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Originally published In Press as doi:10.1074/jbc.M306397200 on July 30, 2003

J. Biol. Chem., Vol. 278, Issue 43, 41749-41755, October 24, 2003
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Bacteriophage T4Dam (DNA-(Adenine-N6)-methyltransferase)

EVIDENCE FOR TWO DISTINCT STAGES OF METHYLATION UNDER SINGLE TURNOVER CONDITIONS*

Ernst G. Malygin{ddagger}, William M. Lindstrom, Jr.§, Victor V. Zinoviev{ddagger}, Alexey A. Evdokimov{ddagger}, Samuel L. Schlagman¶, Norbert O. Reich§, and Stanley Hattman¶||

From the {ddagger}Institute of Molecular Biology, State Research Center of Virology and Biotechnology Vector, Koltsovo 630559, Novosibirsk Region, Russia, the §Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93103, and the Department of Biology, University of Rochester, Rochester, New York 14627-0211

Received for publication, June 17, 2003 , and in revised form, July 30, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-L-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5'-GATC/5'-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA « 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-L-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages. First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (kmeth1 = 0.21 s–1); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination. In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (kmeth2 = 0.023 s–1). We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. The hemimethylated duplex 5'-GATC/5'-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions. The first stage (kmeth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation. The slower second stage (kmeth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain. Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions. When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Type II DNA-methyltransferases (MTases)1 usually recognize short (4–6 bp) palindromic sequences and catalyze methyl group transfer from donor S-adenosyl-L-methionine (AdoMet) to position N6 of adenine (Ade) and either the C5 or N4 of cytosine (Cyt) (1); the reaction products are methylated DNA and S-adenosyl-L-homocysteine (AdoHcy). The elucidation of the mechanisms of action of these enzymes remains an important task in the field of biological DNA methylation. In addition, because of their relatively high specificity and simple structural organization, Type II MTases are attractive objects for detailed studies of specific protein-DNA interactions.

The greatest advances in studying the mechanism of action were achieved with the (Cyt-C5)-MTases, for which not only the chemical mechanism of catalysis is known (2), but also three-dimensional structures of enzyme-substrate complexes were solved (3–6). A most surprising and important finding was that the target deoxycytidine "flips out" of the double helix (5), an event that is presumed to precede methyl transfer (7). Among (Ade-N6)- and (Cyt-N4)-MTases, three-dimensional structures are published only for the TaqI, PvuII, DpnM, and RsrI enzymes (8–11). Co-crystallization of these enzymes with DNA and cofactor has not been successful, with the exception of the TaqI MTase. The crystal structure of an AdoMet analog-DNA-TaqI MTase ternary complex (12) revealed flipping of the target deoxyadenosine. Flipping of the target deoxynucleoside has not been shown directly with other (Ade-N6)-MTases. However, alternative methodologies, in which the target base is replaced with the fluorescent analog 2-aminopurine (N), and modeling of spatial structures confirm such a DNA deformation upon interaction with (Ade-N6)-MTases (13–18).

Another important line of study is on kinetic mechanisms of DNA methylation, because the detailed understanding of recognition specificity and catalysis requires knowledge of the rate constants for individual reaction stages. DNA MTases utilize polymeric DNA as their natural substrates in vivo. However, analysis of in vitro methylation of polymeric DNA, even if it contains a single target sequence, is complicated by the need for linear diffusion of the DNA-bound enzyme to find its specific recognition site. The use of relatively short oligodeoxynucleotide (ODN) duplexes (usually 12–30 bp) containing a single specific site allows for simplification of the experimental conditions and acquisition of more precise data for calculating reaction parameters. In addition, substitutions of chemical groups or other modifications can be introduced, and the effect of these defined alterations on the reaction course can be assessed (19–22). Because a natural DNA may contain some structural defects such as nicks, gaps, deletions, or chemical modification of nucleotide residues, it is interesting to know how these alterations affect the methylation process.

