endonucleases K or P and S-adenosylmethionine renders the DNA ... enable bacteria to recognize and rapidly destroy DNA in- ... tion checkedby isotope dilution). ... X [32P]DNA (400 cpm) was added to each tube as ... Samples were extensively dialyzed .... of 0.1%, and each sample was extracted with water-saturated.
Proc. Nat. Acad. Sci. USA Vol. 69, No. 11, pp. 3138-3141, November 1972
DNA Modification Methylase Activity of Escherichia coli Restriction Endonucleases K and P (S-adenosylmethionine/6-methylaminopurine/multifunctional enzymes) ALLAN HABERMAN, JANET HEYWOOD, AND MATTHEW MESELSON The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138
Contributed by Matthew Meselson, August 21, 1972 The highly purified restriction endoABSTRACT nucleases of E. coli K and coliphage P1 transfer methyl groups from S-adenosylmethionine to adenine residues of unmodified DNA. Incubation of unmodified DNA with endonucleases K or P and S-adenosylmethionine renders the DNA resistant to restriction. The enzymes, therefore, have both restriction endonuclease and modification methylase activities.
ever, the methyl transferase reactions require only SAM. This makes it possible to study methylation and modification without the occurrence of the nucleolytic reaction. Specific methylation
Fig. 1 shows a time course of methylation of bacteriophage X DNA by restriction endonuclease P. The maximum number of methyl groups transferred per X DNA molecule after prolonged incubation is about 30. Further incubation with fresh enzyme gives no further increase. The high specificity of the
Restriction endonucleases are strain-specific enzymes that enable bacteria to recognize and rapidly destroy DNA introduced from foreign strains (1). They make double-chain scissions at a limited number of specific sites on the DNA molecule, rendering it susceptible to further degradation by nonspecific nucleases. Cells possessing a restriction enzyme of given specificity are also furnished with a specific methylase that transfers methyl groups from S-adenosylmethionine (SAM) to specific adenine residues located at or near the sites where the restriction enzyme acts. By this means, cells are protected against their own restriction enzyme, for such methylation protects a site against cleavage. DNA introduced from any strain lacking the specific methylase of the host will be degraded. DNA made insensitive to restriction is said to have undergone modification. Methylases that perform this reaction are called modification methylases. Modification and restriction are highly specific, in the sense that the modification imparted to DNA by a given bacterial strain protects it only against the restriction endonuclease of the same strain. Several different restriction and modification specificities are known, and several of the enzymes involved have been isolated. The experiments reported here deal with the specificities designated K and P (2-8). These are determined by genes of Escherichia coli strain K and coliphage P1, respectively. We wish to present evidence that DNA restriction endonucleases K afid P are also modification methylases. This is shown by the ability of the purified enzymes selectively to transfer the methyl group from SAM to unmodified DNA and by the specific resistance the DNA thereby acquires to endonucleolytic attack by the same enzyme. DNA cleavage by restriction endonuclease K requires the presence of Mg++, ATP, and SAM. Cleavage by endonuclease P requires Mg++ and ATP and is stimulated by SAM. How-
Hours of incubation FIG. 1. Time course of methylation by restriction endonuclease P. The reaction mixtures contained 0.1 M PIPES (pH 6.8) (30°), 0.2 mM EDTA, 10 mM 2-mercaptoethanol, 1.3 jM [3H]methyl-SAM (Amersham/Searle, 8.5 Ci/mmol, concentration checked by isotope dilution). To each 0.1 ml of reaction mixture were added 1010 phage units of unmodified or modified X DNA, and about 0.3 j.g of restriction endonuclease P from the glycerol gradient fraction (2). The tubes were incubated at 300 for the times indicated. X [32P]DNA (400 cpm) was added to each tube as a recovery marker, and 250 lig of calf-thymus DNA per reaction mixture was added as a carrier. The DNA was then precipitated in 5% trichloroacetic acid-10 mM sodium pyrophosphate-1 M NaCl at 00. After centrifugation at 6000 X g for 15 min, the supernatants were discarded, and the pellets were dissolved in 0.2 ml of 0.5 M NH40H. Samples were extensively dialyzed against 10 mM Tris (pH 8.0)-10 mM NaCl-0.2 mM EDTA and reprecipitated as described above. Each precipitate was collected on a glass-fiber filter, and washed with 10 ml trichloroacetic acid-sodium pyrophosphate-NaCl and 10 ml methanol, both at 00. The filters were then counted in Aquasol (New England 0, unmodified X DNA; Nuclear) by liquid scintillation. 0 A* P modified X DNA.
