©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phage T4 DNA N-adenineMethyltransferase
OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION (*)

Valeri G. Kossykh (§) , Samuel L. Schlagman , Stanley Hattman

From the (1)Department of Biology, University of Rochester, Rochester, New York 14627

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The bacteriophage T4 dam gene, encoding the Dam DNA [N-adenine]methyltransferase (MTase), has been subcloned into the plasmid expression vector, pJW2. In this construct, designated pINT4dam, transcription is from the regulatable phage p and p promoters, arranged in tandem. A two-step purification scheme using DEAE-cellulose and phosphocellulose columns in series, followed by hydroxyapatite chromatography, was developed to purify the enzyme to near homogeneity. The yield of purified protein was 2 mg/g of cell paste. The MTase has an s of 3.0 S and a Stokes radius of 23 Å and exists in solution as a monomer.

The K for the methyl donor, S-adenosylmethionine, is 0.1 10M, and the K for substrate nonglucosylated, unmethylated T4 gtdam DNA is 1.1 10M. The products of DNA methylation, S-adenosyl-L-homocysteine and methylated DNA, are competitive inhibitors of the reaction; K values of 2.4 10M and 4.6 10M, respectively, were observed. T4 Dam methylates the palindromic tetranucleotide, GATC, designated the canonical sequence. However, at high MTase:DNA ratios, T4 Dam can methylate some noncanonical sequences belonging to GAY (where Y represents cytosine or thymine).


INTRODUCTION

DNA methyltransferases (MTases)()are ubiquitous in living cells and present an interesting example of sequence-specific DNA-protein interaction. DNA MTases recognize specific sequences in DNA and transfer methyl groups from the methyl donor, AdoMet, to adenine or cytosine residues in the recognition sequence. Three kinds of methylation product are known, N-methyladenine, N-methylcytosine, and 5-methylcytosine. Most of the known DNA MTases are part of restriction-modification systems, but there are MTases that exist in the cell without a corresponding cognate endonuclease(1) . Bacteriophage T4 encodes a DNA [N-adenine]MTase (T4 Dam) that methylates adenine in the sequence GATC in cytosine, 5-methylcytosine, or 5-hydroxymethylcytosine-containing DNA(2) . At a much lower efficiency, T4 Dam also recognizes some noncanonical sites in sequences derived from GAY (where Y represents cytosine or thymine)(3) . The dam gene, which encodes T4 Dam, was cloned, and its nucleotide sequence was determined(4, 5) . We have been especially interested in defining the region(s) of T4 Dam responsible for sequence-specific DNA recognition as well as the functional roles of conserved motifs present in all DNA [N-adenine]MTases(6) . In this paper, we describe the overexpression, purification, and characterization of kinetic parameters for T4 Dam.


EXPERIMENTAL PROCEDURES

Materials

[methyl-H]AdoMet was purchased from DuPont NEN. Unlabeled AdoMet (Sigma) was purified by high pressure liquid chromatography on a C18 reversed-phase column. The concentration was calculated using a molar extinction coefficient at 256 nm of 14,700 (in acid)(7) . AdoHcy, DTT, and N-ethylmaleimide (Sigma); DE81 filters, phosphocellulose (P11) and DEAE-cellulose (DE-52) (Whatman), hydroxyapatite (Bio-Rad), Sephacryl S-100 (Pharmacia Biotech Inc.), restriction enzymes and DNA ligase (New England Biolabs), and Nonidet P40 (BDH Chemicals) were purchased from the companies in parentheses.

Bacterial Growth

Escherichia coli GM 2971 (Fmrrhsd S20 rm- ara14 proA2 lacY1 galK2 rspL20 (str) xyl5 mtl1 supE44 dam13::Tn9 Cm) was from Dr. M. G. Marinus, Department of Pharmacology, University of Massachusetts Medical School, Worcester. Plasmid-containing cells were grown in LB-ampicillin broth (8) at 30 °C to an A = 0.8-1.0. The temperature was raised to 42 °C, and incubation continued for 3 h. The cells were collected by low speed centrifugation and stored frozen at -20 °C.

DNA MTase Assay

Methyl transfer assays were carried out by a DE81 ion exchange filter assay(9) . Assay mixtures (50 µl) contained 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, and 200 µg/ml bovine serum albumin. Substrate DNA and [methyl-H] AdoMet concentrations were as noted in each experiment.

