THE Joun~u OF B~LOGLCAL Cmmwm~ Vol. 252, No. 20. Issue of October 25, pp. 7265-7212, 1977

Prrnted rn U.S.A

EcoRI Methylase PHYSICAL AND CATALYTIC PROPERTIES OF THE HOMOGENEOUS ENZYME*

(Received for publication, May 31, 1977)

ROBERT A. RUBIN AND PAUL MODRICH

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Escherichia coli RI methylase has been isolated by a procedure which is suitable for large scale use and which yields enzyme with a specific activity IO-fold higher than previous methods. The purified methylase is homogeneous as judged by polyacrylamide gel electrophoresis, isoelectric focusing, and analytical sedimentation. The methylase is a basic protein composed of a single polypeptide chain of molecular weight 39,000, the stable form in solution being a 3.0 S monomer. No aggregation has been observed at con- centrations up to 0.3 mg ml-’ in the temperature range of 4- 20”, and the presence of S-adenosyl-L-methionine is without effect.

Catalytic studies have demonstrated that the enzyme functions as a monomer. Initial rates of methyl transfer are first order in methylase concentration, and the enzyme obeys Michaelis-Menten kinetics with respect to both sub- strates. At 37”, the K, for the EcoRI site of ColEl DNA is 1.3 nn, that for S-adenosyl+methionine is 0.26 PM, and the turnover number is three methyl transfers per min. The mechanism of methyl transfer to unmodified DNA is also consistent with the functional form of the enzyme being a monomer. The enzyme transfers methyl groups to theEcoR1 sequence one at a time and dissociates from the DNA prior to any subsequent catalytic events. Furthermore, the ki- netic parameters for addition of a second methyl group to a site which is already methylated on one strand are not more favorable than those for addition of the first.

These properties of the methylase are in marked contrast to those of the endonuclease (Modrich, P., and Zabel, D. (1976) J. Biol. Chem. 251, 5866-5874). Thus, we suggest that the two proteins interact with their common recognition sequence in different ways.

The Escher-i&a coli RI (EcoRI)’ DNA restriction and modi-

* This investigation was supported by United States Public Health Service Grant GM23719 and Grant PCM76-04914 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

i The abbreviations used are: EcoRI, in the nomenclature pro- posed by Smith and Nathans cl), a plasmid-specified DNA restric- tion and modification system initially identified in Escherichia coli strains bearing the drug resistance transfer factor RTF-1 (6). Like- wise, EcoB indicates the restriction-modification system determin-

fication enzymes recognize a common, 2-fold symmetrical hex- anucleotide sequence in duplex DNA:

d(GfA-ii-T-T-0 d(C-T-T-G-A-G).

t

Restriction is a consequence of double strand cleavage within this sequence (arrows), while modification is the result of methylation of the 2 adenine residues adjacent to the axis of symmetry (asterisks) to yield 6-methylaminopurine (2, 3).

Biochemically theEcoR1 enzymes are relatively simple. The methylase and endonuclease are separable proteins (4, 5) which are specified by distinct genes (6) and which have simple catalytic requirements. In vitro modification requires only unmodified DNA and S-adenosyl-L-methionine, while restriction of unmodified DNA requires only Mg2+. These properties together with the limited size of the recognition sequence make the EcoRI system attractive for investigation of sequence-specific DNA-protein interaction. For this reason, we as well as others have undertaken study of these proteins (4-7). The physical and catalytic properties of the endonucle- ase have been the subject of extensive study (5, 7); however, the properties of the modification methylase have not been investigated in detail. To this end, we have developed a high yield isolation procedure which results in homogeneous meth- ylase with a specific activity lo-fold higher than that previ- ously reported (4, 7) and have examined the physical and enzymological properties of the pure enzyme.

EXPERIMENTAL PROCEDURES

Materials

Bacteriophage and Bacterial Strains-Escherichia coli strains HB129 end1 gal str and RY13 were obtained from Dr. H. Boyer (University of California, San Francisco). RY13 is isogenic with HB129 except for the presence of plasmids specifying multiple drug resistance and EcoRI host specificity (6). Both strains also possess EcoB host specificity. E. coli C600 thr leu thi Zac (pVH51) was provided by Dr. D. Helinski (University of California, San Diego). E. coli W3110 ArrpE5 tna2 (pVH153) was provided by Dr. V. Hershlield (Duke University). Bacteriophage hb189c was obtained from Dr. R. W. Davis (Stanford University). Stocks of Ab189c carrying EcoB or both EcoB and EcoRI modification (designated h. B or A. RI) were isolated after passage of phage through HB129

ing host specificity of E. coli strain B. KPO,, potassium phosphate; AdoMet, S-adenosyl+methionine; DTNB, 5,5’-dithiobis(2-nitroben- zoic acid); SDS, sodium dodecyl sulfate.

7265

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7266 Physical and Catalytic Properties of EcoRI Methylase

and RY13, respectively. Preparative growth and isolation of hb189c was performed as described by Thomas and Davis (8).