Earlier we studied the influence of various defects in synthetic ODN duplexes upon binding of the bacteriophage T4Dam (DNA-(Ade-N6)-MTase) and on the steady-state parameters of methylation (23–26). We showed that the addition of T4Dam to the reaction mixture led to an initial reaction "burst," which was then followed by a slower steady-state phase of methylation. This result indicated that the rate-limiting stage of the reaction is the release of product(s) from the complex with the enzyme. Certain defects in duplex structure reduced the kcat value but had only small effects on the Km or the ability of T4Dam to bind the duplex. Other alterations reduced T4Dam binding ability but retained good substrate characteristics, viz. relatively high kcat and low Km values. Thus, diverse effects on the interaction with T4Dam were exerted by different changes of the specific site. In these steady-state studies, methylation was carried out under conditions where substrate DNA was in excess (enzyme/DNA « 1) and T4Dam has a monomeric structure; in contrast, when enzyme/DNA > 1, a dimeric enzyme structure is favored (27). In this regard, the x-ray crystal structure of a T4Dam-DNA-AdoHcy ternary complex (28) showed that the enzyme:DNA (a 12-mer ODN duplex) ratio was 2:1. Hence, it is important to examine the catalytic characteristics of dimeric T4Dam and compare them with the properties of the monomeric form. Therefore, we have extended our studies on T4Dam to analyses of pre-steady-state kinetics under single turnover conditions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Enzymes and Chemicals—[3H]CH3-AdoMet was purchased from Amersham Biosciences. Unlabeled AdoMet (Sigma) was purified further by chromatography on a C18 reversed-phase column, as described previously (29). ODNs were synthesized and purchased from Midland Certified Reagent Co. (Midland, TX). They were purified further as described (13); concentrations were determined spectrophotometrically from the molar extinction coefficients of individual nucleotides and the known sequence of each ODN. T4Dam MTase was purified to homogeneity, as described previously (30). Protein concentrations were determined by the Bradford method (31).

Defined synthetic duplexes were obtained by heating and annealing individual ODN chains (Table I) from 90 to 20 °C over a 7–12-h period. For example, annealing complementary ODN I (designated as the upper strand) and ODN II produced duplex 1 with a centrally located T4Dam recognition site GATC/GATC (Table II). The hemimethylated variant duplex 1m was produced by annealing ODN Im (contains an M residue in the recognition site) with ODN II. Other target site variants contained a modified base substitution in the "bottom" strand II; e.g. G was replaced by 7-deazaG (G -> Z, ODN IIz), or Ade was replaced by 2-aminopurine (A -> N, ODN IIn). Structurally defective duplexes lacking some element of the recognition site (either a nucleotide or phosphate) were prepared by annealing equimolar mixtures of upper strand ODN I with one or more of ODN III-IX (Table I).


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  		TABLE I
Synthetic ODNs

 

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  		TABLE II
Kinetic parameters for T4Dam methylation of 20-mer duplexes with canonical GATC/GATC or modified sites

^, absence of a phosphate; (.), absence of a nucleotide; ND, not determined. Single turnover parameters kmeth1, kmeth2, P1, and P2 were derived from results plotted according to Equation 2. Values in parentheses are the standard deviations of the parameters.

 

Single Turnover Assays—Methyl transfer assays were carried out at 25 °C, as described previously (25). Reaction mixtures contained 100 mM Tris-HCl, pH 8.0, 10 mM EDTA, 10 mM dithiothreitol, and 0.2 mg/ml bovine serum albumin. The microvolume rapid quench instrument RQF-3 (KinTek Corp.) was used for pre-steady-state assays. Syringes, mixers, and age-loops were equilibrated to 25 °C. The feeding syringe containing the enzyme preparation was kept at 4 °C to avoid inactivation of the T4Dam MTase during the experiment. SDS (0.05%, w/v) in 25 mM Tris-HCl (pH 8.3) was used as the quench solution. The quenched samples were collected in Eppendorf tubes and evaporated to a volume of 100 µl using an Eppendorf vacuum concentrator. Duplicate 50-µl aliquots were spotted onto DE81 anion-exchange filter paper (Whatman, 2.0 cm) for counting 3H counts/min. The molar concentration of [3H]CH3 groups incorporated into DNA was quantitated as described earlier (32). Each experiment was performed at least twice, and the mean values were used for analysis. Kinetic parameters were obtained by using the computer program "Scientist" (MicroMath Inc.) for a regression analysis to fit the experimental data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
Single Turnover Kinetics of Methylation of Duplexes Containing a Canonical T4Dam Recognition Site: a Two-stage Reaction—T4Dam methylation of defined synthetic duplexes (Table II) was assayed under single turnover conditions; i.e. AdoMet (8 µM) was saturating relative to T4Dam (2.7 µM), and both were saturating relative to the ODN duplex (0.2 µM). Under these conditions, two T4Dam (presumably bound with AdoMet) are bound per duplex (32). In the classic case of single turnover, a substrate molecule has a single reactive group that is converted to product. The course of the reaction can be described by a simple exponential function

