Vol. 129, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Mar. 1977, p. 1330-1334 Copyright ©) 1977 American Society for Microbiology

Partial Purification of the Escherichia coli K-12 mec+ Deoxyribonucleic Acid-Cytosine Methylase: In Vitro Methylation Completely Protects Bacteriophage Lambda Deoxyribonucleic Acid Against Cleavage by REcoRII STANLEY HATTMAN Department of Biology, University of Rochester, Rochester, New York 14627

Received for publication 27 September 1976

A procedure is described for the partial purification of the deoxyribonucleic acid (DNA)-cytosine methylases controlled by the RII plasmid and by the Escherichia coli mec+ gene. The two enzymes exhibit similar but distinct chromatographic behavior on diethylaminoethyl-cellulose and phosphocellulose. Preliminary studies on the two methylases indicate that they are indistinguishable with respect to their K,, for S-adenosylmethionine and their pH [in tris(hydroxymethyl)aminomethane buffer] and NaCl concentration optima. In vitro methylation of various phage X DNA substrates by the mec+ or RII enzyme modifies the DNA to a form that is completely resistant to double-stranded cleavage by the RII restriction endonuclease (R EcoRII). These results are consistent with our earlier proposal that the mec+ methylase recognizes RH host specificity sites.

RII (or hspll) host specificity, controlled by certain fi- R-factors (15), is mediated by a ctyosine-specific deoxyribonucleic acid (DNA) methylase (2, 6, 12). Most strains of Escherichia coli also contain a DNA-cytosine methylase (3, 4), which is controlled by the mec (or dem) locus (7, 9). Evidence has accumulated which indicates that the RII modification and mec+ methylation have overlapping sequence specificity (10, 11, 13). For example, the distribution of 5-methylcytosine (MeG) among oligopyrimidine tracts is the same for mec+ and RIImethylated DNA; the base sequence of the major tract is C-MeC-T for both enzymes (10, 11). More convincing evidence came from in vitro studies (13) on the susceptibility of phage and bacterial DNAs to cleavage by purified RII restriction endonuclease (R EcoRHI): (i) bacterial DNA from mec+ strains was completely resistant to R-EcoRII, whereas DNA (lacking MeG) from mec- strains was extensively degraded; (ii) covalently closed double-stranded circular fd DNA (RFI) from infected mec+ cells was resistant to R EcoRII, whereas fd * RFI from mec- cells was cleaved at least twice (13, 14). Taken together, the above findings indicate that the mec+ methylase can confer RII modification. In contrast to these data was the observation that the DNA from mature A * mec+ phage is cleaved by R -EcoRII (8) and has only half as much as MeC as A * mec+ RII+ or A * mecRII+ (5, 7); however, A * mec+ DNA is partially

protected against R EcoRH compared to X-mec- DNA (8; S. Schlagman, Ph.D. thesis, University of Rochester, Rochester, N.Y., 1976). To unify these findings, it was suggested that the mec+ methylase does act as an RII modification enzyme, but that the in vivo rate of methylation is lower in mec+ versus RII+ cells (8, 10, 13); consequently, maturation of vegetative DNA into viral particles occurs prior to methylation saturation of all the RH sites. Consistent with this model was the observation that X * mec+ DNA could be further methylated in vitro by a crude extract of mec+ cells (10); moreover, the additional MeC produced was quantitatively the same for both mec+ and mec-RII+ extracts. Finally, mec+ and mec-RII extracts produced the same number of MeC residues per X *mec- DNA, and the sites were apparently not independent (10). It still remained to be shown, however, that the sites methylated in vitro by the mec+ enzyme are, in fact, RII recognition sites. The present communication describes a procedure for the partial purification of the mec+ and RII methylases. It is also shown that mec+ and RII methylation modifies X mec+ and XAmec- DNA to complete resistance against double-stranded cleavage by R EcoRII. MATERIALS AND METHODS Bacterial strains. E. coli strains F+ 1100 rK- mK+ sup+ endI- thi- mec+ or mec- were described previ1330

