THE OF CHEMISTRY Vol. 257, No. 5, Issue of March 10. pp ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 5, Issue of March 10. pp. 2605-2612, 1982 Printed in U.S.A. Escherichia

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 5, Issue of March 10. pp. 2605-2612, 1982 Printed in U.S.A.

Escherichia coli dam Methylase PHYSICAL AND CATALYTIC PROPERTIES OF THE HOMOGENEOUS ENZYME*

(Received for publication, August 19, 1981)

Gail E. Herman4 and Paul Modrich8 From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

The Escherichia coli dam methylase has been purified 3000-fold to a purity of 95% from a clone which over- produces the enzyme 10- to 20-fold. Physical properties of enzyme purified from the overproducing clone were identical with those of enzyme previously obtained from a non-overproducing E. coli strain (Geier, G. E., and Modrich, P. (1979) J. BioL C h m . 254, 1408-1413). The methylase is comprised of a single polypeptide chain of M, = 31,000, has an s20,w of 2.8 S, a Stokes radius of 24 A, and exists in solution as a monomer. Its aggregation state is not affected by the presence of S- adenosyl-L-methionine.

The simple kinetic behavior of the methylase indi- cates that it functions as a monomer. Initial rates of methyl transfer are first order in enzyme concentra- tion, and Michaelis-Menten behavior is obeyed with respect to both substrates. At 37 “C, in the presence of saturating DNA, the enzyme has a turnover number of 19 methyl transfers/min with a K, for S-adenosyl-L- methionine of 12.2 p ~ . At half-saturating S-adenosyl-L- methionine, the apparent K,,, for d(G-A-T-C) sites in ColEl DNA is 3.0 m. The mechanism of methyl transfer is also consistent with the monomer being the func- tional form of the enzyme. Studies with G4 RFI DNA (two d(G-A-T-C) sites) indicate that the methylase transfers 1 methyl group to a recognition site and then dissociates from this DNA prior to subsequent cataly- sis. It appears that kinetic parameters for methyl trans- fer to sites already modified on one DNA strand may be slightly more favorable than those for transfer to sites in which both strands are unmethylated.

The dam methylase is responsible for the majority of meth- ylated adenine residues in Escherichia coli DNA (1, 2). The enzyme recognizes the symmetric tetranucleotide d(G-A-T-C) and introduces 2 methyl groups/duplex site, with the product of methylation being 6-methylaminopurine (3). Mutants of E. coli deficient in dam methylase activity in vitro, and contain- ing markedly reduced levels of 6-methylaminopurine in their DNA in uiuo, show a variety of phenotypic alterations. These include increased spontaneous mutability (2, 4-6), increased sensitivity to some chemical mutagens including the base analogs 2-aminopurine and 5-bromouracil (5), increased levels of genetic recombination (7, 8), and lethality in conjunction with non-lethal recA or recBC mutations (2, 4, 9). Two pos-

* This work was supported by Grant GM 23719 from the National Institues of Health and Grant PCM 7823036 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 “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Training Grant GM 07184. 9 Recipient of Development Award CA 00495 from the National

Cancer Institute.

sible functions of dam methylation have been suggested. Meselson et al. have provided evidence that methyl groups introduced by the enzyme function in strand discrimination during posreplication mismatch repair (10). Further, identifi- cation of 18 dam recognition sites within the E. coli origin of DNA replication (11,12) and conservation of 17 of these sites in the Salmonella typhimurium origin (13) has suggested a role for methylation of this enzyme in origin function or maintenance.

Although some information has accumulated concerning the biology of DNA methylation (14-17), little is known about structures and mechanisms of enzymes involved. To date, the only DNA methylases that have been obtained in pure form have been several modification enzymes which confer resist- ance to certain restriction endonucleases. These enzymes ap- pear to fall into two classes (16, 17). Type I DNA methylases, as typified by Eco B and Eco K enzymes,’ are large multi- subunit enzymes which have been isolated in two forms (17). Type I complexes comprised of polypeptide products of hsds and hsdm genes possess modification methylase activity while complexes also containing the hsdR product possess both modification and restriction activity (17,19-21). Methyl trans- fer to unmodified DNA by Type I enzymes requires only AdoMet; although this reaction is extraordinarily slow (19). The methyl transfer reaction is at least 70 times faster on DNA methylated on one strand of the duplex (hemimeth- ylated DNA), and this rate is enhanced another 10-fold by ATP and M$+ (21).

In contrast, Type I1 modification methylases are relatively simple with respect to both cofactor requirements and struc- ture. AdoMet is the only cofactor required for methyl transfer by Type I1 modifkation enzymes (16). Furthermore, the Eco RI and Hpa I1 enzymes, which have been obtained in pure form, have been shown to be comprised of single polypeptide chains and to function as the monomer (22,23). In the case of Eco RI methylase it has also been demonstrated that the enzyme transfers a single methyl group per DNA binding event and that methyl transfer to hemimethylated DNA is not more favorable than transfer to unmodified sites (22).

In view of the biological role of the dam methylase and in order to compare its mechanism of methyl transfer with those of enzymes discussed above, we have undertaken isolation and characterization of the dam methylase of E. coli K12. We have previously purified dam methylase from a cytosine meth- ylase-deficient (dcm-) strain of E. coli and have determined the recognition sequence of the enzyme in duplex DNA (3). However, analysis of the structure and mechanism of the protein was hampered by the extremely low levels of enzyme present in wild type E. coli. In order to obtain larger quantities

’ Restriction endonucleases and modification methylases are des-

’ The abbreviations used are: AdoMet, S-adenosyl-L-methionine: ignated as proposed by Smith and Nathans (18).

SDS, sodium dodecyl sulfate; KPO, potassium phosphate.