Abbreviations: SAM, S-adenosylmethionine; PIPES, piperazineN,N'-bis(2-ethanesulfonic acid); TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.
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Proc. Nat. Acad. Sci. USA 69
(1972)
DNA Methylation by Restriction Endonucleases
reaction is shown by the smallness of the amount of methyl transfer to DNA already bearing the modification imparted by passage through E. coli (P1). Methylation by restriction endonuclease K is depicted in Fig. 2. The maximum degree of specific methylation seen in experiments with increasing amounts of enzyme is 10-15 methyl groups per X DNA molecule. Upon hydrolysis of DNA methylated by restriction endonuclease K or P the radioactivity transferred from [3H]methyl-SAM is found in 6-methylaminopurine. This is in agreement with the formation of 6-methylaminopurine in other studies of DNA modification (8, 9). The DNA was hydrolyzed in trifluoroacetic acid (10) and analyzed by twodimensional thin-layer chromatography on cellulose plates in n-butanol-water-33% aqueous ammonia 86:13:1 and then in isopropanol-12 N HCl-water 170:41:39. The radioactivity migrated with added 6-methylaminopurine and not with 5-methylcytosine, 7-methylguanine, 6-dimethylaminopurine, or 1-methyladenine. The pH optimum for methylation by endonvclease K in Tris buffer at 370 is 8.0 and that for endonuclease P in PIPES buffer at 30° is 6.8. These values are close to the respective pH optima for restriction under the same conditions.
3139
fully susceptible to cleavage. In the reciprocal experiment, DNA methylated by endonuclease K was readily broken down upon incubation under restriction conditions with endonuclease P. Identity of the enzymes responsible for modification and restriction It is unlikely that the modification methylase activity associated with restriction endonucleases P and K is due to con-
N'~
Specific modification
As may be seen in Figs. 3 and 4, unmodified DNA incubated with endonuclease K or P and MAMX becomes largely resistant to attack by the same enzyme in the full restriction system containing Mg++, ATP, and SAMI. As suggested earlier (2), the modification activity of endonuclease K probably accounts for its inability to cleave unmiodified X DNA uniformly, since SAM is present in the restriction reaction mix-
(.0
)tK
ture.
In order to test the specificity of the modification reaction, DNA methylated by endonuclease P was challenged with endonuclease K under restriction conditions and found to be -
8
Distance from meniscus 4
l
10
20
Hours of incubation FIG. 2. Time course of methylation by restriction endontuclease K. The reaction mixtures contained 0.1 M TES (pH 7.8) (370), 0.2 m'M EDTA, 10 mMl 2-mercaptoethanol, and 6 MAI [3H]methyl-SA.M (Amersham/Searle, 7.9 Ci/mmol, concentration checked by isotope dilution). To each 0.1 ml of reaction mixture were added either 3.0 X 1010 phage units of unmodified X l)NA or
3.8 X 1010 phage units of modified DNA and 0.3 yg of restric-
tion endonuclease K purified by polyacrylamide gel electrophoresis. Reaction mixtures were incubated at 37° for the times indicated. X [32P] DNA (2500 cpm) and 250 pig of calf-thyrhus l)NA were then added to each tube, and the DNA was precipitated,
centrifuged, dissolved and dialyzed as described in the legend were then counted in Aquasol by liquid A, K modified scintillation. 0 unmodified X DNA; * to Fig. 1. The samples 0,
XDNA.