VOL. 129, 1977 ously (7); derivatives lysogenic for phage AcI857indor harboring the RII factor, N-3 (15), were prepared in this lab. Media and buffers. L-broth contained per liter: 10 g of tryptone (Difco Laboratories), 5 g of yeast extract (Difco), 10 g of NaCl (adjusted to pH 7.0 with NaOH). The extract buffer contained: 10 mM sodium phosphate buffer (pH 7.2), 1 mM disodium ethylenediaminetetraacetic acid (Na2EDTA), and 5 mM 2mercaptoethanol. D-buffer contained extract buffer supplemented with 50 mM NaCl and 5% (vol/vol) glycerol. P-buffer contained the same as D-buffer except for 20% (vol/vol) glycerol. Isotopes and chemicals. Phosphocellulose (Whatman P11) and diethylaminoethyl (DEAE)-cellulose (CELLEX-D; BioRad) were prepared according to the specifications of the manufacturer. S-adenosyl[methyl-3Hlmethionine ([3H]SAM) was obtained from Amersham/Searle Corp.; [32P]H3P04 was obtained from New England Nuclear Corp. Protamine sulfate (U.S.P. injection grade) was obtained from Eli Lilly Corp. Partial purification of the DNA-cytosine methylase controlled by the E. coli mec+ gene. E. coli mec+ or E. coli mec- RII+ cultures were grown to approximately 5 x 108 per ml at 37°C in L-broth, harvested by centrifugation, and stored frozen at -20°C until ready for use. The cells were disrupted by grinding in a prechilled mortar with twice their weight in Alumina (Alcoa). All subsequent operations were at 4 to 5°C. The homogeneous paste was suspended in 3 volumes of extract buffer and centrifuged for 30 min at 15,000 rpm in a Sorvall SS-34 rotor. The supernatant was collected and centrifuged for 2 h at 50,000 rpm in an IEC B60 A-321 rotor. The supernatant was collected, and neutral 1% (wt/vol) protamine sulfate was added slowly with stirring (the final concentration was brought to 0.05% [wt/volI). After 30 min, the mixture was clarified by centrifugation for 10 min at 10,000 rpm in a Sorvall SS-34 rotor. The supernatant was dialyzed against D-buffer. The dialysate (350 mg of protein) was applied to a DEAE-cellulose column (2.5 by 25 cm) that was equilibrated in D-buffer. The column was washed with D-buffer until no more ultraviolet-absorbing material was observed in the effluent; elution was carried out with a 1-liter linear gradient of NaCl (0.05 to 0.35 M) in D-buffer. The DNA-cytosine methylase peak fractions (peak I, Fig. 1) were pooled and dialyzed against P-buffer and applied (30 mg of protein) to a phosphocellulose column (1.1 by 28 cm) which was equilibrated in P-buffer. The column was washed until there was no ultravioletabsorbing material in the effluent. Elution was carried out with a 200-ml linear gradient of NaCl (0.05 to 0.95 M) in P-buffer. The fractions containing enzyme activity were pooled and dialyzed against Pbuffer containing 50% glycerol and stored at -20°C. A similar procedure was utilized to partially purify the DNA methylase controlled by the RII factor. DNA methylase assays. The assay mixture (0.25 ml) contained per ml: 120 yg of calf thymus DNA; 100 jg of bovine serum albumin; 100 ,tmol of

E. COLI DNA-CYTOSINE METHYLASE

1331

,mol of 2-mercaptoethanol; 1.5 ,umol of [3H]SAM (specific activity, 400 ,uCi/,umol); and 0.05 or 0.10 ml of enzyme. The reactions were terminated after 40 min at 37°C by addition of cold 5% trichloroacetic acid and filtration onto GF/A glass-fiber disks (10). DNA-adenine and DNA-cytosine methylase activities were distinguished by analyzing the 6-[3H]methylaminopurine ([3H]MeA) and 5-[3H]methylcytosine ([3H]MeC) produced by methylation of calf thymus DNA. For example, a 2-h incubation with purified mec+ and RII enzyme produced 4,000 and 6,000 cpm in MeC, respectively, while the radioactivity in MeA was not significantly above background. Under similar conditions, the mec+ crude extract produced a ratio of [3H]MeA to [3H]MeC of 0.4. Preparation of 32P-labeled A DNA. Log phase cultures of E. coli lysogenic for prophage XcI857 were grown at 31°C in L broth; 50 ,ul of neutralized H3PO4 (5 mCi/ml; carrier-free) was added to 25 ml of culture (2 x 108 cells per ml). After 10 min of incubation at 31°C, the culture was transferred to 42°C and incubated with aeration for 18 min (to inactivate the thermolabile repressor and induce the prophage). Following this induction period, the cells were incubated at 37°C until lysis was completed. The phage were harvested by centrifugation and purified by equilibrium centrifugation in a CsCl gradient; the phage were deproteinized by phenol extraction and the DNA was dialyzed against 50 mM Tris-hydrochloride, pH 7.5 plus 0.1 mM Na2EDTA. The final preparation had an absorbancy ratio (A26./ Am) of 1.90; from a 25-ml lysate, the yield of X DNA was aproximately 40 itg at a specific activity of 1.4 x 104 cpm/,ug.