(Eq. 1)
where Pmax is the maximum level of substrate conversion, and kmeth is the rate constant of the chemical stage of the reaction. However, the curves did not obey a simple exponential dependence (representative results are presented in Fig. 1, and statistical evidence is presented under "Appendix"). All data sets were best described by using Equation 2 below for two-step methylation of duplexes; however, it is not ruled out that some other more complex model might fit the data even better. Duplex 1 (Table II) contains GATC on both strands of the target site. During a single turnover reaction, T4Dam should catalyze a methyl transfer to every duplex, and it might even methylate both Ade residues.



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  		FIG. 1.
Single turnover kinetics of T4Dam methylation of canonical unmethylated (A), hemimethylated (B), and several representative defective ODN duplexes (C–F). Shown in inserts are the recognition site structures, where "{wedge}" and "(.)" designate absence of a phosphate and a nucleotide, respectively. Dashed curves in A and B represent fittings to the one-step reaction mechanism (Equation 1); solid curves represent fittings to the two-step model (Equation 2). Residual plots (data – fit) for the one-step (dashed line) and two-step (solid line) mechanisms are shown in the panels below A and B. The reaction parameters for two-step methylation of all substrate duplexes are presented in Table II and described in the text. The concentrations in the resulting mixtures were: 2.7 µM T4Dam, 8 µM AdoMet, 0.2 µM duplex, 100 mM Tris-HCl, pH 8.0, 10 mM EDTA, 10 mM dithiothreitol, and 0.2 mg/ml bovine serum albumin.

 

Because the overall DNA-MTase reaction is irreversible (2, 26), it can be represented as a two-stage conversion of the initial oligonucleotide duplex containing two target Ade residues. where E is T4Dam bound with AdoMet and/or AdoHcy, D is the initial duplex, mD is the half-methylated duplex; mDm is the fully methylated reaction product, kmeth1 is the rate constant of the first stage of the methylation reaction, and kmeth2 is the rate constant of the second stage. Because the technique used only allows registration of the sum of the products of reaction p = mD + mDm, the kinetic scheme (Scheme 1) is described by Equation 2 (33):

(Eq. 2)
where P1 is the number of Ade residues methylated per duplex during the first stage of the reaction, and P2 is the number of Ade residues methylated per duplex in the final product. For duplex 1, containing the canonical palindromic GATC/GATC site, the first stage of the single turnover reaction involves methylation of one of the two Ade residues (P1 = 1.03 [3H] methyl groups transferred per duplex) with rate constant kmeth1 = 0.21 s–1 (Fig. 1A and Table II). This value is 14-fold higher than the kcat under steady-state conditions (24, 25). This finding confirms that, as in the case of the EcoRI MTase (34), the chemical step of the reaction (methyl group transfer) is not rate-limiting. In the first stage after binding, T4Dam methylates one strand only. The second methylation stage proceeded at a rate that was an order of magnitude slower than in the first stage, and the P2 was 1.90 methyl groups per duplex. Thus, both Ade residues in the canonical GATC/GATC site were methylated under single turnover conditions, where two Dam-AdoMet complexes can be bound to the duplex (27). This result suggests that the two distinct kmeth values reflect differing methylation conditions; e.g. the second methylation is rate-limited by the dissociation of T4Dam-AdoHcy or its clearance from the site on the opposite strand.