DNA-E. coli HB129 DNA was extracted by the method of Marmur (9). Bacteriophage A-DNA was isolated from purified viri- ons by extraction with redistilled phenol (5). SV40 DNA was a gift from Dr. John Newbold (University of North Carolina, Chapel Hill). Covalently closed circular pVH51, pVH153, ColEl, and PM2 DNA’s were isolated by procedures described previously (5). Plasmid pVH51 is derived from the colicinogenic plasmid El (ColEl) by deletion and contains a single EcoRl site (10). AM, of 2.1 x 10” has been reported for pVH51; however, we have determined the molecu- lar weight to be 2.3 x lo6 by agarose gel electrophoresis of the EcoRl nuclease generated linear form in the presence of EcoRl frag- ments of hcl857s7 DNA whose molecular weights are accurately known (8). The larger value is used for calculations in this paper. Plasmid pVH153 contains two EcoRl sites (11). We have determined the EcoRI fragment molecular weights to be 2.3 x lo6 and 5.0 X 106, and hence have employed a M, of 7.3 x lo6 for calculations.

Strand separation of hb189c DNA was performed by the method of Szybalski et al. (121, using poly[r(U,G)l (P-L Biochemicalsl. Purity of separated single strands was judged to be approximately 90% by analytical CsCl equilibrium centrifugation in the presence of poly[r(U,G)l. Heteroduplex molecules were formed by mixing approximately equal amounts of separated A. B and A. RI strands as isolated from preparative CsCl/poly[r(U,Gl1 gradients, heating the mixture at 100” for 2 min after adjusting to pH 8 to 9 with 1 M NaOH, and annealing at 70” for 30 min. DNA of native density was then isolated by CsCl equilibrium density gradient centrifugation. Control homoduplex A. B molecules were prepared by the same procedure used to make heteroduplex DNA, except that the 100” heat treatment was omitted.

Enzymes and Proteins-EcoRl endonuclease was Fraction V as described (5). Chymotrypsinogen A was from Worthington. Other proteins were described previously (51.

Other Materials-S-Adenosyl-L-[methyZ-3Hlmethionine (18 Ci/ mmol) was obtained from New England Nuclear. Radiochemical purity as determined by descending chromatography on Whatman No. 1 paper in 1-butanob0.1 N HCl/ethanol (50:50:20) exceeded 96%. Unlabeled AdoMet (Sigma) was purified according to Mudd (131 to a purity of at least 98%. The concentration was determined spectro- photometrically according to Zappia et al. (14). The concentration of [3HlAdoMet was determined by an isotope dilution procedure in the methylase assay with pVH51 DNA as substrate. The concentration of unlabeled AdoMet required to reduce the specific activity of 3H- methyl groups by 50% was determined and taken to be equal to the concentration of the isotopically labeled compound.

Bio-Gel A-0.5m was purchased from Bio-Rad. Dithiothreitol was from P-L Biochemicals. Spectral grade glycerol was Baker Photrex. Other materials were as described previously (5).

Methods

EcoRI Methylase Reactions - The standard assay for methylase activity measures transfer of 3H-methyl groups to HB129 or pVH51 DNA as specified. Reactions (100 ~1) contained 0.1 M Tris/HCl (pH 8.0), 5 mM EDTA (pH 8.01, 400 pglml of bovine serum albumin, 0.42 rnM DNA (as nucleotide), 2.5 to 5 m&r dithiothreitol, and 1.1 PM AdoMet. After incubation at 37” for 10 min, 80-~1 samples were spotted on 1.5-cm squares of Whatman DE81 paper. Papers were washed five times with 0.2 M NH,HCO,, three times with ethanol, and once with diethyl ether, dried under a heat lamp, and tritium was determined in 5 ml of a toluene-based scintillation fluid. One unit of methylase activity converts 1 pmol of 3H-methyl groups/min to a form which binds to DE81. The assay was linear with enzyme to approximately 0.1 unit and with time for at least 10 min. The enzyme was diluted as required into 0.02 M KPO, (pH 7.41, 0.2 M NaCl, 0.2 mM EDTA, 2 mM dithiothreitol, 0.2 mg/ml of bovine serum albumin, 10% (w/v) glycerol (methylase diluant).

EcoRI Endonuclease Digestions - Reaction mixtures contained 0.1 M Tris/HCl (pH 7.61, 0.05 M NaCl, 0.2 rnM EDTA, 5 mM MgCl,, typically 10 to 20 pg/ml of DNA, and 1 unit/ml of EcoRl endonucle- ase. Incubation was for 20 min at 37”. When DNA products were to be analyzed by gel electrophoresis, reactions were quenched by addition of an equal volume of 20 rnM EDTA (pH 8.01, 20% (w/v) sucrose, 0.025% bromphenol blue.

Gel Electrophoresis-The method of Weber and Oshorn (15) was used for protein electrophoresis in the presence of 0.1% SDS. lsoelec- tric focusing under native conditions was performed on 5% polyacryl- amide gels containing 2% Bio-Rad ampholytes (Biolyte 3/10, pH 3 to

101 according to the procedure recommended by the manufacturer (Technical Bulletin 1030) except that gels contained 1 mM dithiothre- itol. Gels were cut into approximately 30 segments of 3.7 mm. Gel slices to be assayed were soaked in 0.5 ml of methylase diluant for 24 h at 0”. For pH measurements slices prepared from parallel gels were soaked in 0.40 ml of degassed 0.10 M KC1 for 6 h at 4” and pH was determined at 4”.