RESULTS Partial purification of the DNA-cytosine methylase controlled by E. coli K-12 mec+. DEAE-cellulose chromatography of an E. coli K-12 mec+ extract partially resolved two DNAmethylase activities (Fig. 1). The results shown represent the best chromatographic separation we have achieved to date; generally, we observe a single broad peak of methylase activity. Fractions 183 to 194 (peak I) and 204 to 215 (peak II) were pooled (represented by the horizontal bars) and assayed for DNA-adenine methylase and DNA-cytosine methylase activities. Peak II contained exclusively DNA-adenine methylase activity, whereas peak I contained primarily DNA-cytosine methylase activity (see Materials and Methods). The ratio of DNA-adenine methylase to DNA-cytosine methylase activity is usually greater than unity; we do not know why this particular preparation had such a low ratio (Fig. 1). It should also be noted that other workers have reported difficulty in separating DNA-adenine and DNA-cytosine methylase activities from E. coli (1, 4). The peak I enzyme was purified further by tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 8.0; 50 ,umol of KCI; 5 ,tmol of Na2EDTA; 7 chromatography on phosphocellulose (Fig. 2).

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HATTMAN 700- -7o0 600

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225

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FIG. 1. DEAE-cellulose chromatography of the E. coli DNA methylases. Conditions for preparation of the mec+ extract, chromatography, and enzyme assay are described in Materials and Methods. The NaCI x); concentration was determined by titration of the CI- ion with mercuric nitrate. Methylase activity (x *). Cl- concentration (O-- -O); absorbance at 280 nm (@

700

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In contrast to the above results, the RH methylase eluted from DEAE-cellulose at a lower NaCl concentration (0.09 to 0.12 M) than the mec+ methylase (0.13 to 0.16 M); consequently, the RII methylase is reproducibly well resolved from the host DNA-adenine methylase activity (data not shown). In addition, the RII methylase eluted from phosphocellulose at an NaCl concentration (0.51 to 0.57 M) higher than that observed for the mec+ enzyme (data not shown). Thus, the DNA-cytosine methylases controlled by the RH plasmid and E. coli mec+ genes are chromatographically distinct. The two enzyme preparations were also assayed for deoxyribonuclease activity. 32P-labeled A DNA was incubated at 37°0 for 120 min in 10 mM MgCl2 in the presence or absence of added DNA methylase. The reaction mixtures were analyzed by zone sedimentation through neutral sucrose gradients; there was no observed change in sedimentation profile for the methylase-treated versus untreated control DNA (data not shown). Under the same conditions, however, purified RH restriction endonuclease (R EcoRII) produced extensive fragmentation of X * mec+ and X * mec- DNAs, but did not degrade RH-modified (X * mec-RII+) DNA. We conclude that the methylase preparations do not contain detectable endonuclease activity. In vitro methylation of calf thymus DNA. By using calf thymus DNA as the substrate, conditions were sought to optimize the activity of the two DNA methylase preparations. We observed that Na2EDTA and KCl could be

50

75

100

Fraction number

FIG. 2. Phosphocellulose chromatography of the peak I fraction (Fig. 1). See legend to Fig. 1.

A single sharp peak of DNA methylase activity was eluted at 0.42 to 0.48 M NaCl; fractions 84 to 94 were pooled and dialyzed against P-buffer. This enzyme fraction contained little or no DNA-adenine methylase activity; 30% of the enzyme activity and less than 1% of the protein were recovered from the peak I fraction. The enzyme remained stable after several months of storage at -20°C.