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  		SCHEME 1
 

Single Turnover Kinetics of Methylation with Duplexes Containing a Nucleotide Substitution in the T4Dam Recognition Site—Duplexes 1m, 3, 4, and 5 (GMTC/GATC, GMTC/ZATC, GATC/GNTC, and GATC/GTAC, respectively) contain only a single Ade residue as a potential methyl acceptor. Thus, for these duplexes, random T4Dam binding should result in one of two mutually exclusive orientations (productive and non-productive) to the substrate, resulting in modification of only 50% of the duplexes. In all cases we observed a rapid exponential phase of methyl transfer followed by a slower increase in product accumulation; the results for duplex 1m are shown in Fig. 1B. Nevertheless, the two-exponential equation (Equation 2) adequately described the methylation of these duplexes, indicating that their methylation also proceeds in two stages. Fig. 1B shows the fitting of the experimental points for duplex 1m, and it worked satisfactorily in all the other cases, allowing determination of all four reaction parameters, kmeth1, kmeth2, P1, and P2 (Table II). However, it must be pointed out that here kmeth2 applies to a different process. For example, consider methylation of the hemimethylated duplex 1m, where the rate of methyl group transfer was almost 2-fold higher (kmeth1 = 0.37 s–1) than for duplex 1 (Fig. 1B, Table II). The extent of methyl transfer during the first-stage P1 was 0.45 [3H]methyl groups per duplex. This value is close to the theoretical value of 0.50, assuming that Dam-AdoMet molecules bound to the productive and nonproductive strands of the hemimethylated target have an equal probability of flipping a target A or N6-methyladenine deoxynucleoside and that interacting with the methylated Ade precludes methylation during the first stage. During the second methylation stage (which was also faster than with duplex 1), the P2 value attained the theoretical maximum of 1.01 [3H]methyl groups per duplex. Therefore, we propose that under single turnover reaction conditions, methylation of the hemimethylated duplex 1m (and other duplexes having one Ade residue capable of methylation) also occurs in two stages. Here, kmeth1 reflects the rate of the methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the Ade residue capable of methylation, whereas the slower kmeth2 reflects methylation by enzyme molecules that were oriented first to the non-productive GMTC chain and have to reorient (27) to the opposite productive chain.

The substitution of Z for G (in duplexes 2 and 3) represents only a minor structural change, which does not alter normal hydrogen bonding. Duplex 2 contains two Ade residues accessible for methylation, whereas duplex 3 has one. With both duplexes 2 and 3, there was a 1.5- to 2-fold lower kmeth1 than for canonical duplex 1 (Table II). Earlier it was shown that the G -> Z substitution disrupts an important contact between T4Dam and guanine atom N7, which is exposed in the major groove (24). In particular, the Kd value (according to gel retardation data) increased from 18 nM for the canonical duplex to 950 nM for the Z-substituted duplex. Nevertheless, the theoretical extent of methylation (P2) of each duplex (2 and 3) was attained (Table II). An -> N substitution (duplex 4) shifts the location of the exocyclic amino group from C6 to C2 (as well as shifting hydrogen bonding with thymine to this position). Although duplex 4 exhibits a 4-fold decrease in the kcat value, the chemical stage rates kmeth1 and kmeth2 increased 2-fold (Table II). The extent of methylation at both stages (P1 = 0.58, P2 = 1.12) corresponded to the expected values.

Introduction of a double-base mismatch opposite the central upper strand AT (duplex 5) decreased the kcat value 30-fold (Table II). However, kmeth1 was not affected, and kmeth2 showed only a 2-fold decrease. Because the target Ade residue in the upper strand cannot form a hydrogen bond, it might be expected that its flipping out of the helix would be facilitated. However, because kmeth1 was not increased, we conclude that either the stacking effect continues to stabilize the structure, or that flipping of the target base is not rate-limiting. The latter would be in agreement with earlier findings (35). The expected extents of methylation P1 and P2 were observed with duplex 5.