2605

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2606 E. coli dam Methylase

of enzyme, an overproducing strain has been constructed by transfer of the dum locus from a Clarke-Carbon plasmid (24) into pBR322 (25). The enzyme has been isolated from one overproducing strain and physical and catalytic properties of purified enzyme from overproducing and wild type strains of E. coli examined.

EXPERIMENTAL PROCEDURES

Materials Bacterial Strains and Bacteriophage-E. coli K12 strains SKI031

(F- mtl-1 xyl-7 argHl his-4 ilvD188 lacMS286 @3OdIIlacBK1 metE46 thi spcR tsx-3 SU- dam+) and SKt036 (as SK1031 but containing the dum-4 allele of E. B. Konrad (7)) were obtained from S. Kushner, University of Georgia, Athens. E. coli K12 strain GM31 (dcm-6) has been described (26). E. coli K12 strain RS5033 (Hfr Hayes metBl rel- 1 str-100 azi7 lac MS286 ~OdIIZucBK1 thi dam-4) was obtained from E. B. Konrad (7). E. coli K12 strain JC4583 (F- endA gal thi thy lac SUII) was obtained from Dr. A. J . Clarke, University of California, Berkeley. E. coli C strain CGHl was prepared by P1 vir transduction of the dam-4 allele from RS5033 into strain C-10 (27). Transductants were selected for streptomycin resistance (28) and screened for 2-aminopurine sensitivity (5). E. coli K12 strain JA200 (F+ AtrpE5 recA thr leu lac) containing a Clarke-Carbon hybrid plasmid pLC13-42 (24) was provided by J. Carbon, University of California, Santa Barbara. Bacteriophage G4 (29) was obtained from J . Scott, Stanford University, Stanford, CA.

Enzymes and Proteins-The dam methylase purified from the E. coli dcm strain GM31 (26) was the preparation previously described (3) and is referred to here as wild type enzyme. Dpn I endonuclease (3), Eco RI endonuclease (30), and T4 DNA ligase (31) were prepared as described. Bacterial alkaline phosphatase (Worthington BAPC) was further purified according to the method of Weiss et al. (32). Bum HI endonuclease, Sal I endonuclease, and SI nuclease were from Bethesda Research Laboratories and Pst I endonuclease from New England Biolabs. Egg white lysozyme, catalase, ovalbumin, and chy- motrypsinogen A were fromworthington. Horse heart myoglobin, bovine hemoglobin, horse heart cytochrome c, and yeast alcohol dehydrogenase were from Sigma. RNase A (Worthington) was heated for 10 min at 80 "C before use.

Nucleic Acids-Bacteriophage T7 DNA, T7 r3H]DNA (10,000 cpm/nmol of nucleotide), and unmethylated ColEl DNA (>95% covalently closed circles) were prepared as previously described (30). Bacteriophage G4 RFI r3'P]DNA (29) (>95% covalently closed circles, 2600 cpm/nmol of nucleotide) lacking the dam modification was isolated as above after infection of E. coli strain CGHl (33). Plasmid pBR322 (25) was obtained from D. Stafford, University of North Carolina, Chapel Hill. Plasmid pLC13-42 DNA was isolated as above.

OtherMaterial~-(~~P)Inorganic phosphate (carrier-free), [methyl- 3H]AdoMet (>IO Ci/mmol), and Aquasol 2 were from New England Nuclear. For kinetic analyses and G4 RFI methylation experiments, AdoMet was purified on prewashed Whatman No. 3MM paper using I-butanol/t.O N HCl/ethanol (505020) or I-butanol/acetic acid/wa- ter (601520) to >97% purity, and concentration determined by iso- tope dilution. E. coli tRNA (Boehringer Mannheim, phenol-ex- tracted) was used to prepare tRNA-agarose (3 mg of tRNA/ml of agarose) as described (34). N-Ethylmaleimide was from Sigma. Other materials have been described (3).

Methods Growth of Cells-T plates (28) (1.5%, w/v, agar) spread with 0.1

ml of a 1:10 dilution of crude colicin E l (35) were used to test for resistance to this bacteriocin. Selective T plates contained 10 p g / d of tetracycline (Sigma), 50 pg/ml of ampicillin (Wyeth), or 400 pg/ml of 2-aminopurine (Calbiochem). Liquid cultures of transformants were grown in L broth (28) without added glucose and contained 5-10 pg/ml of tetracycline. Cells to be utilized for enzyme purification were grown as described (3).

Recombinant DNA Methods-Restriction endonuclease hydrolysis of plasmid DNA with commercial enzymes was performed as recom- mended by the supplier, while Eco RI endonuclease reactions were as described previously (30). Plasmid pBR322 and pLC13-42 DNA's were each cleaved with restriction endonuclease Eco RI, Pst I, Bum HI, or Sal I, and reactions heated for 15 min at 75 "C. Restriction products were ligated at 10 "C overnight in reactions (204) containing 0.02 M Tris/HCl (pH 7.6), 0.5 mg/ml of bovine serum albumin, 66

pM ATP, 10 m~ dithiothreitol, 2 mM MgC12, 25 pg/ml of each DNA, and 5 units/ml of T4 DNA ligase (31). DNA was heated at 37 "C for 15 min and quick chilled on ice prior to addition of ligase. In some experiments, after Pst I cleavage, the vector pBR322 was treated with bacterial alkaline phosphatase (32, 36) for 60 min at 60 "C in the presence of 0.1 m~ ZnCb. Phosphatase-treated DNA was phenol- extracted and dialyzed versus three changes of 0.02 M Tris/HCl (pH 7.6), 0.05 M NaCl, 1 m~ EDTA prior to ligation.

Ligase reactions were used directly to transform a dam4 strain (SK1036), with selection for resistance to tetracycline or ampicillin (37). Transformants were screened for resistance to 2-aminopurine (51, and extracts of base analog-resistant clones were assayed for dam methylase activity (3). Plasmid DNA (pGG503) from one such clone was isolated and transformed into E. coli K12 hosts GM31 (dcm-6) and JC4583 (endA).