E. COLI DNA-CYTOSINE METHYLASE

VOL. 129, 1977

omitted from the reaction mixture with no loss of enzyme activity. Both methylases showed a broad pH optimum (in Tris-hydrochloride buffer) between pH 6.8 and 8.5. Both enzymes were stimulated slightly by added NaCl; up to a twofold increase in methylation rate was observed at 30 mM NaCl. In the presence of 30 mM NaCl, the K,,, for SAM was 11 uM for both enzymes; the K,,, was calculated from a Lineweaver-Burk double-reciprocal plot (data not shown). In vitro methylation of phage A DNA. Since it had been shown that the mec+ methylase can act in vivo as an RII modification enzyme (13), it was important to determine whether the in vitro methylation of X DNA (10) occurs at RH sites. This was tested by methylating 32P-labeled X DNA with purified E. coli mec+ enzyme and then testing the DNA for its susceptibility to cleavage by R EcoRII. It can be seen (Fig. 3) that untreated (MeC-deficient) 32P-labeled X * mec- DNA was extensively degraded by R EcoRII. In contrast, 32P-labeled X mec- DNA that was methylated for 15 min with the mec+ enzyme exhibited some resistance to cleavage by R-EcoRII; by increasing the methylation period to 60 min, it was possible to modify X mec DNA to complete resistance to R EcoRII. Complete protection was also observed using 32p_ X mec+ DNA as the substrate; similar labeled A results were obtained with the RII methylase (data not shown). In a control experiment, we observed a bimodal profile (representing intact and degraded X DNA) following R EcoRII treatment of a mixture of unmethylated and in vitro methylated 32P-labeled X mec- DNA; moreover, the relative amount of the two peaks corresponded to that for the input DNA species (data not shown). This rules out the possibility that the in vitro methylation reaction mixture contains an inhibitor of R EcoRII. Finally, the presence of DNA methylase itself does not interfere with R EcoRII activity; e.g., following an incubation with DNA methylase in the absence of added SAM, 32P-labeled X * mec- DNA was extensively degraded by R EcoRII. Thus, modification of X DNA to a form that is resistant to R EcoRII cleavage requires DNA methylation. The ability to completely protect X DNA against R-EcoRII indicates that mec+ methylation occurs at RII sites. -

DISCUSSION Previous reports (11-13) from this laboratory showed that the RII and E. coli mec+ methylases have overlapping sequence specificity; e.g., in vivo methylation by the mec+ enzyme modified host cell DNA and fd RFI DNA to

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FIG. 3. Sucrose gradient centrifugation analysis of 32P-labeled X - mec- DNA methylated by the mec+ enzyme and then treated with R *EcoRII. Conditions for the in vitro methylation and nuclease digestion are as follows: reaction mixtures (0.5 ml) were prepared at 4°C containing 6 pg of 32P-labeled XA mec DNA (7 x 103 cpmlpg), 5 nmol of SAM, 50 p,mol of Tris-hydrochloride (pH 7.5), 5 mM sodium phosphate, pH 7.2,25 mM NaCl, 0.5 mM Na2EDTA, 2.5 mM 2-mercaptoethanol, 25% glycerol, and E. coli mec+ methylase. The reaction mixture was then transferred to 37°C, and after 0,15, and 60 min, 150pl portions were transferred into dialysis bags containing 0.5 ml of water. After overnight dialysis against cold water, the solution was concentrated to approximately 100 pI by dialysis against solid G-200 Sephadex. Duplicate 50-pl samples were taken and adjusted to about 90 mM Tris-hydrochloride, pH 7.5, plus 5 mM MgCl2, 5 p1 of purified R -EcoRII was added to only one sample. After 2 h of incubation at 37°C, the digestion was stopped by the addition of 50

td of 20 mM Na2EDTA. The reaction mixture

was

layered on a 3.6-ml linear (5 to 20%o) sucrose gradient (in 20 mM Tris-hydrochloride, pH 7.5, plus 1 mM Na2EDTA plus 100 mM NaCl) and centrifuged for 2 h at 55,000 rpm in an IEC SB405 rotor. The tubes were punctured at the bottom, and 10-drop fractions were collected directly into scintillation vials for determination of 32p radioactivity. The times in the panels indicate the duration of the methylase reaction. Each sample was centrifuged (from right to left in the figure) in a separate bucket; the figure shows superimposed profiles for the R -EcoRIItreated DNA (O) and the undigested control DNA (0).