Electrophoresis of DNA was performed on 0.3-cm thick slabs of 1% agarose as described previously (5) except voltage gradients were 2.7 V/cm. After electrophoresis for 16 h, DNA was visualized by staining with 1 pg/ml of ethidium bromide. DNA content of bands was determined by microdensitometer tracings of photo- graphic negatives according to Depew and Wang (161, except peak areas were determined in all cases, Film response was calibrated for each gel by electrophoresis of a sample of Acl857s7 DNA frag- ments generated by digestion with EcoRl endonuclease. A correction was made for the reduced fluorescence yield of covalently closed circular DNA by running standards containing known amounts of closed circular and linear DNA on each gel. For example, with pVH51 DNA the fluorescence of closed circles relative to linear DNA was found to be 0.75 + 0.04 (n = 6) as judged by the microdensitometry procedure. Circular DNA containing one or more single strand breaks was assumed to have the same fluorescence yield as linear DNA.

Sedimentation Analysis - Analytical centrifugation employed a Beckman model E centrifuge with a photoelectric scanner. Prior to sedimentation the enzyme was dialyzed exhaustively against 0.02 M

KPO, (pH 7.4), 0.25 M KCl, 0.50 or 0.75 mM dithiothreitol. Boundary sedimentation was performed at 44,953 rpm at 20.0” at initial protein concentrations of 22 to 337 pglml. A scanning wavelength of 278 nm was used at protein concentrations above 90 pglml, while 230 nm was employed at lower concentrations. The sedimentation coefficient was corrected to standard conditions (17). A protein partial specific volume of 0.741 at 25” was calculated from amino acid composition (18) and corrected for temperature dependence as described (19).

Sedimentation equilibrium centrifugation was performed accord- ing to Chervenka (17) at initial protein concentrations of 58 to 316 pg/ml. Rotor speeds were 12,000 to 15,000 rpm and temperatures were 8.5-12”.

For sucrose density gradient sedimentation, enzyme solution (0.20 ml) was layered on a 4.8-ml sucrose gradient (10 to 30%, w/v) containing 0.02 M KPO, (pH 7.41, 0.25 M KCI, 1 mM dithiothreitol. Centrifugation was at 39,000 rpm at 4” for 24 h in a Beckman SW 50.1 rotor. Sedimentation coefficients were determined by the method of Martin and Ames (20) using standards described previ- ously (51. Recovery of methylase activity was typically greater than 95%.

Amino Acid Analysis-A Beckman model 121 analyzer was em- ployed. We are grateful to Dr. T. Vanaman (Duke University) for use of his instrument. Enzyme samples were hydrolyzed for 41, 59, and 68 h in 6 N HCl containing a fresh crystal of phenol at 110” ? 1 in uocuo. Values for serine, threonine, and tyrosine were corrected to zero hydrolysis time. Cysteine was determined after reduction as carboxymethylcysteine according to Crestfield et al. (21) and by titration of the native enzyme with DTNB by minor modifications of the procedure of Colman (221.

For DTNB titration, Fraction V was dialyzed exhaustively at 0 under N, versus 0.1 M Tris/HCl (pH 7.61, 0.2 M NaCl, 1 mM EDTA which was rendered oxygen free by prior boiling. To 4 volumes of dialyzed enzyme (which retained full activity) in a cuvette at 23” was added 1 volume of 2.9 mM DTNB in 0.05 M sodium acetate (pH 5.1). After flushing with N,, the cuvette was sealed and the change in A,,, was monitored at 23”. Blank determinations were performed analogously except that the final change of dialysis buffer was used in place of enzyme.

Other Methods-E, coli RY13 was grown as described (51. Protein content of partially purified fractions was determined according to Lowry et al. (23). An E::, of 200 at 260 nm was assumed in calculating DNA concentrations. Other methods were those used previously (5).

RESULTS

Purification of EcoRI Methylase - Purification of EcoRI methylase from 225 g of Escherichia coli RY13 is summarized in Table I. Unless indicated otherwise, all steps were carried out at O-4” and centrifugation was at 12,000 x g for 20 to 30 min in a Sorvall GS3 rotor.

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Physical and Catalytic Properties of EcoRI Methylase 7267

Purification of EcoRI methyl FIW- tion SkP

TABLE I

Streptomycin super- natant

Ammonium sulfate, dialyzed pool

Phosphocellulose Hydroxylapatite

, Bio-Gel A-0.5m

e from 225 g ofEscherichia coli RY13

Protein Recov- Specific activity erv

(91,000; 76,0001b

% (100)

71

37 25 16

a Protein determined by amino acid analysis. b Assayed on pVH51 DNA and SV40 DNA, respectively. Other

assays utilized E. coli HB129 DNA (see “Methods”).

Extract Preparation and Streptomycin Fractionation -E. coli RY13 cell paste (225 g) was thawed overnight at 4” and suspended in 675 ml of 0.02 M KPO, (pH 7.0), 20 mM 2- mercaptoethanol, 1 mM EDTA with the aid of a Waring Blendor. The suspension was sonicated in 225ml portions with 10 30-s pulses at 100 watts using a Branson sonicator equipped with a large probe. Temperature was maintained at 5” or less with an ice-salt bath. The extract was centrifuged and the supernatant was diluted with the above buffer to a final volume of 742 ml to yield an A,,, of 200. Freshly prepared 25% (w/v) streptomycin sulfate (148 ml) was added with stirring and after an additional 30 min the precipitate was removed by centrifugation. The supernatant (Fraction I, 812 ml) had a ratio APXO/AafiO of 0.86 and an A,,, of 27.1.