Enzyme Assays-The dam methylase assay has been described (3) and measures transfer of r3H]methyl groups from AdoMet to calf thymus DNA. One unit of dam methylase is defined as that which catalyzes conversion of 1 pmol of r3H]methyl groups into a form which binds to DE81 in 30 min at 37 "C. SI nuclease assays were performed as recommended by the supplier. Nonspecific nuclease assays (100 pl) contained 0.02 M Tris/HCl (pH 8.0), 0.1 M NaCl, 5 m~ MgC12,400 pg/ml of bovine serum albumin, 5 pg/ml of RNase (50 pg/ ml in crude extracts), and 20 pm of T7 r3H]DNA or 150 p~ pBR322 DNA. RNase A was included in nuclease assays to eliminate any inhibition by contaminating tRNA since the major nuclease contam- inant was endonuclease I (38). After incubation at 37 "C for 30 min, reactions with T7 DNA were terminated by addition of carrier DNA and trichloroacetic acid-soluble radioactivity determined (39). Reac- tions with pBR322 DNA were electrophoresed on 1% agarose gels using a Tris-borate buffer system (40) and visualized as described (30).

Purification of the dam Methylase-dam methylase was purified from the E. coli K12 strain GM31/pGG503. The method is a modif- cation of that used previously (3). A summary of the purification from 1.0 kg of cell paste is presented in Table I. AU steps were performed at 4 "C and centrifugation was at 12,000 X g for 10 to 30 min in a Sorvall GS3 rotor.

After thawing overnight at 4 "C, cells were suspended in 3 liters of 0.05 M Tris/HCl (pH 7.6), 10 m~ 2-mercaptoethanol, and disrupted in 250- to 300-ml portions with five I-min pulses at 85 to 95 watts using a Branson sonifier. Temperature was maintained below 7 "C. After clarification by centrifugation, the extract was diluted with the above buffer to yield an A ~ M ) of 280 (Fraction I, 4300 ml).

To fraction I were added 350 ml of a 5% (v/v) solution of Polymin P. After stirring for 15 min, the precipitate was removed by centrifu- gation and discarded. An additional 560 ml of Polymin P were then added to the supernatant, and after centrifugation, the pellet was extracted with 1600 ml of 0.02 M KPO4 (pH 7.4), 0.3 M KCl, 1 mM EDTA, 10 m~ 2-mercaptoethanol, 5% (w/v) glycerol. This eluate was clarified by centrifugation and diluted by addition of 3200 ml of 0.02 M KPO, (pH 7.4), 10 m~ 2-mercaptoethanol, 5% glycerol. Alumina Cy gel (350 ml, 4.7% solids) was added, the solution stirred for 20 min, and insoluble material collected by centrifugation. The pellet was extracted sequentially with 1600-ml portions of 0.2 M and 0.8 M KPO4 (pH 7.4), containing 10 m~ 2-mercaptoethanol and 5% glycerol, with activity eluting in the latter buffer (Fraction 11, 1600 ml).

Fraction I1 was dialyzed versus 2 changes of 25 liters of 0.02 M KPO, (pH 7.4), 0.12 M KC1, 1 m~ EDTA, 10 m~ 2-mercaptoethanol, 10% glycerol (2 h/change). After removal of insoluble material by centrifugation, the supernatant (1960 ml) was diluted with 680 ml of 0.02 M KPO, (pH 7.4), 1 m~ EDTA, 10 mM 2-mercaptoethanol, 10% glycerol, and applied at 750 ml/h to a phosphocellulose column (14 cm X 22 cm2) equilibrated with 0.02 M KPO4 (pH 7.4), 0.2 M KCl, 1 m~ EDTA, 10 m~ 2-mercaptoethanol, 5% glycerol. The column was washed with 600 ml of this buffer and eluted with a 3.0-liter linear gradient of KC1 (0.2 to 1.0 M) in 0.02 M KPO4 (pH 7.4), 1 mM EDTA, 10 mM 2-mercaptoethanol, 5% glycerol. Fractions containing dam methylase activity, which eluted at approximately 0.60 M KC1, were pooled (Fraction 111, 200 ml).

Fraction I11 was diluted with 440 ml of 0.02 M KPO4 (pH 7.4), 1 mM EDTA, 10 m~ 2-mercaptoethanol, 10% glycerol and applied at 150 ml/h to a blue dextran-Sepharose column (28 cm X 2.9 cm2) equili- brated with 0.02 M KPO, (pH 7.4), 0.25 M NaC1, 5 mM EDTA, 10 mM 2-mercaptoethanol, 10% glycerol. The column was washed with 2 liters of this buffer, and eluted with a 750-ml linear gradient of NaCl (0.25 to 1.1 M) in 0.02 M KPO, (pH 7.4), 5 m~ EDTA, 10 mM 2- mercaptoethanol, 10% glycerol. Active fractions, which eluted at

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approximately 0.69 M NaCI, were pooled (Fraction IV, 67 ml). Fraction IV was dialyzed uersus 2 liters of 0.02 M KPOJ (pH 7.4),

0.12 M NaCI. 5 mM EDTA, 10 mM 2-mercaptoethanol, 10% glycerol for 2 h (final volume, 74 ml), diluted by addition of 34 ml of this buffer without NaCI, and applied to a tRNA-agarose column (11 cm X 0.75 cm') at 25 ml/h. The column was washed with 10 ml of 0.02 M KP0.t (pH 7.4), 5 mM EDTA, 2.5 mM dithiothreitol, 10% glycerol containing 0.15 M NaCl (spectral grade glycerol was employed in this and subsequent steps), and enzyme eluted with this buffer containing 0.4 M NaCl (Fraction V, 11 ml). Fraction V was diluted with an equal volume of glycerol prechilled to 0 "C. and dithiothreitol added to 2.5 mM. The enqyme was stored at -20 "C and lost no detectable activity ((10%) in 12 months.

