The Mechanism of DNA Cytosine-5 Methylation

KINETIC AND MUTATIONAL DISSECTION OF HhaI METHYLTRANSFERASE*

Giedrius Vilkaitis, Egle Merkiene, Saulius Serva, Elmar WeinholdDagger , and Saulius Klimasauskas§

From the Institute of Biotechnology, Laboratory of Biological DNA Modification, LT-2028 Vilnius, Lithuania and Dagger  Institut für Organische Chemie der RWTH Aachen, D-52056 Aachen, Germany

Received for publication, February 14, 2001, and in revised form, March 28, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-L-methionine (AdoMet) and product S-adenosyl-L-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. The M.HhaI·AdoMet complex (koff = 22 s-1, KD = 6 µM) is partially converted into products during isotope-partitioning experiments, suggesting that it is catalytically competent. Chemical formation of the product M.HhaMeDNA·AdoHcy (kchem = 0.26 s-1) is followed by a slower decay step (koff = 0.045 s-1), which is the rate-limiting step in the catalytic cycle (kcat = 0.04 s-1). Analysis of reaction products shows that the hemimethylated substrate undergoes complete (>95%) conversion into fully methylated product during the initial burst phase, indicating that M.HhaI exerts high binding selectivity toward the target strand. The T250N, T250D, and T250H mutations, which introduce moderate perturbation in the catalytic site, lead to substantially increased K<UP><SUB>D</SUB><SUP>DNA(ternary)</SUP></UP>, k<UP><SUB>off</SUB><SUP>DNA(ternary)</SUP></UP>, K<UP><SUB>M</SUB><SUP>AdoMet(ternary)</SUP></UP> values but small changes in K<UP><SUB>D</SUB><SUP>DNA(binary)</SUP></UP>, K<UP><SUB>D</SUB><SUP>AdoMet(binary)</SUP></UP>, kchem, and kcat. When the target cytosine is replaced with 5-fluorocytosine, the chemistry step leading to an irreversible covalent M.HhaI·DNA complex is inhibited 400-fold (k<UP><SUB>chem</SUB><SUP>5FC</SUP></UP> = 0.7 × 10-3 s-1), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation kchem. We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (kchem) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Methylation of cytosine residues in DNA occurs in diverse organisms from bacteria to humans. Cytosine methylation in DNA is catalyzed by DNA methyltransferases (MTases)1 that transfer methyl groups from the ubiquitous donor S-adenosyl-L-methionine (AdoMet) producing modified cytosines with a methyl group at either C-5 or N-4 (1). In higher organisms, where only 5-methylcytosine is found, DNA methylation is essential for controlling a number of cellular processes including transcription, genomic imprinting, developmental regulation, mutagenesis, DNA repair, and chromatin organization (2). Aberrations in cytosine-5 methylation correlate with human genetic disease, and therefore, the MTases are potent candidate targets for developing new therapies (3). In prokaryotes, MTases are usually but not exclusively found as components of restriction modification systems (1).

Besides their important physiological role, the MTases are attractive models for the study of protein-DNA interactions, a central event in many biological processes. The major advantages of bacterial C5-MTases as model systems are as follows: (a) wide diversity of targets recognized (over 200 specificities known); (b) ability to promote covalent reactions within the DNA; (c) their relatively simple molecular organization; and (d) high level of sequence and structural homology with eukaryotic enzymes. It is not surprising that most evidence of the catalytic mechanism of cytosine-5 methylation has been obtained from the studies of prokaryotic MTases. A particular example is HhaI MTase, a component of a type II restriction-modification system from Haemophilus haemolyticus. M.HhaI recognizes the tetranucleotide sequence GCGC and methylates the inner cytosine residue (boldface) and is one of the smallest in the C5-MTase family. This enzyme has been extensively examined by employing a variety of methods. Interaction with the substrates was shown to lead to dramatic conformational changes in both the bound DNA and the enzyme itself. MTase-mediated rotation of the target nucleotide out of the DNA helix (base-flipping) serves to deliver the base into a concave catalytic site in the enzyme (4). Subsequent massive movement of the 20-residue catalytic loop toward the DNA envelopes the flipped out base and creates the environment necessary for the covalent reaction to occur.