Ammonium Sulfate Fractionation -Solid (NHJ2S04, 390 gl liter, was added over a period of 10 min with stirring. After stirring an additional 30 min, the precipitate was collected by centrifugation and extracted successively with solutions of 45%, 40%, and 35% saturation in (NH&SO, (prepared by dissolving 277 g, 242 g, and 208 g of (NHJSO,, respectively, in 1 liter of 0.02 M KPO, (pH 7.4), 20 mM 2-mercaptoethanol, 1 mM EDTA and adjusting the pH of each solution to 7.1 (at 0”) with KOH). For each extraction, the precipitate was suspended in 244 ml of the appropriate solution, the suspen- sion was stirred for 30 min, and insoluble material removed by centrifugation. The 45, 40, and 35% (NH&SO, solutions contained 24%, 51%, and 26%, respectively, of the activity present in Fraction I.* These fractions were pooled (765 ml) and concentrated by the addition of 143 g of (NH&SO,. The precipitate was dissolved in 135 ml of 0.02 M KPO, (pH 7.4), 20 mM 2-mercaptoethanol, 1 mM EDTA and dialyzed for 8 h uersus 8 liters of 0.02 M KPO, (pH 7.4), 20 mM 2-mercaptoeth- anol, 10% (w/v) glycerol (Buffer A) containing 0.2 M KC1 and 0.5 mM EDTA to yield Fraction II.

Phosphocellulose Chromatography -Fraction II (145 ml) was diluted to 1560 ml with Buffer A containing 0.09 M KCl, 0.5 mM EDTA and applied at 160 ml/h to a phosphocellulose column (33.4 cm x 12.6 cm? equilibrated with Buffer A containing 0.1 M KC1 and 0.5 mM EDTA. The column was washed with 425 ml of Buffer A containing 0.1 M KCl, 0.5 mM EDTA and then eluted with a 3.8-liter linear gradient of KC1 (0.1 to 0.5 M) in Buffer A, 0.1 mM EDTA. Methylase activity eluted at 0.32 M KC1 (Fraction III).

Hydroxylapatite Chromatography -Fraction III (385 ml) was applied at 13 ml/h to a column of hydroxylapatite (13.6

‘EcoRI endonuclease is recovered in washes of 35% saturation and below (51.

cm x 1.77 cm’) equilibrated with 0.02 M KPO, (pH 7.4), 0.2 M

KCl, 10% (w/v) glycerol, 2 mM dithiothreitol. The column was washed with 36 ml of 0.08 M KPO, (pH 7.4), 10% (w/v) glycerol, 2 mM dithiothreitol and then eluted at 8.6 ml/h with a 190-ml linear gradient of KPO, (pH 7.4, 0.08 to 0.30 M)

containing 10% (w/v) glycerol and 2 mM dithiothreitol. Activ- ity eluted in two adjacent peaks at 0.18 M and 0.19 M

phosphate. While this elution pattern was always observed, subsequent chromatography on different media always re- sulted in a single activity peak. The pooled fractions (19 ml) were dialyzed against 2 x 400 ml (4 h/change) of 0.08 M

KPO,, 10% (w/v) glycerol, 2 mM dithiothreitol and the dialy- sate (21 ml) immediately applied at 4 ml/h to a column of hydroxylapatite (5.94 cm x 0.64 cm”) equilibrated with the same buffer. The column was eluted with 0.5 M KPO, (pH 7.4), 2 mM dithiothreitel, 10% (w/v) glycerol and fractions absorbing at 280 nm were pooled (Fraction IV).

Bio-Gel A-0.5m Chromatography -Fraction IV (2.3 ml) was applied to a column of Bio-Gel A-0.5m (49.2 cm x 2.81 cm*) equilibrated with 0.02 M KPO, (pH 7.4), 0.25 M NaCl, 1 mM EDTA, 10% (w/v) spectral grade glycerol, 2.5 rnM dithio- threitol. The column was eluted at 14.7 ml/h with this buffer, and fractions of 1.4 ml were collected. Methylase activity and most of the protein eluted in symmetrical and coincident peaks at 0.76 column volume. The eluate was dialyzed twice against 300 ml (4 h/change) of 0.02 M KPO, (pH 7.4), 0.25 M

KCl, 1 mM EDTA, 2.5 mM dithiothreitol, 10% (w/v) spectral grade glycerol and then dialyzed for 12 h against 250 ml of the same buffer containing 50% (v/v) spectral grade glycerol (Fraction V, 3.4 ml).

Fraction V lost no activity (~5%) over a period of at least 12 months at -20”. Under standard methylase assay condi- tions it was free of exonuclease activity on T7 [“HIDNA and was free of endonuclease activity on ColEl DNA (50.5-pmol single strand breakslmglmin) when assayed under either EcoRI methylase or endonuclease conditions. Activity on PM2 DNA, which has no EcoRI recognition site, was approximately 0.3% of the activity on pVH51 DNA, indicating the absence of contaminating methylases.

Polyacrylamide Gel Electrophoresis -After reduction and denaturation, Fraction V yielded a single major protein band on 5% or 7.5% polyacrylamide gels containing 0.1% SDS (Fig. 1). Mobilities relative to bromphenol blue were 0.73 and 0.52, respectively. When compared with mobilities of proteins of known molecular weight, these values yield an apparent molecular weight of 39,000 for the reduced and denatured enzyme. A trace impurity (52%) with an R, of 0.86 was detected on the 7.5% gel.