Gel Electrophoresis-SDS-polyacrylamide gel electrophoresis was performed by the method of Weber and Osborn (41). except samples were heated a t 100 "C for 20 min in the presence of 2% (w/v) dithiothreitol, 1% SDS, and dialyzed versus 10 mM NaP0, (pH 7.1). 0.1% SDS, 1% (w/v) dithiothreitol, 10% (w/v) glycerol prior to elec- trophoresis. Nonreduced samples were prepared in the same manner except dithiothreitol was omitted. Molecular weight standards were ovalbumin (M, = 44,000), Eco RI endonuclease ( M , = 31,000), chy- motrypsinogen (M, = 24,500), horse heart myoglobin (M, = 16,900). and lysozyme (M, = 14,300).

Isoelectric focusing under native conditions was performed for 24 h at 4 "C on 7.5% polyacrylamide gels containing 2% ampholytes (Bio- Rad Biolytes 3/10) as recommended by the manufacturer except that gels contained 2.5 mM dithiothreitol. Gels (10 cm) to be assayed were cut into approximately 50 slices of 0.21 cm which were soaked for 24 h at 0 "C in 0.2 ml of 0.02 M KPO, (pH 7.4). 0.2 M NaCI, 0.2 mg/ml of bovine serum albumin, 2 mM dithiothreitol, 1 mM EDTA, 10% (w/v) glycerol. For pH measurements, slices from parallel gels were incu- bated in 0.4 ml of degassed 0.1 M KC1 for 7 h at 4 "C and pH determined a t 4 "C. Gels to be stained were soaked in 10% isopropyl alcohol-lO% acetic acid for several days to remove ampholytes and stained for 30 min in 0.25% Coomassie brilliant blue.

Other Methods-Protein was estimated by the procedure of Lowry et al. (42). An of 200 a t 260 nm was assumed in calculation of DNA concentrations. A molecular weight of 3.7 X 10" for G4 RFI DNA was employed for determining molar yields (29). Other proce- dures have been described previously (3).

RESULTS

Cloning of the dam Methylase-Since the dam locus is 90% cotransdusible with trpS (26), a plasmid of the Clarke-Carbon collection (pLC13-42) (24) which bears trpS was tested for the presence of the dam locus. Strains bearing the dam-4 allele grow poorly on T plates containing 400 pg/ml of 2-aminopu- rine, while dam' strains grow normally (5). Introduction of pLC13-42 DNA into strain SK1036 (dam-4) rendered the strain resistant to 2-aminopurine indicating the presence of a wild type dam locus. C o n f i a t i o n of this conclusion was provided by in vitro assay of dam-mediated methyltransferase activity. SK1036/pLC13-42 had a specific activity of 45 units/ mg as compared with 0.3 unit/mg for the plasmid-free dam-4 strain.

In an attempt to obtain clones which overproduced the methylase, the dam locus was recloned into pBR322 using several restriction endonucleases. While 2-aminopurine-resist- ant clones were obtained after ligation of Eco RI or Pst I restriction products, only latter clones contained elevated in vitro levels of the enzyme. One such clone, SK1036/pGG503, overproduced the enzyme 10- to 20-fold relative to wild type (data not shown). Comparable levels of overproduction were found in the dcm strain GM31 (Table I)." Pst I endonuclease hydrolysis of pGG503 revealed that it contained two frag- ments: the vector pBR322 and the largest Pst I fragment in the Clarke-Carbon plasmid pLC13-42 (Fig. 1). Restriction

'' The elevation of in vitro dam activity is approximately 50-fold ir strain JC4588/pGG503 (43) which is a rec A host. In all othe backgrounds (strains JC4583, SK1036, GM31) elevations of 10- to 20 fold were found. Similar levels of overproduction were found usin either calf thymus or dam- E. coli DNA as substrates.

TABLE I Purification of dam methylase from I kg of E. coli K12 GM.31/

pGG503

Fraction and step Protein Specific activitv Recovery

mg units/mg protein I. Crude extract 78.000 286

11. Alumina Cy gel 1,500 12,100 82 111. Phosphocellulose 33.2 298.000 44 IV. Blue dextran-Seph- 8.6 580.000 23

V. tRNA agarose arose

3.0 900,000-1,000,000 12

FIG. 1. Cleavage of plasmid DNA with Pst I endonuclease. Samples were pLC13-42 (wells I and Z) , pGG.503 (wells 3 and 4) . and pBR322 (ujells 5 and 6). Within each set the left sample contained undigested DNA while the right sample was treated with Pst I endonuclease. Electrophoresis was on a 1% agarose gel using a Tris- borate buffer system (40) for 4 h at 100 V. DNA bands were visualized by staining for 1 h in 1 pg/ml of ethidium bromide. There are two additional Pst I fragments of pLC13-42 which have run off the gel shown. Material not entering the gel in well 1 was not observed in all preparations and may represent aggregated or nicked DNA.

enzyme hydrolysis with Eco RI and double digests with Pst I and Eco RI (not shown) were also consistent with incorpora- tion of the large Pst I fragment (M, = approximately 12 X lo6) from pLC13-42 into the pBR322 vector.

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2608 E. coli dam Methylase

Purification of the dam Methylase from a n Overproducing Clone-E. coli K12 dam methylase was purified from the dcm strain GM31 containing the hybrid plasmid pGG503 (“Meth- ods’’). In this background, the enzyme is overproduced ap- proximately 10- to 20-fold in crude extracts (Table I). Yields of methylase obtained form the overproducing clone were consistent with the degree of overproduction of enzyme (Table 11) indicating that the increased activity resulted from a physical overproduction in vivo.

