjected to denaturation and renatura- RII, and isolated fragment EcoRII-B by gel electrophoresis tion, concentrated by ethanol precipitation, and centrifuged in a 5-20% neutral sucrosegradient (24) (45,000 rpm for 3 hr at 180 in a in a 1.4% agarose slab. The viral strand of EcoRII-B was iso- Spinco SWpblc rotor). Cerenkov counting of the collected fractions lated by denaturation and renaturation in the presence of an showed that approximately half the radioactivity layered had excess of full-length viral DNA followed by subsequent neu- moved about three-tenths the length of the gradient, while the' tral sucrose gradient centrifugation. When the viral strand other half moved about three-quarters the length of the gradient. of EcoRII-B thus obtained was incubated in the presence of The slower sedimenting radioactive fractions were combined, di- endo R-HaeIII, it was cleaved into two fragments as shown alyzed, and precipitated in ethanol in the presence of 1 1Ag of lambda phage DNA as carrier. The precipitate of the viral strand in Fig. Sa versus b. In addition, if the isolated viral strand of of EcoRII-B thus obtained (0.15 pmol) was dissolved in 40 ,lA of EcoRII-B is hybridized to an excess of alkali-denatured, reaction buffer (7 mM Tris-HCl, pH 7.4, 7 mM MgCl2). (a) A 10 M1 nonradioactive EcoRII-B, it shows on gel electrophoresis the aliquot of the viral strand of EcoRII-B was electrophoresed on the mobility of authentic EcoRII-B, and gives, upon cleavage gel. (b) Another 10 Ml aliquot was run after treatment with 7.5 Ml of with endo R.HaeIII, two fragments which co-migrate with endo R-HaeIII at 370 for 2 hr. The remaining 20 1A of 32P-labeled the double-stranded fragments obtained by the cleavage of viral strand of EcoRII-B was mixed with 3.0 pmol of 3H-labeled double-stranded EcoRII-B, subjected to denaturation and renatu- RFI with a mixture of endo R.HaeIII and endo R.EcoRII ration, and precipitated in ethanol. The precipitate was dissolved (Fig. Sc-f). These results indicate that endo R.HaeIII can in 20 ,ul of the reaction buffer. (c) A 10 1M aliquot of the renatured cleave a single-stranded DNA molecule that contains only EcoRII-B thus obtained was treated with 7.5 M1 of endo R-HaeIII, one HaeIII site. We cannot completely exclude the possibili- and then run on the gel. (d) The other 10 Md of the renatured Eco- ty that fragment EcoRII-B contains a second HaeIII site that RI-B was run directly. (e) 32P-labeled RFI treated with a mixture is located close to the first one, although such a of endo R-HaeIII and endo R-EcoRII. (f) 32P-labeled RFI treated hypothetical with endo R-EcoRII. The positions of the origin (0) and double- short HaeIII fragment has not been detected. The simplest stranded EcoRII-B are indicated by arrows. Conditions for the gel interpretation of our results is that intramolecular pairing electrophoresis were identical to those in Fig. 2. The figure is a ra- between two HaeIII sites is not required for the cleavage of dioautograph of a single slab gel. single-stranded DNA. The relationship betwen recognition mechanisms of double-stranded and single-stranded DNAs duced by endo R-HaeII plus endo R-Hind (see Fig. 1) were by this enzyme remains to be further elucidated. digested with endo R-HaeIII and subsequently subjected to One of the obvious questions of interest is whether the denaturation and renaturation in the presence of an excess cleavage of single-stranded DNA is a general characteristic amount of intact viral strand have indicated that the single- of restriction endonucleases. Endo R-EcoB, which cleaves stranded fragments 1, 2, 3, 4, 5, 6, 7, 8, and 9 are viral DNA at sites different from the DNA recognition sites (24), strands of HaeIII fragments A, B, C, D, F, E, G, H, and I, is inactive on single-stranded DNA (21, 31). Among the re- respectively (data not shown). striction endonucleases that produce site-specific cleavages, Parenthetically it should be noted that in most gel systems endo R-Hind appears not to cleave alkali-denatured T7 double-stranded fragments of similar size are difficult to re- DNA (4). However, our preparation of endo R-HpaII isolat- solve (see fragments D, E, and F in Fig. 2a). However, the ed from H. parainfluenzae (7, 8) produces discrete frag- gel electrophoretic separation of their single-stranded coun- ments with fl viral DNA as the substrate (data not shown), terparts can be much better (see fragments 4, 5, and 6 in although, in this instance, we have not yet tested whether Fig. 2b), probably because the mobility of single-stranded the cleavage is due to endo R-HpaII itself or another con- molecules reflects structure and base composition in addi- taminating enzyme. It, therefore, appears rather likely that tion to size. the cleavage of single-stranded DNA is not peculiar to endo Many restriction endonucleases have been shown to cleave R.HaeIII. double-stranded DNA within specific base sequences that have a 2-fold rotational symmetry (14-19). For instance, endo R.HaeIII, the enzyme used in the present study, cleaves double-stranded DNA within the following base sequence We thank Drs. P. Model and G. F. Vovis for helpful discussions and critical reading of the manuscript. Superb technical assistance (5') GC-G-C-C (3') of Mrs. Gillian Turgeon is gratefully acknowledged. This work was supported in part by grants from the National Science Foundation (3') C-C-G--G (5') to K.H. and N.D.Z. Downloaded by guest on September 28, 2021 2558 Biochemistry: Horiuchi and Zinder Proc. Nat. Acad. Sci. USA 72 (1975) 1. Arber, W. (1974) "DNA modification and restriction," Progr. restriction endonuclease and modification methylase," Na- Nucl. Acid Res. Mol. Blol. 14,1-37. ture New Biol. 244,40-43. 2. Meselson, M., Yuan, R. & Heywood, J. (1972) "Restriction and 18. Sugisaki, H. & Takanami, M. (1973) "DNA sequence restrict- modification of DNA," Annu. Rev. Biochem. 41, 447-466. ed by restriction endonuclease AP from Haemnophlus aphiro- 3. Boyer, H. W. (1971) "DNA restriction and modification philus," Nature New Blol. 248,138-140. mechanisms in bacteria," Annu. Rev. Microbiol. 25, 153-176. 19. 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