Steady-state kinetic studies of M.HhaI (5) and M.MspI (6) indicated an ordered binding of substrates (DNA before AdoMet). However, biochemical and crystallographic analyses demonstrated the existence of a binary MTase·AdoMet complex (7-9), hinting that a mechanism with random substrate binding might be valid. Recent attempts to resolve this question employing isotope partitioning and single turnover analysis, contrary to claims by other authors (10), have not provided the final answer (see below).

The chemistry of enzymatic conversion of cytosine into 5-methylcytosine is relatively well studied and largely follows that earlier proposed for thymidylate synthase (Fig. 1A). The key step is the formation of a Michael adduct between the sulfhydryl of a conserved cysteine (Cys-81 in M.HhaI) and C-6 of the pyrimidine ring; this appears to be coupled with protonation of N-3 to yield an activated enamine intermediate (Fig. 1A, step 42) (5, 11, 12). The carbon 5 becomes nucleophilic for the SN2-type attack onto the methyl sulfonium center of AdoMet resulting in the capture of the methyl group by the ring (Fig. 1A, step 53). The resulting "dihydro-cytosine" derivative is resolved by beta -elimination of the C-5-proton and C-6-thiolate to give the methylated cytosine (Fig. 1A, step 44 or 45). The latter step is blocked, and the corresponding covalent intermediate is trapped when the target base is replaced with the mechanism-based inhibitor 5-fluorocytosine (13). Covalent complexes involving 5-fluorocytosine have been characterized (14) and visualized in subsequent crystal structures (4, 15). Much less is known about the time scale of these reactions and relative contributions of elementary steps to the overall process of cytosine methylation.

In the present work, we sought to obtain kinetic insights into events underlying interactions of enzyme with the substrates and their relationship with catalysis. Recently, we have demonstrated that substitutions of Thr-250 with bulkier residues introduce structural perturbations in the catalytic center leading to pronounced effects on the catalytic properties of the enzyme (16). This conserved residue is located in the target recognition domain with its side chain pointing toward the catalytic residues that intimately interact with the target base. The above mutants were used as structural probes to obtain information on elementary catalytic steps of the enzymatic C-5 methylation of cytosine.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Expression and Purification-- Mutant and wild type M.HhaI proteins were prepared and assayed as described previously (16). The 37-mer DNA oligonucleotides were obtained from MWG-Biotech AG (Germany), HPSF grade (named as shown in parentheses, the HhaI recognition site is underlined, residues at the target base position are in boldface), as follows: upper strand, 5'GACTGGTACAGTATCAGGCGCTGACCCACAACATCCG (GCGC) and 5'GACTGGTACAGTATCAGG(5FC)GCTGACCCACAACATCCG (GFGC); lower strand, 5'TCGGATGTTGTGGGTCAG(5mC)GCCTGATACTGTACCAGT (GMGC) and 5'TCGGATGTTGTGGGTCAGCGCCTGATACTGTACCAGT (GCGC). DNA duplexes were produced by annealing appropriate oligonucleotides as described previously (17). 5'-32P labeling of oligonucleotides was performed with a DNA labeling kit (MBI Fermentas) as described previously (18).

Electrophoretic Gel Mobility Shift Assays-- Titrations were performed at room temperature in Reaction buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM EDTA, 0.2 mg/ml of bovine serum albumin) containing 10% glycerol. For studies involving binary complexes, 5'-32P-labeled hemimethylated 37-mer duplex (GCGC/GMGC, 0.2 nM) was titrated with increasing protein concentrations (0.125-64 nM). Ternary complex formation was monitored with 2 pM DNA, 100 µM AdoHcy, and 3.9-125 pM MTase (WT and T250C, T250G, T250N, and T250S mutants) or 3.9 pM to 64 nM (T250D and T250H). Samples of 15 µl were incubated for 30 min, and aliquots were loaded onto a running 8% polyacrylamide gel at 10 V/cm. Gels were dried on Whatman 3MM paper, and radioactive bands were visualized by autoradiography either to an x-ray film followed by scanning with a ScanMaker E6 densitometer (Microtec) or to a Cyclone PhosphorImager (Packard Instrument Co.). Bound and free DNA bands were quantitated with OptiQuant software (Packard Instrument Co.), and data were fit to the full quadratic equation for single-site binding using the data analysis program GraFit (19).