The use of tRNA-agarose as the terminal step in purifica- tion (Table I) served to remove more than 99% of a contami- nating endonuclease which cofractionated with dam methyl- ase through much of the procedure, and reproducibility was improved over the previous method which used phenylalanyl- Sepharose for this purpose (3). Nevertheless, Fraction V did contain residual endonuclease activity. Although the prepa- ration did not detectably convert T7 [”HIDNA to an acid- soluble form, it did introduce single strand breaks into plasmid pBR322 DNA at a low rate (100 pmol min” mg”). This endonuclease activity was nonspecific and inhibited by tRNA suggesting that it was endonuclease I (38). This was confirmed by isolation of the methylase from JCt583/pGG503 (EndA dcm’). Although this preparation was contaminated at the 3% level by DNA cytosine methylase activity due to the dcm’ genotype of JC4583, it was free of contaminating endonuclease (<1.0 pmol min” mg”). Thus, the residual endonuclease is a contaminant and not associated with the dam methylase.

Polyacrylamide Gel Electrophoresis a n d Purity-The elec- trophoretic mobility of dam methylase on SDS-polyacryl- amide gels is dependent on the state of reduction of the enzyme. Conventional sample preparation of Fraction V from the overproducing clone (heating 20 min a t 70 “C in the presence of 2% 2-mercaptoethanol) produced varying ratios of two bands with mobilities of 0.59 and 0.65 on 7.5% gels (not shown). More vigorous reduction (boiling 20 min in the pres- ence of 2% dithiothreitol) resulted in a single protein zone with mobility of 0.59. Since the unreduced protein migrates with a mobility of 0.65 (not shown) the reduced mobility of the denatured protein observed only upon vigorous reduction indicates the presence of one or more intrachain disulfides which are resistant to reduction. The mobility of the com- pletely denatured and reduced enzyme was identical with that observed for methylase isolated from wild type E. coli (Fig. 2). Densitometer tracings of the SDS-polyacrylamide gels from the overproducing clone indicate a purity in excess of 95%. Mobilities relative to bromphenol blue were 0.79 and 0.59 on 5% and 7.5% SDS-polyacrylamide gels, respectively (not shown). When compared with mobilities of proteins of known molecular weight, these values yield an apparent M, of 31,000 k 1,000 (five measurements) for the reduced and de- natured enzyme.

Isoelectric focusing of Fraction V on 7.5% polyacrylamide gels under native conditions revealed a major protein zone

TABLE I1 Comparison of specific activities and yields of dam methylase from

E. coli K12 strains GM31 and GM31/pGG503 Protein concentration was estimated by the method of Lowry et

al. (42) as opposed to earlier determination based on A?YH (3). Minor differences in purification procedure for the two preparations are described in the text.

Extract, specific activity

Purified enzyme

Soecific activitv Yield units/mg units/mg mg/&

Wild type 29 9mOOO 0.34

Ovemroducer 286 1.OOO.OOO 3.0

which possessed a low level of dam methylase activity (Fig. 3). Although recovery of activity was only 0.04%, the major protein zone and activity peak coincided at an isoelectric pH of 5.1. Minor protein zones appeared a t pH 3.9 (8% of the

FIG. 2. SDS-polyacrylamide gel electrophoresis of darn methylase. Six pg of Fraction V wild type methylase (3) (left) or methylase from the overproducing strain (right) were subjected to electrophoresis on a 7.5% polyacrylamide gel containing SDS follow- ing denaturation and reduction (“Methods”). Gels were stained with Coomassie brilliant blue.

9 4 d 80

7 70 5 60

50 40 30

-

02 0.4 0.6 .I 1

08

60 5 0 40 30 20 IO

IO RELATIVE DISTANCE

FIG. 3. Isoelectric focusing of durn methylase on 7.5% poly- acrylamide gels. Fraction V (40 pg of protein in 0.2 ml of 50% glycerol (v/v)) was diluted with an equal volume of 4% ampholytes, 2.5 mM dithiothreitol and layered onto each of two gels (“Methods”). Protein solutions were overlaid with 2% ampholytes in 10% glycerol (v/v) and tubes fdled with 0.06 N H2.304. Parallel gels for pH meas- urements contained no enzyme. Electrophoresis was at 200 V for 24 h at 4 “C. Upper and lower reservoirs contained 0.06 N H2SO4, and 0.02 N Ca(OH)2 and 0.04 N NaOH, respectively. Gels were stained for protein (photographed and scanned on a Joyce-Loebl densitometer), or sliced for pH measurement and assay of darn methylase as de- scribed under “Methods.” Recovery of methylase activity was 0.04%. An arrow indicates the position taken as isoelectric pH.

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stainable protein) and pH 4.5 (2.5%). The former had dam methylase activity and may represent aggregated material not entering the gel. Due to consistently low recovery of activity upon electrophoresis under nondenaturing conditions, Frac- tion V sedimented through sucrose gradients (below) was analyzed on polyacrylamide gels in the presence of SDS. Only a single protein species of M, = 31,000 was observed across the profile of activity (not shown). This finding, together with the increased yield of the M, = 31,000 protein in the overpro- ducing clone (Table 11), indicate that Fraction V is near homogeneous with methylase activity being associated with the major protein species.

Native Molecular Weight Determination-Samples of pu- rified methylase from wild type E. coli and the overproducing clone were subjected to sucrose density gradient centrifuga- tion and gel filtration under native conditions(44,45). An ~ 2 0 , ~

and R, for each preparation were determined by comparison with known standards (Fig. 4). Both enzyme preparations had an S Z O , ~ of 2.8 S, and a Stokes radius of 24 A. Assuming a partial specific volume of 0.725, a calculated molecular weight for the preparations is 28,000 (44,45). Moreover, the presence of 50 p~ AdoMet during sedimentation did not alter the S Z O , ~

of methylase. Thus, the native enzyme is a monomer, and its aggregation state is not affected by the presence of AdoMet.