Purification of T4 Dam

Frozen cells (18 g) were suspended in 60 ml of PEM buffer (20 mM KPO, 1 mM EDTA, 7 mM 2-mercaptoethanol) with 0.1 mM phenylmethylsulfonyl fluoride, 0.1% of Nonidet P-40, and 0.4 M NaCl. Lysozyme was added to a concentration of 500 µg/ml and, after a 1-h incubation at 4 °C, the cells were disrupted by sonication. Insoluble debris was removed by centrifugation at 100,000 g for 1 h. The supernatant was diluted 2-fold in PEM buffer and passed through a DEAE-cellulose (DE52) column connected to a 1.5 10-cm P11 phosphocellulose column (equilibrated in buffer PEM with 0.2 M NaCl). Proteins bound to the P11 phosphocellulose were eluted in a 200-ml, 200-800 mM NaCl gradient in PEM buffer. DNA MTase activity was monitored as described above; appropriate fractions were pooled, diluted 2-fold with buffer PEM, and applied to a 1 4-cm hydroxyapatite column (equilibrated in PEM buffer with 0.2 M NaCl). Proteins bound to the matrix were eluted with a 50-ml gradient of 20-500 mM potassium phosphate with 1 mM EDTA, 7 mM 2-mercaptoethanol, and 0.2 M NaCl, pH 7.4. Active fractions were concentrated on a 1.0-ml phosphocellulose column. The enzyme was dialyzed against PEM with 0.1 M NaCl and 50% glycerol and stored at -20 °C. Protein was determined by the method of Bradford(10) .

Gel Filtration

Purified T4 Dam MTase was subjected to gel filtration on Sephacryl S-100 in PEM buffer with 0.1 M NaCl. The void volume was determined with blue dextran. Marker proteins bovine serum albumin (R = 37.0 Å), ovalbumin (R = 27.6Å), bovine carbonic anhydrase (R = 24.3Å), and lysozyme (R = 20.6Å) were used to calibrate the column.

Glycerol Gradient Sedimentation

Centrifugation was performed in 12.5-ml glycerol gradients (10-30%, w/v) containing PEM buffer with 0.1 M NaCl and 50-100 µg of T4 Dam MTase. Centrifugation was for 48 h at 40,000 rpm at 4 °C in a Beckman SW41 rotor. Sedimentation coefficients were determined using bovine serum albumin (4.31 S), ovalbumin (3.55 S), bovine carbonic anhydrase (2.85 S), and bovine heart cytochrome c (1.83 S) as standards.

Steady State Studies

Apparent K (DNA), K (AdoMet), and k values were determined by monitoring the initial velocities of [H]methyl transfer from labeled AdoMet to nonglucosylated, unmethylated T4 gtdam DNA. Kinetic parameters were obtained using the EnzymeKinetics program from Trinity Software.

Gel Electrophoresis

SDS-PAGE was carried out according to Laemmli(11) , and agarose gel electrophoresis was performed in Tris acetate buffer(12) .


RESULTS

Construction of a T4 Dam-overproducing Plasmid

Fig. 1shows the overall scheme for cloning the T4 dam gene into the expression vector, pJW2(13) . As a source of this gene, we used the plasmid pSSH10, which contains T4 dam on a 1.6-kilobase fragment between the HindIII and BamHI sites in pBR322(14) . We eliminated the NdeI site in the T4 dam fragment and created a SmaI site in its place. The SmaI-BamHI fragment was cloned into pGC7 (a derivative of pGC1 (15) with a polylinker). This construct was then used to create an NdeI site at the ATG start of the gene (by oligonucleotide site-directed mutagenesis). Finally, the NdeI-BamHI fragment was excised and cloned into the vector pJW2. The resulting plasmid, pINT4dam, is shown in Fig. 1.


Figure 1: Construction of plasmid pINT4dam for overexpression of T4 Dam. Expression of the T4dam gene is under control of the tandemly arranged phage promoters, p and p. bla, -lactamase gene; ori, colE1 origin of replication; cI, phage gene encoding a thermolabile repressor; T7 RBS, translation initiation region of the T7 phage gene 10; and fdtt, fd transcription terminator.



T4 Dam MTase induction and overproduction is illustrated in Fig. 2. SDS-PAGE analysis revealed a strongly enhanced band at the position expected for T4 Dam (molecular mass = 30.4 kDa) (Fig. 2). The maximum amount of T4 Dam protein was observed after 4 h and remained constant at this level for at least another 4 h (Fig. 3). In contrast, the amount of soluble T4 Dam MTase activity reached a maximum at 3 h and then declined slowly thereafter.


Figure 2: Overexpression of T4 Dam. E. coli cells carrying a T4 Dam-overproducing plasmid were grown as described under ``Experimental Procedures.'' An equivalent number of cells were removed at various time intervals and lysed, and proteins were analyzed by SDS-PAGE (12.5%). The time (hrs. after induction) after induction is shown above each lane. Lanes denoted by MW each contained molecular mass protein standards (given in kDa); the lane denoted as T4 Dam contained purified enzyme.