Single Turnover Kinetics of Methylation with Duplexes Containing a Deletion in the T4Dam Recognition Site—Absence of a single internucleotide phosphate in the target site influences methylation in a position-dependent fashion. For example, deletion of a phosphate between T and C (duplex 6) had no effect on kcat, kmeth1, and kmeth2 (Fig. 1C, Table II), and the extent of methylation in the first stage was as expected, although slightly lower after the second stage. In contrast, absence of the phosphate between A and T (duplex 7) lowered kmeth2 by 1.5-fold and kcat by 2.5-fold (Fig. 1E, Table II). Absence of a phosphate between G and A (duplex 8) had the largest effect on kinetic parameters (Table II). Thus, the kcat value was reduced almost 10-fold, whereas the kmeth1 increased more than 2.5-fold, but P1 decreased. The latter change indicates that the proportion of catalytically competent complexes was lower than expected. Finally, kmeth2 was lowered 3-fold, but the P2 = 1.2 suggests there was complete methylation of the native strand.

Duplex 9, which lacks C and additional 3'-nucleotides in the bottom strand, had single turnover parameters quite close to those for duplex 6 (Fig. 1D, Table II), which lacks a phosphodiester bond between the T and C residues of the bottom strand. In contrast, duplex 10, which lacks G and additional 5'-nucleotides in the bottom strand, gave entirely different single turnover parameters (Table II). For example, the P1 value was less than 0.1; nevertheless, the kmeth1 of 1.17 s–1 is 6-fold higher than that for the native duplex. On the other hand, the second stage of methylation showed an 8-fold lower kmeth2 compared with the native duplex. Still, the P2 value of 1.10 suggests complete methylation of the native strand. The kmeth2 and kcat values were identical and at least five times lower than the respective values for native duplex 1. Duplex 11, which lacks T in the bottom strand, had a 4-fold higher kmeth1 value compared with native duplex 1 (Fig. 1F, Table II), yet it had a low P1 value of 0.16. The sharp reduction in the proportion of stage 1 productive complexes for duplexes 7, 8, 10, and 11, each devoid of some structural element, might be attributed to some distortion in the normal bending of DNA in the recognition site, which was shown to accompany interaction with DNA-MTases (36).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
APPENDIX
 REFERENCES
 
Under Single Turnover Conditions T4Dam Acts as a Dimer and Methylates Both Strands of Canonical GATC/GATC Duplex 1—We showed earlier (by gel filtration and sucrose gradient ultra-centrifugation) that two T4Dam molecules are present in complexes formed with 20- and 32-mer ODN duplexes under conditions of single turnover (37). To study this further, we took advantage of the ability of glutaraldehyde to cross-link protein subunits, because of the formation of covalent bonds between closely located lysine residues (27). We showed that the formation of glutaraldehyde-cross-linked T4Dam dimers increased in the range of 20-mer duplex/enzyme = 0.1–0.5. Dimers became the predominant form of the enzyme in the oligomeric fraction. Increasing the duplex/enzyme ratio progressively diminished the ability of glutaraldehyde to cross-link T4Dam, eventually disappearing at higher ratios. Thus, under single turnover conditions, when the concentration of T4Dam was an order of magnitude higher than that of the substrate duplex, the enzyme may function as a dimer/oligomer. These results allow T4Dam to be included among a group of dissociating enzyme systems, whose features were considered in detail by Kurganov (38). In contrast, under presteady-state conditions, T4Dam exhibits a monomeric structure (39). Consistent with the notion of varying states of T4Dam is the fact that the kmeth values differed according to experimental conditions (Table II). Thus, with native duplex 1, the kmeth value in the "burst experiment" was 2.5-fold higher than kmeth1 in the first stage of single turnover methylation (see below).