General Requirements of the dam Methylase Reaction- General catalytic requirements of the dam methylase are shown in Table 111. The enzyme, like the Eco RI and Hpa I1 modification methylases (22,23), does not require Mg2' being fully active in the presence of EDTA. The methylase has a narrow pH optimum and is inhibited at ionic strengths greater than 0.2 M. Furthermore, the enzyme methylates denatured

&a METMYLASE ACTIVITY

100 200 ,$00 BOTTOM

DROPS

10 I I I I I I I I 0.2 04 0.6 0.8

KO"

FIG. 4. Sedimentation coefficient and gel filtration of dam methylase. Upper, sucrose density gradient sedimentation was per- formed in 4.8 ml of sucrose gradients (IO to 30% w/v) containing 0.02 M KPOd (pH 7.4), 0.25 M NaC1, 1 m dithiothreitol, and 700-1,OOO units of methylase. Centrifugation was at 40,000 rpm at 4 "C for 24 h in a Beckman SW50.1 rotor. Sedimentation coefficients were deter- mined by the method of Martin and Ames (44) using bovine hemo- globin dimer (2.8 S), yeast alcohol dehydrogenase (7.4 S), and catalase (11.3 S ) as standards. Recovery of methylase activity was typically greater than 50%. Lower, Fraction V methylase (40-50 p g ) was sub- jected to gel filtration on Sephadex G-100 (Pharmacia) equilibrated with 0.02 M KPOd (pH 7.4), 0.25 M NaCI, 5 l ~ l ~ EDTA, 2.5 mM dithiothreitol, 10% glycerol (w/v). The void volume (VO) was deter- mined using blue dextran. V, is the total column volume and V, the elution volume of enzyme or marker protein. K,, is defined as (V, - Vo)/(V, - VO) (45). Marker proteins were bovine serum albumin (R, = 35.5 A), ovalbumin (R. = 27 A), and horse heart cytochrome c (R, = 17 A). Recovery of methylase activity was typically greater than 50%. 0, position taken as R, of wild type methylase; A, position taken as R. of methylase from overproducing clone.

TABLE I11 Requirements of dam methylase for optimal activity

Methylase assays were performed for 10 min at 37 "C with calf thymus DNA (3) except for activity on native and denatured DNA which utilized T7 DNA. Aliquots of denatured T7 DNA were deter- mined to be single-stranded after removal from the methylase assay by their sensitivity to S1 nuclease (>95%). N-Ethylmaleimide sensi- tivity was determined by pretreating enzyme at 8 "C with 10 mM N- ethylmaleimide for various times (30 s to 10 min). Preincubated enzyme samples were quenched by dilution for assay into buffer containing excess dithiothreitol. Control samples were preincubated with 2 rn dithiothreitol.

Native T7 DNA Rate of methylation 100 Extent of methylation 100

Rate of methylation 14 Extent of methylation 60-70

0.02 M Tris (pH 8.0) 100 + 0.05 M NaCl 95 + 0.1 M NaCl 96 + 0.2 M NaCl 35 + 0.3 M NaCl 5 + 0.5 M NaCl 0.5

0.1 M Tris (pH 8.0) 100

pH 7.0 68 pH 7.6 100 pH 8.0 100 pH 8.7 58

Pretreatment with dithiothreitol 100 N-Ethylmaleimide (30 s) <1

Denatured T7 DNA

Ionic strength optima

pH optima, 0.1 M Tris

N-Ethylmaleimide sensitivity

DNA, albeit at a substantially reduced rate and to a lower extent than native DNA. However, we have not determined whether methylation occurred at single-stranded d(G-A-T-C) sequences or within regions of intramolecular secondary struc- tures that may exist in such molecules. The enzyme appears to have one or more essential cysteine residues since activity is abolished by N-ethylmaleimide. A similar requirement has been observed in the case of Eco RI methylase (22).

Catalytic Properties of the dam Methylase-ColEl DNA which contains approximately 15 dam recognition sites (3) was used as substrate for steady state kinetic experiments. Initial rates of methylation were fiit order with respect to enzyme concentration up to 6 I ~ M (not shown), and the enzyme obeyed Michaelis-Menten kinetics with respect to both AdoMet and DNA (Fig. 5). Since the protein is a monomer at concentrations 30-fold higher than those used in these exper- iments, this simple kinetic behavior indicates that the mono- mer is catalytically active.

In the presence of saturating DNA, the K,,, for AdoMet was determined to be 12.2 tm (Fig. 5, upper). Since this experiment was performed in the presence of a saturating concentration of DNA, it permits calculation of a true catalytic constant for the enzyme. The kcat determined from these measurements is 19 methyl transfers/min/monomer at 37 "C. Because of the high K, for AdoMet, experiments performed at concentrations of AdoMet greater than the K,,, resulted in loss of assay sensitivity due to high background. Thus, an apparent K,,, for a dam recognition site in ColEl DNA was determined to be 3.6 nM at an AdoMet concentration of 13.4 PM (Fig. 5, lower). As shown below, the dam enzyme transfers a single methyl group to a DNA recognition site during a binding event. Since these steady state kinetic experiments measured initial rates of methylation, the K, values determined are those for addi- tion of the fiist methyl group to an unmodified site. Moreover, since the ColEl substrate contains approximately 15 dam sites, it is possible that some sites on the DNA molecule are

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2610 E. coli dam Methylase

- 01 0.1 02 0.3 0.4 [Ado Met]-’ pM”

p $ 10.0 6.0

’40 o 2.