Isoelectric focusing on 5% polyacrylamide gels under native conditions also revealed a single major protein zone and demonstrated that this basic species possesses EcoRI methyl- ase activity. As shown in Fig. 2, the major protein zone and the peak of activity coincided at an isoelectric pH of 8.7. Minor protein zones appeared at pH 4.9 (cl%) and pH 7.9 (5%), the former having no detectable methylase activity and the latter incompletely resolved from the peak of activity. Gel electrophoresis thus demonstrated that Fraction V has a purity of 95 to 98% and that methylase activity is associated with the major protein species.

Sedimentation Analysis -The native molecular weight of EcoRI methylase was determined by sedimentation equilib- rium centrifugation at initial enzyme concentrations of 58 to 316 pug/ml. Plots of logarithm of concentration uersus 9 were linear (Fig. 3), indicating physical homogeneity, and yielded

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7268 Physical and Catalytic Properties of EcoRI Methylase

0 - ik

Dye 4

- I 1.0

Rf

7-

Y. 5- -

z,- EcoRl Methylase

2-

I '0

I I I 1 I 02 04 0.6 0.6 1.0 I

Rf

FIG. 1. SDS-gel electrophoresis ofEcoR1 methylase. Upper panel, 9 pg of Fraction V were subjected to electrophoresis on a 7.5% polyacrylamide gel containing sodium dodecyl sulfate following reduction and denaturation (see “Methods”). The gel was stained with Coomassie brilliant blue and scanned at 550 nm. Lower panel, the molecular weight of the reduced and denatured enzyme was determined by electrophoresis in the presence of standards of known molecular weight. Mobilities are relative to bromphenol blue. Stan- dards were Escherichia coli DNA polymerase I (M, = 109,000), bovine serum albumin (M, = 68.0001, ovalbumin (M, = 44,0001, DNase I (M, = 31,000), EcoRI endonuclease (M, = 28,500 (511, chymotrypsinogen A (M, = 25,000), and lysozyme (M, = 14,300).

a M, of 39,000 f 3,500 (four measurements) for the native enzyme. As determined by analytical boundary sedimentation at initial concentration of 22 to 337 pg/ml, the s2,,, u‘ of the enzyme is 3.03 2 0.05 S (seven determinations), in agreement with the sedimentation coefficient of 3.0 2 0.1 S (four deter- minations) determined for methylase activity by sucrose gra- dient sedimentation (data not shown). The s2,,, u1 as determined by the latter method was unaffected by the presence of 10 pM

AdoMet in the gradient. Thus, the native enzyme is a mon- omer with an sZO, u, of 3.0 and a molecular weight of 39,000.

Amino Acid Analysis and Ultraviolet Spectra -The amino acid composition of EcoRI methylase is shown in Table II. With the exception of a high lysine content, the amino acid composition of the protein is not unusual. Although acidic residues predominate, the basic nature of the protein (p1 = 8.7) indicates that a considerable fraction of these residues are in the amide form. These data have been used to calculate a protein partial specific volume (6) of 0.741 at 25” (18). The ultraviolet spectra of the enzyme under native or alkaline conditions were those of a typical protein (not shown). The spectrum of the native enzyme was characterized by an

FIG. 2. Isoelectric focusing of EcoRI methylase on 5% polyacryl- amide gels. Fraction V was diluted into 2% ampholytes, 2.5 mM dithiothreitol, 25% glycerol (v/v) and 51 pg of protein were layered on each of two gels containing 2% ampholytes. Protein solutions were overlaid with 2% ampholytes in 10% glycerol (v/v) and tubes were filled with 0.06 N H,SO,. Parallel gels for pH measurements contained no enzyme. Electrophoresis was at 175 V for 22 h at 4”. Upper and lower reservoirs contained 0.06 N H,SO,, and 1 g of CaO plus 2 g of NaOH in 1.3 liters of H,O, respectively. Gels were sliced for pH measurement and assay ofEcoR1 methylase (see “Methods”). Recovery of methylase activity was approximately 3%. One gel was stained 1 h at 37” in ethanol/H,O/acetic acid (50:45:5) containing 0.2% bromphenol blue and protein quantitated by scanning at 610 nm. An arrow indicates the position taken as isoelectric pH.

FIG. 3 (left). Sedimentation equilibrium analysis of&OR1 meth- ylase. Sedimentation was performed as described under “Methods” at 12,480 rpm at 8.5” for 46 h.

FIG. 4 (right). Linear relationship between initial rate of meth- ylation and concentration of EcoRI methylase. Assays with pVH51 DNA were performed as described under “Methods.”

absorption maximum at 278 nm and a 280 to 260 ratio of 2.0 to 2.1. With a protein concentration based on amino acid analysis, the extinction coefficient (Ej~,,,) at 278 nm was determined to be 10.8.

Thiol groups play a crucial role in the native enzyme. Half- cysteine content determined as carboxymethylcysteine was in good agreement with cysteine content measured by DTNB titration of native methylase, indicating that most if not all cysteine is present in the reduced form. An essential role for thiol groups in catalytic activity was indicated by the require- ment for thiol-reducing reagents in methylase assays and purification procedures. Furthermore, catalytic activity was abolished when the enzyme was treated with 3 mM N-ethyl- maleimide for 5 min at 15” (not shown), demonstrating that thiol groups are required for enzymatic activity.