We propose that T4Dam methylation proceeds in two stages under single turnover reaction conditions. The specific site of native DNA duplex 1, GATC/GATC, interacts consecutively and rapidly with two T4Dam-AdoMet complexes. As shown earlier (27), the presence of AdoMet induces an asymmetric state in the ternary complex in which one target Ade residue is flipped out. The asymmetric orientation of the enzyme dimer toward one strand of the duplex may be caused by steric hindrances due to the simultaneous productive interaction with two target residues (34). A rapid transfer of one methyl group from AdoMet to an Ade residue (kmeth1 = 0.21 s–1) occurs in the first stage. We suggest that the presence of the second T4Dam-AdoMet in the catalytic complex may be responsible for the reduction in kmeth1 in comparison to kmeth = 0.56 s–1, where a single T4Dam-AdoMet binds and catalyzes methylation (39). In the second stage, methylation of the complementary strand occurs, but at a rate that is an order of magnitude slower (kmeth2 = 0.023 s–1). This rate is similar to the steady-state rate constant, kcat (0.015 s–1). Thus, the second stage might be limited by the rate at which AdoHcy dissociates from T4Dam on the methylated strand or by the clearance of T4Dam-AdoHcy from the site.

Effect of Target Site Modification—Base substitutions and deletions in the recognition site variably influenced the single turnover reaction parameters. When the GAT portion of the target sequence was interrupted, the Ade residue could not be methylated, although the Ade residue in the complementary strand GATC could be methylated. In addition, most interruptions caused a sharp decrease in the proportion of the initial, productive enzyme-substrate complexes, especially for duplexes 8, 10, and 11 (Table II). As is shown by the gel retardation method, duplexes 8 and 11 have a greater affinity for T4Dam than the native duplex 1 (23). Therefore, the decrease in the proportion of productive complexes cannot be explained by a weakened affinity for the duplex. Thus, it appears that the GAT sequences in both strands form a second order symmetrical structure that may be necessary for optimal interaction between the duplex and T4Dam-AdoMet. In contrast to interruptions in GAT, deletion of C and the adjoining 3'-sequence in the bottom strand (duplex 9) did not significantly alter the reaction parameters (Table II). These results are consistent with earlier observations on the influence of defects in the GATC/GATC recognition site on the catalytic activity of the EcoDam MTase (20), where the normal functioning of this enzyme required the presence of an intact GA on both strands of the recognition site. Thus, T4Dam is more sensitive to defects in the recognition site than is EcoDam.

It was shown recently that the T4Dam reaction obeys an ordered mechanism (AdoMet{downarrow}DNA{downarrow}DNAme{uparrow}AdoHcy{uparrow}) and that the rate-limiting step of the reaction is the release of AdoHcy from the enzyme-product complex (26). As seen in Table II, the kcat values decreased many fold for defective duplexes 4, 5, 7, 8, and 10, whereas the kmeth1 values did not decline significantly, and some even increased (duplexes 4, 8, and 10). We suggest that for the defective sites after the rapid stage 1 methylation, release of enzyme from the site becomes the rate-limiting step. Similar defective sites in natural DNA, even if rare, might become traps for slowing down in vivo methylation.

Comparison of T4Dam with Other DNA-(Amino)-MTases— Single turnover methylation of a 14-mer specific duplex has been well characterized for the EcoRI DNA-(Ade-N6)-MTase (34), where the chemical step of the reaction has a kmeth = 41 s–1, ~300 times higher than kcat. Thus, the chemical step in the reaction (the methyl group transfer) is not the rate-limiting step; rather, the dissociation of the enzyme-product complex must be rate-limiting. It was also shown that methylation of only one DNA strand occurred per EcoRI MTase binding event. This indicated that EcoRI MTase was asymmetrically oriented toward one of the two duplex strands. Product formation over time was also analyzed under single turnover conditions for the HhaI MTase (40). A kmeth = 0.26 s–1 was obtained for a hemimethylated duplex, ~2-fold greater than that for the unmethylated duplex; in contrast, the ratio of product to substrate was 2-fold higher for the unmethylated duplex. In all the cases described above, the kinetics of product formation under single turnover conditions gave a good fit to a single exponential.