-0 2 0.2 04 0.6 08 [dom SITES]” nM”

FIG. 5. Steady state kinetics of h methylase. Upper, deter- mination of K,,, for AdoMet. Reactions (0.05 ml) contained 0.1 M Tris/ HC1 (pH 8.0), 10 rn EDTA, 400 pg/ml of bovine serum albumin, 2.5 rn dithiothreitol, 10 IIM ColEl DNA (150 nM in dam recognition sites), 700 PM dam methylase, and indicated concentrations of AdoMet. Incubation was at 37 “C for 8 min. Lower, determination of apparent K, for dam recognition sites in CoEl DNA. Reactions were as above except that they contained 13.4 PM AdoMet, 140 PM meth- ylase, and indicated concentrations of DNA (in terms of dam recog- nition sites). DE81 filters for determination of a K, for ColEl DNA were prewashed in 0.5 m~ unlabeled AdoMet before use in assays to decrease nonspecific absorption of [3H]AdoMet. All data were ana- lyzed using a weighted regression (46).

better substrates than others. If this is in fact the case, the K , determined for DNA would reflect methylation of such sites.

Mechanism of Methylation-Since modification of a dam recognition site involves addition of 2 methyl groups, several mechanisms can be postulated. First, the enzyme could trans- fer 1 or 2 methyl groups to a site during a single binding event. Further, it could dissociate from the DNA molecule subse- quent to methylation or remain bound, diffusing along the DNA helix to additional sites.

By analyzing resistance of partially methylated DNA sub- strates to Eco RI endonuclease cleavage, it has been previ- ously established that the Eco RI methylase transfers methyl groups to DNA one at a time with dissociation from the DNA molecule being a prerequisite for subsequent turnover (22). We have utilized a similar approach to investigate the mech- anism of dam methylation by analyzing susceptibility of the two dam sites in G4 RFI DNA (29) to Dpn I endonuclease cleavage. This endonuclease will cleave at d(G-A-T-C) se- quences provided that the internal adenine is methylated on both DNA strands (47). Unmethylated DNA or DNA meth- ylated on only one strand of the duplex is resistant to cleavage. Thus, if the dam enzyme transfers 2 methyl groups at a time to a recognition site, each DNA binding event should produce a Dpn I-susceptible site. However, if the enzyme transfers only 1 methyl group to a recognition site/binding event, then at low extents of methylation under steady state conditions, no sites should contain 2 methyl groups and hence, no sites should be sensitive to Dpn I endonuclease.

As shown in Fig. 6, G4 RFI DNA was methylated under steady state conditions and average extent of methyl transfer determined. Partially methylated samples were isolated and

” IO 2 0 30 40

E Zb z e B 01 AVERAGE 10 PER MOLECULE METHYL 20 GROUPS 30 40

IO 0.9

C

4 0.8 5 07 8 06 z 05

0 4 2 03 f 02

01

10 20 30 4 0

D

~r

10 20 30 40 AVERAGE METHYL GROUPS

PER MOLECULE

FIG. 6. Mechanism of methylation of G4 RFI DNA by the dam methylase. A, susceptibility of G4 RFI DNA to Dpn I endo- nuclease as a function of average extent of methylation. Unmethyl- ated G4 [32P]DNA (11.2 IIM in dam recognition sites) was methylated as in Fig. 5 at 37 “C using 3.6 p~ [3H]AdoMet and 700 PM methylase (0.25 ml final volume). After 20 min, additional methylase (to 2.0 nM final concentration) was added to ensure complete methylation of molecules. Samples (0.005 ml) were removed and average extent of methyl transfer determined. The reaction achieved completion at an an average extent of transfer of 3.92 methyl groups/molecule (based on the ratio of 3H/32P), which was normalized to 4.00. Additional samples (0.02 m l ) were added to 0.03 ml of 0.02 M Tris/HCl (pH 7.6), 0.05 M NaC1, 1 m~ EDTA, and immediately extracted twice with phenol, twice with buffer-saturated ether, and precipitated three times with absolute ethanol. Recovery of DNA was typically greater than 80%. Samples were dissolved in 0.02 ml of 0.05 M Tris/HCl (pH 7.6), 5 m~ MgC12,0.05 M NaC1, and digested with Dpn I endonuclease (520 units/ml) for 17 h at 37 “C. Cleavage products were separated on 1% agarose gels containing 1.5 p g / d of ethidium bromide. Electro- phoresis was for 20 h at 75 V in the presence of unlabeled G4 standards. Fluorescent bands were excised, solubilized in 1 ml of 1.0 N HC1 at 70 “C for 1 h, and 32P and 3H determined in Aquasol 2. Percentage of resistant DNA was calculated as C + L/2 where C and L are the mole percent of circular and full length linear DNA. Percentage of susceptible DNA was calculated as L/2 + F1 + F2 where F1 and F2 are the two fragments of M, = 2.1 X IO6 and 1.6 X IO6, respectively, produced by Dpn I cleavage of G4 DNA methylated on both strands of the duplex by the dam enzyme (29). A background of 2% susceptible DNA in an unmethylated control was subtracted from all values. The theoretical curve, assuming random addition of 1 methyl group/DNA binding event, was calculated using the bino- mial distribution ( n = 4) (48). B, biphasic decay of Dpn I endonucle- ase-resistant molecules. Molecules completely resistant to Dpn I endonuclease (“zero site,” see Fig. C ) contain 0 or 1 methyl group/ dam recognition site. The mole percent of such Dpn I-resistant molecules is plotted on a semilog scale uersus the average extent of methylation. C, mole fraction of 0-, 1-, and 2-site fully methylated molecules as a function of average extent of methylation. ZERO, 0- site circular molecules (C) are resistant to Dpn I endonuclease. They can contain 0 or 1 methyl group/site but no fully methylated sites. ONE, 1-site linear molecules (L) can contain 2 or 3 methyl groups/ molecule with only 1 site being methylated on both strands. TWO, 2- site molecules are those sensitive to Dpn I cleavage at both recognition sites. This class, in which both sites must be methylated on both strands, was estimated as the sum of label in fragments of F1 and F2. D, average extent of methylation of linear molecules as a function of average total extent of methylation. The average extent of methyla- tion of linear molecules was determined by the 3H/32P ratio in the full length linear component observed after Dpn I hydrolysis.