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Physical and Catalytic Properties of EcoRI Methylase 7269

TABLE II

Amino acid composition ofEcoRI methylase Amino acid Moles per M, = 39,000

Alanine 17.2 Arginine 14.2 Aspartic acid 38.4 Half-cystine 7.1” (5.99 Glutamic acid 30.6 Glycine 19.6 Histidine 6.45 Isoleucine 21.6 Leucine 30.1 Lysine 35.3 Methionine 1.8 Phenylalanine 21.0 Proline 11.4 Serine 22.4 Threonine 12.7 Tryptophan 3.2’ Tyrosine 21.6 VaIine 23.0

’ Determined after reduction as carboxymethylcysteine. D Determined by titration of native unreduced enzyme with

DTNB. ’ Determined spectrophotometrically under alkaline conditions

(24).

Catalytic Properties of EcoRI Methylase -Greene et al. (7) have described a preparation of EcoRI methylase which had a specific activity of 7000 units/mg with SV40 DNA as substrate and which was homogeneous as judged by SDS-gel electropho- resis. Under similar conditions our Fraction V has a specific activity of 91,000 units/mg with pVH51 DNA as substrate and 76,000 unitslmg with the SV40 substrate (Table I). As de- scribed above, we have found that high concentrations of thiol-reducing reagents are required during isolation and assay for good recovery and optimal activity. Oxidation of essential cysteine residues during isolation or assay by Greene et al. (7) could account for this discrepancy in specific activi- ties.

As shown above, the stable form of the methylase in solution is a monomer in the presence or absence of AdoMet at concentrations as high as 8 PM (monomer). Since the recognition sequence of the enzyme is 2-fold symmetric (2, 31, we wished to determine whether the enzyme interacts with the sequence as a monomer. If more than one monomer must bind to the EcoRI sequence in order for catalysis to occur, then the dependence of initial velocity on enzyme concentra- tion should be greater than first order and deviation from Michaelis-Menten behavior would be expected.

As shown in Fig. 4, the initial rate of methylation was first order with respect to enzyme up to 500 PM (monomer). Fur- thermore, the enzyme obeyed Michaelis-Menten kinetics with respect to both AdoMet and DNA (Fig. 5). Since the methylase is monomeric at concentrations 3 to 4 orders of magnitude greater than those used in these experiments, we have con- cluded that it can in fact function as a monomer.

In contrast, Greene et al. (7) have suggested that oligomer- ization of the methylase is necessary for catalytic activity. This conclusion was based on their observation of a nonlinear dependence of reaction rate on enzyme concentration. How- ever, as discussed above, the enzyme used by Greene et ul. (7) had a significantly lower specific activity than that employed here, and thiol reductants were not present in their methylase reactions. It is plausible that the nonlinear dependence ob-

35-

30-

-725- I c

.EZO- E

7

/ 5t I I I 1 I I I

-10 -05 0 05 1.0 1.5 2.0 2.5 [PVHSl, nM]-'

FIG. 5. Steady state kinetics of EcoRI methylase. Upper panel, determination of K, for pVH51 DNA. Reactions (0.1 ml) contained 0.1 M Tris/HCl (pH 8.01, 5 mM EDTA (pH 8.0), 500 pg/ml of bovine serum albumin, 2.5 mM dithiothreitol, 2.75 pM [3H-methyllAdoMet, 38.8 PM EcoRI methylase, and indicated concentrations of pVH51 DNA. Incubation was at 37” for 10 min. DNA concentrations are in terms of molecules. Lower panel, determination of K, for AdoMet. Reactions were as above except that they contained 25 nM pVH51 DNA, 120 PM EcoRI methylase, and indicated concentrations of AdoMet.

served by this group reflected selective inactivation at low enzyme concentrations due to oxidation of essential thiols.

The steady state kinetic experiments shown in Fig. 5 were performed in the presence of a saturating concentration of the invariant substrate. Therefore, these data permit calculation of the true catalytic constant for the enzyme. The kcat deter- mined from Fig. 5 (upper panel) is 3.2 methyl transfers/min/ monomer at 37”, while that obtained from the experiment shown in the lower panel is similar, being 2.8 min-I. The corresponding K, values are 0.26 PM for AdoMet and 1.3 nM for pVH51 DNA (molecules).

Mechanism of Methylation -Since complete modification of an unmethylated EcoRI sequence involves addition of two methyl groups (3), several catalytic mechanisms are possible. For example, methyl transfer may occur either singly or in combination. In addition, the enzyme may leave the recogni- tion sequence by dissociation from the DNA or, as is the case for rat liver DNA methylase (25), by diffusion along the substrate in search of additional unmodified sites.

To determine whether methyl transfer occurs singly or in combination, the conversion of pVH51 DNA to an EcoRI

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7270 Physical and Catalytic Properties of EcoRI Methylase

endonuclease-resistant form was examined as a function of average degree of methylation (Fig. 6). DNA was methylated under steady state conditions and methyl group transfer monitored. DNA samples with an accurately known methyl content were isolated and subjected to digestion with an excess of EcoRI endonuclease. As shown in Fig. 6, the fraction of molecules resistant to single and double strand cleavage by the endonuclease increased linearly with methyl content in the range of 0 to 0.4 methyl groups/DNA. Furthermore, this portion of the curve had a slope of 95% resistance per methyl group per DNA, demonstrating that the enzyme transfers methyl groups one at a time and leaves the DNA after each catalytic event.