For dissociation rate (koff) measurement of MTase·DNA·AdoHcy complexes, 1 nM 5'-32P-labeled hemimethylated 37-mer duplex was preincubated with 3 nM MTase and 100 µM AdoHcy in Reaction buffer at room temperature for 60 min. 100 nM unlabeled DNA was then added and aliquots withdrawn at time intervals between 0 and 24 h (0-180 min for T250D and T250H). Reactions were resolved by polyacrylamide gel electrophoresis as described above. Data were approximated to exponential decay equations.

Steady-state Kinetics-- Methylation velocities were determined by measuring catalytic incorporation of 3H-methyl groups from [methyl-3H]AdoMet onto a 37-mer hemimethylated duplex DNA. Reactions were carried out at 37 °C for 6 min in Methylation buffer (50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 6 mM 2-mercaptoethanol, 0.2 mg/ml bovine serum albumin) with 100 nM [methyl-3H]AdoMet (84 Ci/mmol, Amersham Pharmacia Biotech) and 0.1-20 nM GCGC/GMGC; K<UP><SUB>M</SUB><SUP>AdoMet</SUP></UP> measurements were performed with 8 nM GCGC/GMGC, 5-160 nM [methyl-3H]AdoMet, and 19 pM enzyme. Duplicate or triplicate samples were spotted onto 2.3-cm DE-81 filters (Whatman), washed 4 times with 50 mM sodium phosphate for 10 min, 2 times with H2O, and 2 times with ethanol, dried, and counted with 4 ml of CytoScintTM (Fisher) in a Beckman LS 1801 liquid scintillation counter. Background counts (measured at zero time incubation, <20% of total) were subtracted, and data were analyzed by fitting to a Michaelis-Menten equation using GraFit (19).

Pre-steady-state and Transient Kinetics-- Reactions were performed in Methylation or Reaction buffer at 25 or 37 °C. Two reaction components in syringes A and B (15 µl each) were mixed rapidly, and after a specified period were quenched with 15 µl of 2 N hydrochloric acid in a Rapid-Quench-Flow instrument RQF-3 (KinTek). Single-turnover reactions contained (final concentrations) 100 nM hemimethylated 37-mer DNA (GCGC/GMGC) preincubated with 200 nM M.HhaI and 0.67-25 µM [methyl-3H]AdoMet (15 Ci/mmol). Pre-steady-state assays contained 200 nM GCGC/GMGC preincubated with 15 nM M.HhaI and 0.67 or 1.3 µM [methyl-3H]AdoMet. In most experiments, a small amount of 5'-32P-labeled GCGC/GMGC duplex was included in the reaction as an internal standard along with unlabeled DNA. Duplicate aliquots from quenched reactions were spotted on 2.3 cm DE-81 filters and processed as above. 32P radioactivity was independently measured to determine recovery of duplex DNA and used to normalize the 3H counts in samples. Normalized progress curves were analyzed by fitting to single-exponential or pre-steady burst equations (20) as appropriate using GraFit (19) or DynaFit (21).

Analysis of Methylation Products by Restriction Endonuclease Cleavage-- The GCGC strand was 32P-labeled at the 5'-end and annealed with unlabeled lower GMGC strand. Enzymatic methylation reactions were initiated by adding components as described above (total volume 30 µl, final concentrations 670 nM AdoMet (Knoll BioResearch, Switzerland), 100 nM labeled GCGC/GMGC duplex, and 200 nM M.HhaI) at 37 °C followed by hand-quenching with 15 µl of 2 N HCl (or 240 µl of 0.67 N HCl) at time points of 0, 3, 20, and 60 s. To neutralize the quencher, 0.5 volume of 5 M potassium acetate was added to each sample, followed by desalting with a MicroSpinTM G-25 column. The methylated DNA was re-annealed with a 170-fold excess of the unmodified lower strand oligonucleotide GCGC by heating to 85 °C and slow cooling to room temperature. The resulting labeled duplex (2 nM concentration of the modified target strand) was incubated with 10 units of R.Hin6I at 37 °C for 1.5 h in 20 µl of Y+/TangoTM buffer (MBI Fermentas). Reactions were analyzed by 10% polyacrylamide gel electrophoresis and autoradiography as described above.