FIG. 3. Protection of unmodified X DNA by restriction endonuclease K. Modification reaction. Each reaction mixture contained 0.1 M TES (pH 8.0), 5 mM MgC12, 0.2 mM EDTA, 5 mM 2mercaptoethanol, 30 iMI SAM, and 5 X 1010 phage units of unmodified X [32P] DNA per 0.1 ml of reaction mixture. In the complete system, 3 ,ug of restriction endonuclease K from the glycerol gradient fraction were added. After 25 hr of incubation at 370, sodium dodecyl sulfate was added to a concentration of 0. 1%, and each sample was extracted with water-saturated phenol. The aqueous layer containing the DNA was dialyzed extensively against 10 mM Tris-HCl (pH 8.0)-10 mM NaCl-0.2 mMA EDTA. Restriction challenge. The treated [32P] DNAs were incubated at 3 X 109 phage units of X DNA with .5 X 109 phage units of [3Hlthymidine K modified X DNA and 0.4 ,g of endonuclease K per 0.1 ml of restriction reaction mixture [0.1 M TES (pH 8.0), 5 MM MgCl2, 5 mM 2-mercaptoethanol, 0.2 mMI EDTA, 30 MM\I SAM, and 0.5 mMI ATP] for 1 hr at 37°. After incubation, the samples were analyzed by neutral sucrose gradient sedimentation (2). Top panel: complete modification reaction system. Bottom 0, 3H panel: modification reaction system minus enzyme. 0O, 32p radioactivity. radioactivity; 0
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Proc. Nat. Acad. Sci. USA 69
Biochemistry: Haberman et al.
-10
~~~~0
0-
i~20-
20
10
Z
10
0
2-0
0o
Distance from meniscus FIG. 4. Protection of unmodified X DNA by restriction endonuclease P. Modification reaction. Each reaction mixture contained 0.1 M TES (pH 8.0), 0.2 mM EDTA, 10 mM 2-mercaptoethanol, 4.6 X 1010 phage units of [3H]thymidine unmodified X DNA, 3.1 MM SAM per 0.1 ml of reaction mixture. The complete system also contained about 1 ,ug of restriction endonuclease P purified
by polyacrylamide gel electrophoresis. The tubes were incubated for 24 hr at 300. The DNA was then extracted by phenol and dialyzed against 0.1 M TrisHCl (pH 8.0)-0.2 mM EDTA-10 mM NaCl. The DNA from each reaction sedimented as intact X DNA moleculesbn neutral sucrose gradients. Restriction challenge. The [3H]DNA from each reaction was incubated at 3.8 X 109 phage units per 0.1 ml of 0.1 M TES (pH 8.0), 0.2 mM EDTA, 10 mM 2-mercaptoethanol, 6 mM MgCl2, 30 MAM SAM, 0.5 mM ATP with 1.8 X 109 phage units of P modified [32P] X DNA and about 0.3 lg of restriction endonuclease P from the glycerol gradient fraction. The tubes were incubated for 1 hr at 300. The samples were then analyzed by sedimentation on neutral sucrose gradients. Top panel: complete modification reaction system. Bottom 3H panel: modification reaction system minus enzyme. radioactivity; 0-O, 32P radioactivity.
taminating enzymes. The restriction endonucleases have purified several thousand-fold by a series of fractionasteps involving DEAE and phosphocellulose column chromatography and preparative glycerol gradient zone sedimentation (2). The endonuclease and methylase activities
been tion
of restriction endonuclease K sediment at the same rate. Upon acrylamide gel electrophoresis in Tris-barbital buffer at pH 7.8, about three-quarters of the protein in the glycerol gradient fraction of endonuclease K was found in a single narrow band. After a parallel gel was sectioned and the individual slices were eluted, both activities were found to be confined to this protein band, and their ratio was the same as in the
input material. The endonuclease and methylase activities of the P enzyme also migrated in acrylamide gels as a single band
containing about one-third of the protein present in the glycerol gradient fraction. Again, the ratio of the two activities eluted from the gel was no different from that in the input material. Comparison
with other
modification methylases
A modification methylase has been isolated from E. coli (P1) (8). It specifically transfers methyl groups from SAM to un-
(1972)