This result also indicates that a recognition sequence meth- ylated on only one strand is not subject to single or double

go-

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OV 0.5 1.0 1.5 2.0 Methyl Groups per DNA

FIG. 6. Resistance of pVH51 DNA to EcoRI endonuclease as a function of average extent of methylation. 0, pVH51 DNA (25 nM molecules) was methylated under standard assay conditions (see “Methods”) at 37” using 410 PM EcoRI methvlase in a volume of 1.0 ml. Samples (0.01 ml)-were-removed at various times and methyl transfer was determined. Additional samples (0.04 ml) were simul- taneouslv removed. diluted with 0.18 ml of 0.1 M Tris (PH 7.6). 10 mM EDNA (pH 8.‘0), and immediately extracted with an equal volume of phenol equilibrated with this buffer. In the remaining reaction mixture DNA was methylated to completion. The limit value of average methyl content per DNA molecule was determined to be 2.08, and this value was normalized to 2.00. DNA samples of accurately known methyl content were thus obtained. The phenol- extracted samples were dialyzed exhaustively against 0.1 M Tris (pH 7.61, 0.05 M NaCl, 0.1 mM EDTA, digested withEcoR1 nuclease, and cleavage products separated and quantitated as described under “Methods.” The sum of mole per cent of nicked circular (typically 5%) and closed circular species was plotted uers’sus average methyl content. 0, the procedure was as above, except pVH51 DNA and methylase concentration were 935 PM and 47 PM, respectively, in a 12.5-ml reaction. Samples (0.23 ml) were removed at various times and made 0.44 mM in denatured salmon sperm DNA and 7.5% in trichloroacetic acid. After incubation at 0”, precipitates were col- lected on glass fiber filters, washed at 0” with 1 N HCl and 95% ethanol, and tritium determined. Additional samples (0.40 ml) were extracted with an equal volume of phenol, dialyzed, and digested as above. The observed methylation limit of 2.06 was normalized to 2.00.

strand cleavage by the endonuclease. This was confirmed by testing endonuclease sensitivity of hb189c heteroduplex mole- cules containing the RI modification in only one strand (Fig. 7). When tested with EcoRI endonuclease under conditions where unmodified natural or renatured duplexes were subject to complete double strand cleavage, only about 10% of the heteroduplex preparation was attacked. This level of double strand cleavage is consistent with the known quantity of homoduplex contamination present in the preparation (see “Materials”). Furthermore, the heteroduplex was not subject to significant single strand cleavage as demonstrated by electrophoretic patterns of thermally denatured molecules. As can be seen, the size distribution of denatured heteroduplex DNA was, except for the homoduplex impurity, unaffected by prior nuclease treatment.

The experiments presented in Fig. 6 also provide additional information concerning steady state kinetic parameters for the methylase reaction. During early phases of the reaction, at either saturating or subsaturating concentrations of DNA, methylation occurs at sites devoid of methyl groups. There- fore, kinetic parameters based on initial velocity measure- ments (Fig. 5) are those for methyl transfer to unmodified sites. As a consequence of the relatively high K, of the enzyme for DNA and the limited quantities of half-methylated heteroduplex available, we have been unable to determine kinetic parameters for addition of a second methyl group to a partially methylated site. However, the data of Fig. 6 also provide information on the relative rates of addition of the first and second methyl group at high and low DNA concentra- tions. Although methyl transfer during early stages of the reaction occurs at unmodified sites, as the reaction proceeds an increasing fraction of enzymatic events will involve sites that have already been methylated once. As this fraction becomes significant, plots of the type shown in Fig. 6 will

FIG. 7. Resistance of heteroduplex-modified DNA to single and double strand cleavage by EcoRI endonuclease. Samples were: Ab189c renatured unmodified homoduplexes (Wells 1 to 4), renatured “half-modified” heteroduplexes (Wells 5 to 8), and natural unmodi- fied homoduplexes (Wells 9 to 12). Within each set proceeding from left to right, samples were treated as follows: no endonuclease treatment; no endonuclease treatment, heat-denatured UOO”, 3 min) immediately prior to electrophoresis; EcoRI endonuclease-treated; EcoRI endonuclease-treated followed by heat denaturation immedi- ately prior to electrophoresis.

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Physical and Catalytic Properties of EcoRI Methylase 7271

0 1 2 3 4 Methyl Groups per DNA

FIG. 8. Resistance of the two site pVH153 DNA to EcoRI nuclease as a function of average methyl content. Conditions of methylation were as described in Fig. 6. Upper panel, the limit value of methyl content per DNA was found to be 4.42 which was normalized to 4.00. Per cent resistant DNA was calculated as N + C + (L/21, where N, C, and L are mole per cents nicked circular, closed circular, and full length linear DNA. Upper panel, per cent resist- ance (N + C + (L/21 as a function of methyl content. Unmethylated control DNA contained 7.9% full length linear DNA after nuclease treatment. Since (L/2) = 4% for control DNA this amount was subtracted from all values of N + C + (L/2). Lower panel, mole per cent of one site modified (I,). two site modified (N + C). and unmodified (100 - [N + C + L]) molecules as a function of methyl content.

deviate from linearity because the addition of a second methyl group to an RI site does not confer additional resistance to the endonuclease. As can be seen, significant deviation from linearity occurs only after a relatively large fraction (40 to 50%) of the molecules have been methylated once. Thus, the kinetic parameters for addition of the second methyl group apparently are not more favorable than those for addition of the first, and in fact could be less so. This is in contrast to the type I modification methylase of EcoB specificity which meth- ylates half-modified sites much more rapidly than unmodified ones (26).