The methylation of a double-stranded DNA may be mediated by one of several alternative mechanisms (41). For example, an MTase may methylate a target sequence on each strand in two independent binding events, or both strands might be methylated after a single binding event. This issue was examined for EcoRI MTase (41) and the BamHI DNA-(Cyt-N4)-MTase (41). Under steady-state conditions (limiting enzyme), the increase in methyl transfer per substrate (pVH51 or SV40) DNA molecule was related to the increase in its resistance to cleavage by the cognate restriction endonuclease. For EcoRI MTase, a straight line with a slope of 95% (resistance per methyl group transferred) demonstrated that the enzyme transfers methyl groups one at a time and leaves the DNA after each catalytic event (42). In contrast, a straight line with a slope of about 50% was observed for BamHI MTase (41). If these data are expressed as moles of methyl groups transferred per mol of nuclease-resistant DNA, a ratio of 1.95 was found. This suggests that BamHI MTase transferred two methyl groups in a single binding event.

The result for the EcoRI MTase was confirmed in experiments examining methylation of ODN duplex substrates under conditions of single turnover (34). The curves of product formation versus time were well fitted to a single exponential, and EcoRI MTase methylated only one Ade residue of the double-stranded site GAATTC/GAATTC in a single binding event. Thus, this pertains under conditions of both limiting enzyme (steady-state conditions) and limiting DNA substrate (single turnover conditions). In contrast to EcoRI MTase, T4Dam methylates one strand under steady-state conditions but methylates both strands under single turnover conditions (Table II). Moreover, unlike EcoRI MTase, T4Dam single turnover curves fit a double exponential, reflecting two consecutive steps in methylation, which is consistent with EcoRI MTase acting as a monomer and T4Dam as a dimer under these conditions.


   APPENDIX
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 APPENDIX
REFERENCES
 
Kinetic data were analyzed using the program Scientist version 2.01 (MicroMath) for regression analysis. We used the statistical "model selection criterion" (MSC) recommended by program developers to determine the goodness of fit for each kinetic model, and additionally we estimated the standard deviation of data. The MSC is defined by the formula

(Eq. 3)
where n is the number of points, wi are the weights applied to each point (wi = 1 in all of our calculations), obs is the weighted mean of the observed data, and p is the number of parameters. The model that has the largest MSC value is by definition the best or most appropriate model.

The standard deviation is defined by the formula

(Eq. 4)
where n is the number of points, wi are the weights applied to the points, and DOF is the number of degrees of freedom for the problem (the number of data points minus the number of fitted parameters) (Table III).


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  		TABLE III
The goodness of fit for one-step and two-step kinetic models

^, absence of a phosphate; (.), absence of a nucleotide.

 

According to both the MSC and standard deviation values, the kinetic data for all substrate duplexes illustrated in Fig. 1 are significantly better described by Equation 2, which corresponds to the two-step model. We also calculated the relative probabilities of the two models to describe the experimental data using the program DynaFit version 3.22 (BioKin) (43). In all cases, the probability of the two-step model was equal to 1.0.


   FOOTNOTES
 
* This work was supported by the Russian Foundation for Basic Research (Project No. 01-04-49869), U. S. Public Health Service Grant R03 TW05755 from the Fogarty International Center, National Science Foundation Grant MCB-9983125 (to N. O. R.), and U. S. Public Health Service Grants GM463333 (to N. O. R.) and GM29227 (to S. H.). 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. Back

|| To whom correspondence should be addressed. Tel.: 585-275-8046; Fax: 585-275-2070; E-mail: modDNA{at}mail.rochester.edu.

1 The abbreviations used are: MTase, methyltransferase; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; T4Dam, bacteriophage T4 encodes a DNA-(adenine-N6)-MTase; ODN, oligodeoxynucleotide; M, N6-methyladenine; N, 2-aminopurine; Z, 7-deazaguanine; MSC, model selection criterion. Back


   ACKNOWLEDGMENTS
 
We thank A. V. Zinoviev for technical assistance, A. I. Zakabunin for helpful discussions of the results, and M. Buckle for a critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 

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