Figure 3: Kinetics of T4 Dam overproduction. E. coli cells growing at 30 °C were shifted up to 42 °C. At various time intervals, aliquots were removed to determine optical density at 600 nm and MTase activity. Cells were lysed by sonication, and insoluble debris was removed by centrifugation. The supernatants were assayed for MTase activity as described under ``Experimental Procedures.'' Circles, MTase activity; squares, A.



Purification of T4 Dam

A two-step chromatography procedure (see ``Experimental Procedures'') provided an excellent means to obtain highly purified T4 Dam MTase (Fig. 4). SDS-PAGE analysis of fractions from the hydroxyapatite column revealed a single 30-kDa band corresponding to the DNA MTase activity. A summary of purification from 18 g of E. coli cell paste is presented in .


Figure 4: Purification of T4 Dam. Samples collected at each purification step were analyzed by 15% SDS-PAGE as described under ``Experimental Procedures.'' Lane1, crude extract; lane2, supernatant after centrifugation at 100,000 g; lane3, pool fraction of P11 phosphocellulose column; lane4, pool fractions of hydroxyapatite column; lane5, pellet after high speed centrifugation of sonicated cells; lanesMW, protein standards (in kDa).



Molecular Weight Determination

Purified T4 Dam MTase was subjected to glycerol gradient centrifugation and gel filtration under native conditions. It had an s of 3.0 S and a Stokes radius of 23 Å. We calculated a molecular weight of 30,690, using a partial specific volume of 0.7445(16, 17) . The apparent molecular mass of T4 Dam was 30 kDa, as determined by SDS-PAGE. This is in agreement with the value of 30.4 kDa from the deduced amino acid sequence. These results suggest that T4 Dam exists as a monomer in solution.

Requirements for T4 Dam Methylation

T4 Dam, like other DNA MTases, does not require Mg, and it is fully active in the presence of 10 mM EDTA. T4 Dam has a broad pH optimum (7.0-8.5) and is inhibited at ionic strengths greater than 0.2 M. At 30 °C with low enzyme:DNA ratios, T4 Dam maintained a constant transfer rate of labeled methyl groups from AdoMet to DNA over a period of at least 3 h (data not shown). The enzyme has one or two essential cysteine residues, since activity was abolished by N-ethylmaleimide.

Steady State Kinetics

Steady state kinetic parameters of T4 Dam methylation were studied using T4gtdam DNA, which is a natural substrate for the enzyme (Fig. 5). Initial rates of methylation were first-order with respect to enzyme concentrations up to 5 nM, and Michaelis-Menten kinetics were obeyed with respect to both AdoMet and DNA. The K values were 0.1 µM for AdoMet and 1.1 pM for T4 DNA. The V was calculated to be 270 nmolminmg enzyme, and the k was calculated to be 0.14 s. The k/K for T4 gtdam DNA was 0.13 10M s, and for AdoMet it was 1.4 10M s. Methylated DNA was a competitive inhibitor of MTase activity with an inhibition constant, K, of 4.6 pM (Fig. 5B). As with other DNA MTases, product (AdoHcy) generated by the methylation reaction was a competitive inhibitor (with respect to AdoMet); the K was 2.4 µM (Fig. 5A).


Figure 5: Steady state kinetics of T4 Dam MTase. The apparent steady state kinetic parameters were obtained at 37 °C with 0.36-10.0 pM T4 gtdam DNA, 0.05-1.32 µM AdoMet in 100 mM Tris, pH 8.0, 1 mM EDTA, 200 µg/ml bovine serum albumin, 1 mM DTT, 0.3-0.6 nM MTase, and varying concentrations of AdoHcy (A) or methylated DNA (B). Methylated DNA was prepared by extensive methylation of T4gtdam DNA by T4 Dam MTase using unlabeled AdoMet; after deproteinization, the DNA no longer served as methyl group acceptor with T4 Dam MTase and [methyl-H]AdoMet.



In Vitro Methylation of Noncanonical Sites

Phage and pUC18 DNAs were methylated by T4 Dam in vitro and then analyzed by digestion with a set of methylation-sensitive restriction nucleases, viz.ClaI, EcoRV, HinfI, FokI, and HphI, all of which recognize a sequence overlapping GATC, the canonical T4 Dam-methylation site. Previous studies had shown that T4 Dam can methylate noncanonical sites in vivo, provided that high concentrations of enzyme were present(3) . As seen in Fig. 6, in vitro T4 Dam methylation protected against cleavage by ClaI (ATCGAT), EcoRV (GATATC), HinfI (GATT, GACT), and FokI (GATG) but not against HphI (GAA and GAG sites). These results demonstrate that in vitro T4 Dam can methylate some subset of the noncanonical sequence, GAY (where Y represents cytosine or thymine).