The mode of action of the enzyme with pVH51 DNA strongly suggests, but does not prove, that multiple RI sites on the same DNA molecule would behave independently with respect to methylation. To directly test this possibility, the experi- ment with pVH51 was repeated with pVH153 DNA, a plasmid

bearing two EcoRI sites (11) separated by 3.48 kilobase (shorter distance) or 7.56 kb (longer distance). The conversion of pVH153 to endonuclease resistance as a function of average extent of methylation (Fig. 8, upper panel) was very similar to that observed with pVH51. The initial slope was 49% resistance per methyl group per DNA, which for a two site molecule is equivalent to 98% resistance per site. In addition, transient accumulation of single site modified pVH153 mole- cules (subject to only one cleavage by EcoRI nuclease) was observed (Fig. 8, lower panel). This species reached a concen- tration of 12-fold excess over added enzyme and was, therefore, a turnover product and a reaction intermediate. Thus, under steady state conditions the two EcoRI sites of pVH153 DNA behave independently with respect to methylation.” Based on the results with these two plasmids we have concluded that the enzyme catalyzes transfer of a single methyl group to the EcoRI sequence and dissociates from the DNA prior to any subsequent catalytic events.

DISCUSSION

We have developed a simple procedure by which homogene- ous EcoRI methylase of high specific activity can be obtained in good yield. As described previously, the procedure can be used for simultaneous isolation of EcoRI endonuclease, which is resolved at the ammonium sulfate step (5). In contrast to previously described procedures (4, 7), this method does not utilize nonionic detergents which can complicate physical analysis.

Despite their ability to recognize a common hexanucleotide sequence, the physical and catalytic properties of the EcoRI endonuclease and methylase are quite distinct. The nuclease is composed of a subunit with a M, of 28,500 and exists in solution as dimers and tetramers (5). A monomeric form of this enzyme has not been detected even at concentrations as low as several nanograms/ml. The methylase, on the other hand, is composed of a single polypeptide chain and only the monomer has been detected in solution. The two enzymes are also distinguished by their isoelectric points. In contrast to the basic nature of the methylase, the endonuclease is an acidic protein with a p1 of approximately 6.3.’ In addition, although thiol groups are essential for catalytic activity in the methylase, the nuclease has no such requirement as judged by its insensitivity to N-ethylmaleimide.5 Finally, the enzymes do not appear to be immunologically related. High titer rabbit antiserum directed against the nuclease does not cross-react with the methylase as judged by Ouchterlony immunodiffusion tests and lack of inhibition of methyl trans- fer.”

Our kinetic studies indicate that the methylase can func- tion as a monomer and, in particular, are inconsistent with enzyme oligomerization as a prerequisite for catalytic activity. This conclusion is also consistent with the studies on mecha- nism of methylation which indicate that methyl transfer occurs to only one DNA strand of the EcoRI sequence prior to

3 The two EcoRI sites of pVH153 DNA are also attacked independ-. ently by the endonuclease. Under steady state conditions at 37” (5) with 2.7 nM pVH153 DNA and 0.14 nM endonuclease, singly cleaved molecules accumulated to concentrations 20 times that of the en- zyme.

4 R. A. Rubin and P. Modrich, unpublished experiments. 5 The endonuclease was unaffected bv incubation with 10 mM N-

” ethylmaleimide for 30 min at 15”. ’ EcoRI methylase activity was unaffected by incubation with 60

times as much- antiserum as required to completely inhibit an equivalent weight of endonuclease.

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7272 Physical and Catalytic Properties of EcoRI Methylase

enzyme dissociation. In contrast, under the dilute conditions employed for catalytic assay, the active form of the endonucle- ase is most likely the dimer (5). Furthermore, at physiological temperature the endonuclease cleaves both strands of the recognition sequence during a single binding event, although at lower temperatures the enzyme does dissociate after cleav- age of just one strand of the helix (5).

The simplest interpretation of these results is that the two enzymes interact with the EcoRI site in different ways. It has been proposed that the 2-fold symmetry characteristic of many DNA recognition sequences reflects corresponding symmetry in the proteins involved (27). While the structural and func- tional properties of the endonuclease are consistent with this hypothesis, those of the methylase are not, unless the poly- peptide chain comprising this enzyme contains regions of internal symmetry. In the absence of such symmetry, the interaction of methylase with its recognition site must be asymmetric. In this regard, it should be noted that while the biological substrate of the endonuclease is truly 2-fold sym- metric, the presumed in vim substrate for the methylase is modified on one strand and hence is inherently asymmetric.

Additional evidence indicating different modes of interac- tion for the two proteins with the EcoRI sequence is provided in the accompanying paper (28) where it is shown that although required for recognition by the methylase, the 2- amino group of guanine within the EcoRI site is not necessary for recognition and catalysis by the endonuclease.

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R A Rubin and P ModrichEcoRI methylase. Physical and catalytic properties of the homogeneous enzyme.

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