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E. coli dam Methylase 2611

subjected to Dpn I endonuclease digestion. At average extents of methylation of less than 0.4 methyl group/molecule, the susceptibility curve closely paralleled that expected for trans- fer of 1 methyl group/binding event. The observed initial slope was approximately 4% as compared with expected slopes of 0% for addition of 1 methyl group/binding event and 25% for addition of 2 methyl groups/binding event: This conclu- sion is further substantiated by noting that the biphasic decay curve of Dpn I-resistant “zero site” molecules (Fig. 6 0 ex- trapolates to 2 when plotted in the the semilog form (Fig. 6B). This indicates that two independent methylation events are required to produce one Dpn I-susceptible site.

It is also apparent that at average extents of methylation in excess of 0.8 methyl group/molecule, results deviate from the theoretical curve calculated from the binomial distribution (a), for completely random addition of 1 methyl group/bind- ing event. Although the results cited above indicate that addition of the first and second methyl groups to a recognition site is mediated by different enzyme molecules, the deviation from the binomial prediction indicates weak coupling between the two events. This behavior is readily explained if there is a kinetic preference of the enzyme for methyl transfer to a site already methylated on one DNA strand. However, given the magnitude of the deviation, this preference cannot be large.

The above results demonstrated that an enzyme molecule transfers a single methyl group to a recognition site during a binding event. It was possible, however, that subsequent to methyl transfer, the methylase would diffuse along the DNA to additional sites rather than dissociate from the DNA mol- ecule. Thus, the independence of methylation of the two dam sites in G4 RFI DNA was investigated. If the dam enzyme transferred 1 methyl group to a site and then diffused along the same DNA molecule to another site, one would not expect accumulation of molecules sensitive to Dpn I cleavage at just one of the two d(G-A-T-C) sites. As shown in Fig. 6C, this species did accumulate in significant excess over added meth- ylase. In addition, by analyzing amounts of methyL3H label associated with linear molecules, an average of 2.15 methyl groups/linear molecule was found when the total average extent of methylation varied from 0.2 to 2.3 methyl groups/ molecule (Fig. If the majority of enzyme diffused along the helix methylating a second site after transfer of a methyl group to a first site, one would expect an average of 3 methyl groups/linear molecule. This expectation was realized only at average extents of methylation in excess of 2.3. Since the methylase turned over at least 5 or 6 times during this period, the majority of the enzyme must have dissociated from the DNA subsequent to each methyl transfer event. Thus, the two dam sites within G4 RFI molecule behave independently with respect to methylation.

It is apparent, however, the shortest distance between the two sites on G4 DNA (2455 base pairs) (29) is much larger than the average distance of 256 base pairs expected to sepa- rate d(G-A-T-C) sites in a random sequence. While the data reported here cannot exclude coupling of methyl transfer to sites separated by several hundred base pairs, it is evident that methylation events on the two strands of a given site are mediated by different enzyme molecules.

The observed initial slope of the experimental curve was taken as the average slope over the range of addition of 0 to 0.4 methyl group/ molecule. “he corresponding average slope of the theoretical curve for addition of 1 methyl group over this range is 2.5%/methyl group, which is in excellent agreement with the observed value.

Essentially all of the 3H label associated with DNA remains bound to the DNA after Dpn I endonuclease treatment (3) and, thus, the average methyl content measured represents the true value.

DISCUSSION

Purification of the dam methylase has previously been hampered by the low level of enzyme in the cell and the presence of contaminating DNA cytosine (dcm) activity. A recombinant plasmid resulting in a 10- to 50-fold elevation of in vitro dam activity has been obtained by transfer of the dam locus into the Pst I site of pBR322. Introduction of this plasmid into a dcm- strain of E. coli has enabled purification of milligram quantities of near homogeneous dam methylase free of contaminating methylase activities.

Physically the dam methylase is similar to the Type 11 methylases of Eco RI and Hpa I1 specificities (22, 23). All three enzymes are small monomeric proteins which function as such. Furthermore, both Eco RI (22) and dam methylases transfer a single methyl group to a recognition site per DNA binding event, findings consistent with assignment of func- tionality to the monomeric state. However, in contrast to the Eco RI enzyme, the dam methylase exhibits a slight prefer- ence for hemimethylated sites. There are other differences between the two proteins as well. The Eco RI methylase is a basic protein while the dam methylase is slightly acidic. Further, the dam methylase will transfer methyl groups to denatured DNA, although at a greatly reduced rate and to a lesser extent than to native DNA. Although it is possible that the dam enzyme may be acting at transient duplex regions within the DNA (49), the Eco RI methylase is not active under similar conditions (16).

Available evidence thus indicates that Eco RI, Hpa 11, and dam methyltransferases each interact in the monomeric state with their individual recognition sites. Barring regions of internal symmetry within the proteins, each of these enzymes must interact with its 2-fold symmetric recognition sequence in an asymmetric manner. This is not necessarily surprising since the presumed in vivo substrate for each enzyme is a hemimethylated duplex site which would possess inherent asymmetry.

Although the dam methylase is similar to Type I1 modifi- cation methylases with respect to structure and mechanism, it does not appear to function as an element of a restriction- modification system. Rather, compelling evidence has accu- mulated implicating the dam enzyme in postreplication mis- match repair (4-6, 10, 43) and perhaps in maintenance or function of the E. coli origin of DNA replication (11-13). It is possible that availability of the cloned dam locus and the purified protein will facilitate further analysis of the role of the enzyme in such processes.

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G E Herman and P Modrichhomogeneous enzyme.

Escherichia coli dam methylase. Physical and catalytic properties of the

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