Figure 6: Methylation of noncanonical sites in heterologous DNAs (phage (A) and plasmid pUC18 (B). Methylation was performed under standard assay conditions using unlabeled AdoMet as methyl donor. Following incubation (for 1 h) with increasing concentrations of T4 Dam (from 0 to 1 µg/µg of substrate DNA, lanes 1-8 (A); from 2 pg to 1 µg/µg of substrate DNA, lanes 1-7 (B)), the DNA was extracted with phenol/chloroform and precipitated in ethanol. Methylated DNAs were resuspended in appropriate buffers, digested with endonuclease ClaI (ATCGAT), EcoRV (GATATC), FokI (GATG), or HinfI (GATT, GACT), and then subjected to agarose gel electrophoresis. Undigested phage DNA and HaeIII-digested phage X174 DNA were run as molecular weight markers.




DISCUSSION

We have constructed a plasmid that overexpresses the T4 Dam DNA MTase in a regulatable fashion. This was accomplished by using an expression vector, pJW2(13) , containing the strong phage promoters, p and p, arranged in tandem, to drive transcription following heat inactivation of the thermolabile cI repressor; in addition, the ribosome binding site of the phage T7 gene 10 was present to enhance translation initiation. Following induction, T4 Dam constituted more than 10% of total cellular protein, but a major portion of the enzyme sedimented as inclusion bodies along with insoluble cellular debris. This situation is similar to that observed with several other MTases, including M.MspI(18) , M.HhaI(19) , and M.EcoRV(20) ; in contrast, a number of MTases have been overproduced in soluble form, such as M.HhaI(21) , M.HhaII(22) , M.EcoRI(23) , and M.RsrI (24). The formation of inclusion bodies is often dependent upon the overexpression system and growth conditions. The fraction of soluble T4 Dam reached a maximum by 3 h postinduction, and then slowly declined. We describe a two-step purification procedure, which yielded T4 Dam in apparently homogeneous form. With this method, we were able to recover active T4 Dam MTase corresponding to about 3% of the total cellular soluble protein.

T4 Dam, at a molecular weight of 30.4 kDa, is one of the smallest MTases that exists as monomer in solution, such as M.EcoDam (32 kDa)(25) , M.EcoRI (39 kDa)(8) , and M.HhaI (37 kDa)(21) . Most characterized MTases exist as monomers in solution (26), although recently it has been reported that M.RsrI shows partial dimerization(24) ; and two MTases from Streptococcus pneumoniae appear to exist as dimers(27) . In the case of M.MspI, the enzyme appears to dimerize at high protein concentrations, which may reflect a tendency to aggregate rather than having functional significance(18) . The sensitivity of T4 Dam to N-ethylmaleimide indicates that one or more cysteine residues is important for activity. Similar requirements have been observed for the adenine MTases, M.Eco Dam and M.EcoRI(25, 28) .

T4 Dam is also similar to bacterial Type II DNA MTases with respect to kinetic parameters, except that it has lower Kvalues for substrates and a higher catalytic rate constant. The relatively high specificity constant of T4 Dam (k/K = 1.4 10M s) also indicates that it is a more efficient enzyme than the other MTases studied so far. Methylated DNA is a competitive inhibitor of MTase activity (K = 4.6 pM), indicating that T4 Dam exhibits (as for other MTases) stronger (almost 4-fold) affinity for unmethylated DNA compared with methylated DNA. The other product of methylation, AdoHcy, also acts as a competitive inhibitor of AdoMet (K = 2.4 µM); the ratio K/K = 24 indicates that T4 Dam has a higher affinity for AdoMet.

The ability of T4 Dam to methylate noncanonical sites in vitro was demonstrated by conferring protection against cleavage by methylation-sensitive restriction endonucleases that recognize sequences partially overlapping GATC. These results are consistent with studies on in vivo methylation by T4 Dam(3) . It was previously shown that phage T2 Dam (which differs from T4 Dam in three amino acid residues) can methylate sites other than GATC in vitro(29) . The bacterial MTases, M.EcoRI(30) , M.EcoRV (31) and M.EcoDam(32) , can methylate noncanonical sites to a limited extent, but only under special conditions in vitro.

The availability of highly purified T4 Dam will facilitate crystallization trials and further analyses of the enzyme, particularly with respect to interaction with its target site(s) on DNA.

  
Table: Purification of T4 Dam MTase



FOOTNOTES

*
This work was supported by Grant GM 29227 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed. Tel.: 716-275-3846; Fax: 716-275-2070; E-mail: VKSY@dbv.cc.rochester.edu.

The abbreviations used are: MTase, methyltransferase; AdoHcy, S-adenosyl-L-homocysteine; DTT, dithiothreitol; DE81, diethylaminoethyl ion exchange filters; KPO, potassium phosphate; PAGE, polyacrylamide gel electrophoresis.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.