modified DNA, giving rise to 6-methylaminopurine. In doing so, it confers specific resistance against attack by restriction endonuclease P but not against restriction endonuclease K. However, this enzyme is not the same as restriction endonuclease P. It possesses no restriction endonuclease activity. The sedimentation coefficient of this unifunctional enzyme is 6 S (J. P. Brockes, personal communication), while that for endonuclease P and its associated methylase activity is 9.2 S. These results suggest the possibility that the unifunctional enzyme is a component of the restriction endonuclease, capable of methylation activity whether free or combined. A similar situation appears likely for restriction endonuclease K. Although no unifunctional K modification methylase has yet been isolated, the restriction endonuclease is a large multimer, composed of polypeptide subunits of three different sizes (1). The implication that the modification methylase activity of restriction endonuclease K may be separable as an independent unifunctional enzyme is strengthened by comparison of the properties of endonuclease K with those of restriction and modification enzymes of E. coli strain B. The K and B systems are allelic, showing complementation in diploids (11-13). The proteins involved in the two systems are therefore likely to be closely homologous. In line with this, restriction endonuclease B is reported to have a sedimentation coefficient close to that of endonuclease K and to contain polypeptide subunits of about the same three sizes (14). Although no modification methylase activity has yet been reported for restriction endonuclease B, there is a B modification methylase without restriction activity (5). This unifunctional enzyme is reported to contain polypeptide subunits of only two of the three sizes found in restriction endonuclease B and to have a considerably lower sedimentation coefficient (14). Thus, the picture emerges that endonuclease P and probably endonucleases K and B contain a modification methylase as a separable component. In this regard it is of interest that the maximum observed specific methylation activity of the unifunctional modification methylases B and P and of restriction endonucleases P and K are all about the same. As noted, these reaction rates are remarkably low (1). To complete this general picture we would expect a number of predictions to be borne out. The polypeptide(s) of the unifunctional methylase P ought to be present in restriction endonuclease P. There may exist a K modification methylase containing only two of the three types of subunit found in restriction endonuclease K. Finally, the B restriction endonuclease may well possess modification methylase activity. This work was supported by a grant from the National Science Foundation and by an NSF Predoctoral Fellowship held by J. H. 1. Meselson, M., Yuan, R. & Heywood, J. (1972) "Restriction and modification of DNA," Annu. Rev. Biochem. 41, 447-466. 2. Meselson, M. & Yuan, R. (1968) "DNA restriction enzyme from E. coli," Nature 217, 1110-1114. 3. Linn, S. & Arber, W. (1968) "Host specificity of DNA produced by Escherichia coli. X. In vitro restriction of phage fd replicative form," Proc. Nat. Acad. Sci. USA 59, 1300-1306. 4. Roulland-Dussoix, D. & Boyer, H. (1969) "The Escherichia coli B restriction endonuclease," Biochim. Biophys. Acta 195, 219-229. 5. Kuehnlein, U., Linn, S. & Arber, W. (1969) "Host specificity of DNA produced by Escherichia coli. XI. In vitro modification of phage fd replicative form," Proc. Nat. Acad. Sci. USA 63, 556-562. 6. Smith, H. 0. & Wilcox, K. W. (1970) "A restriction enzyme from Hemophilus influenzae, I. Purification and properties," J. Mol. Biol. 51, 379-391.
DNA Methylation by Restriction Endonucleases
Proc. Nat. Acad. Sci. USA 69 (1972) 7. Yoshimori, R. (1971) A Genetic and Biochemical Analysis of the Restriction and Modification of DNA by Resistance Transfer Factors. Ph.D. thesis, Univ. California, San Francisco. 73pp. 8. Brockes, J. P., Brown, P. R. & Murray, K. (1972) "The deoxyribonucleic acid modification enzyme of bacteriophage P1," Biochem. J. 127, 1-10. 9. Kuehnlein, U. & Arber, W. (1972) "Host specificity of DNA produced by Escherichia coli. The role of nucleotide methylation in in vitro B-specific modification," J. Mol. Biol. 63, 9-19. 10. Razin, A., Sedat, J. W. & Sinsheimer, R. L. (1970) "Struc-
11. 12.
13. 14.
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ture of the DNA of bacteriophage OX174, VII. Methylation," J. Mol. Biol. 53, 251-259. Boyer, H. & Roulland-Dussoix, D. (1969) "A complementation analysis of the restriction and modification of DNA in Escherichia coli," J. Mol. Biol. 41, 459-472. Arber, W. & Linn, S. (1969) "DNA modification and restriction," Annu. Rev. Biochem. 38, 467-500. Glover, S. W. (1970) "Functional analysis of host-specificity mutants in Escherichia coli" Genet. Res. 15, 237-250. Lautenberger, J., Eskin, D. & Linn, S. (1972) "The DNA modification methylase and restriction endonuclease of Escherichia coli B," Fed. Proc. 31, 474Abstr.