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HATTMAN

complete resistance to in vitro cleavage by the RII restriction nuclease. However, DNA from mature phage X grown in mec+ cells (= X * mec+) was cleaved in vitro by R EcoRII (8); nevertheless, this DNA was partially resistant to R EcoRll cleavage (compared to X * mecDNA). Partial resistance to R EcoRII was attributed to the fact that X DNA is not methylated to saturation prior to phage maturation (7, 8); i.e., X *mec+ DNA contains only one half as many MeC residues as X *mec- RII+. Further studies revealed, however, that X - mec+ DNA could serve as a substrate for in vitro methylation by a crude extract of mec+ cells; moreover, the MeC level attained was the same as that produced by an RII+ extract (10), and it appeared that the sites methylated were not independent. It remained to be shown that the sites methylated by the mec+ enzyme are, in fact, true RII recognition sites. If this were the case, then in vitro methylation by the mec+ enzyme should modify X DNA to complete resistance against cleavage by R-EcoRII. The experiments reported in the communication confirm this prediction (Fig. 3). Since the R-EcoRIIX mec+ DNA are not sensitive sites on mature A intrinsically refractory to modification by the mec + enzyme, we believe that a relatively lower in vivo rate of mec+ versus RII methylation is the most likely explanation for the partial protection (methylation) of mature X *mec+ DNA (10, 13). In addition to possessing overlapping sequence specificity, the two methylases exhibit similar, though distinct, chromatographic behavior on DEAE-cellulose and phosphocellulose. Furthermore, the two methylases have the same pH and NaCl concentration optima and the same K,,. for SAM. Taken together, our findings suggest the possibility that the two proteins are evolutionarily related.

ACKNOWLEDGMENTS The excellent technical assistance of Diane Kuharik is gratefully acknowledged. The author is deeply indebted to Samuel Schlagman for providing the purified R-EcoRII preparation. This study was supported by Public Health Service grant no. AI-10864 and by Public Health Service Research Career Development award no. AI-28022 from the National Institute of Allergy and Infectious Diseases.

LITERATURE CITED 1. Bouche, J.-P., and J.-M. Dubert. 1972. DNA methyl-

2.

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9. 10.

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14.

15.

ases ofEscherichia coli K12. Evidences for changes in their state of association following purification. Eur. J. Biochem. 27:53-59. Boyer, H. W., L. T. Chow, A. Dugaiczyk, J. Hedgpeth, and H. M. Goodman. 1973. DNA substrate site for the EcoR,, restriction endonuclease and modification methylase. Nature (London) New Biol. 244:40-43. Fujimoto, D., P. R. Srinivasan, and E. Borek. 1965. On the nature of deoxyribonucleic acid methylases. Biological evidence for the multiple nature of the enzymes. Biochemistry 4:2849-2855. Gold, M., and J. Hurwitz. 1964. The enzymatic methylation of ribonucleic and deoxyribonucleic acid. V. Purification and properties of deoxyribonucleic acidmethylating activity of Escherichia coli. J. Biol. Chem. 239:3858-3865. Hattman, S. 1972. Plasmid-controlled variation in the content of methylated bases in bacteriophage lambda deoxyribonucleic acid. J. Virol. 10:356-361. Hattman, S., E. Gold, and A. Plotnik. 1972. Methylation of cytosine residues in DNA controlled by a drug resistance factor. Proc. Natl. Acad. Sci. U.S.A. 69:187-190. Hattman, S., S. Schlagman, and L. Cousens. 1973. Isolation of a mutant of Escherichia coli defective in cytosine-specific deoxyribonucleic acid methylase activity and in partial protection of bacteriophage X against restriction by cells containing the N-3 drugresistance factor. J. Bacteriol. 115:1103-1107. Hughes, S. G., and S. Hattman. 1975. The sensitivity of bacteriophage X DNA to restriction endonuclease RII. J. Mol. Biol. 98:645-647. Marinus, M. G., and N. R. Morris. 1973. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J. Bacteriol. 114:1143-1150. May, M. S., and S. Hattman. 1975. Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes. J. Bacteriol. 122:129-138. May, M. S., and S. Hattman. 1975. Analysis ofbacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes. J. Bacteriol. 123:768-770. Schiagman, S., and S. Hattman. 1974. Mutants of the N-3 R-factor conditionally defective in hsplI modification and deoxyribonucleic acid-cytosine methylase activity. J. Bacteriol. 120:234-239. Schlagman, S., S. Hattman, M. S. May, and L. Berger. 1976. In vivo methylation by theEscherichia coli K-12 mec+ deoxyribonucleic acid cytosine methylase protects against in vitro cleavage by the RH restriction endonuclease (R EcoRII). J. Bacteriol. 126:990-996. Vovis, G. F., K. Horiuchi, and N. D. Zinder. 1975. Endonuclease R-EcoRII restriction of bacteriophage fl DNA in vitro: ordering of genes V and VII, location of an RNA promotor for gene VmI. J. Virol. 16:674684. Watanabe, T., T. Takano, T. Arai, H. Nishida, and S. Sato. 1966. Episome-mediated transfer of drug resistance in Enterobacteriaceae. X. Restriction and modification of phages by fi- R factors. J. Bacteriol. 92:477486.