Cofactor Exchange Assay-- Four methylation reactions (A-D) were performed essentially as described above by mixing pulse and chase solutions by hand (10 µl each) or in a rapid-quench device (15 µl each). For reaction A, the pulse mix contained 2 µM M.HhaI and 40 µM [methyl-3H]AdoMet (4.7 Ci/mmol), chase mix contained 2 µM GCGC/ GMGC and 5 mM cold AdoMet; for reaction B, the pulse mix contained 2 µM M.HhaI and 40 µM [methyl-3H]AdoMet (4.7 Ci/mmol), chase mix contained 2 µM GCGC/GMGC; and for reaction C, the pulse mix contained 2 µM M.HhaI and 40 µM [methyl-3H]AdoMet (3.8 mCi/mmol), chase mix contained 2 µM GCGC/GMGC and 5 mM [methyl-3H]AdoMet (3.8 mCi/mmol). All reactions were incubated at 37 °C for 30 s and quenched with 0.5% SDS. Reaction D (background control) was identical with reaction A, except that it was quenched immediately after mixing. Experiments were performed in duplicate, and duplicate samples from each reaction were processed as described above and analyzed. Background counts obtained in reaction D (typically 60-90 cpm) were subtracted from those in reactions A-C.

Methylation of DNA Containing 5-Fluorocytosine-- Reactions containing 2.7 µM of MTase, 400 µM AdoMet, and 7 µM 37-mer hemimethylated duplex (GFGC/GMGC) were incubated at 37 °C in Methylation buffer. Aliquots were withdrawn at time intervals from 0 to 25 h and quenched by adding SDS gel loading buffer (MBI Fermentas) and heating the samples at 100 °C for 5 min. Samples were analyzed by electrophoresis in 10% polyacrylamide gel containing SDS and stained with Coomassie Brilliant Blue. Gels were scanned with a BioDocII video documentation system (Biometra). Data were analyzed by fitting to a single exponential equation using GraFit (19).

Fluorescence Spectroscopy Analysis of MTase-Cofactor Interaction-- Fluorescence emission spectra and fluorescence intensities from titrations were measured at 25 °C on an SLM Aminco-6 spectrofluorimeter at an excitation wavelength (lambda Ex) of 280 or 290 nm (slitwidths 1 and 2 mm) and emission wavelength (lambda Em) of 350 nm (slitwidths 8 and 16 mm). M.HhaI (1 µM) was titrated by incremental addition of AdoMet or AdoHcy in Reaction buffer with no albumin. Titration data (fluorescence intensities as a function of total cofactor concentration) were analyzed with the equilibrium solver routine of DynaFit (21).

Stopped-flow experiments were performed on a Hi-Tech Scientific SF 61MX apparatus (single mixing mode) equipped with a Xe-Hg UV lamp. The excitation wavelength was 296 nm (monochromator slitwidth 2 mm); emission light was passed through a 320 nm cut-on filter. In association experiments, 0.5 µM M.HhaI was rapidly mixed with 0.5-40 µM AdoMet or 0.5-5 µM AdoHcy (final concentrations) in the buffer above. Progress curves were collected for each cofactor concentration in a time window from 1.4 to 261 or 1.4 to 60 ms for AdoMet and AdoHcy, respectively. Multiple time courses (at least 6 runs) were averaged and analyzed by fitting to exponential equations using KinetAsyst2, version 2.0 (Hi-Tech) (22). Multicurve fitting and confidence interval search was performed with DynaFit (21). A combined data set of different cofactor concentrations (485 × 11 data points for AdoMet and 347 × 11 data points for AdoHcy) were fitted to a one-step single-mode binding mechanism (E + S iff  ES) or one-step dual-mode mechanism (E + S iff  ES + ES'). The rate constants, fluorescence intensity factor(s) of protein, and offsets of progress curves (background fluorescence intensities) were refined.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have recently described a steady-state kinetic comparison of M.HhaI and six Thr-250 mutants using multisite poly(dG-dC) DNA as a substrate (16). However, more detailed information on interactions between M.HhaI and DNA can be obtained with shorter single-site DNA duplexes. In the present work we employed a 37-mer hemimethylated duplex to study DNA binding, reactive complex formation, and methylation kinetics by HhaI MTase. In addition, Thr-250 mutations were employed as structural probes that perturb catalytic parameters of the enzyme.

DNA Binding Activities of Thr-250 Mutants-- Association of M.HhaI with its DNA substrate is a key step in the catalytic cycle. Therefore, the capability of WT MTase and its Thr-250 mutants to bind a 37-mer hemimethylated duplex was studied. The 32P-labeled DNA was titrated with increasing amounts of enzyme and analyzed by gel electrophoresis under non-denaturing conditions. Binding data followed a single-site binding isotherm (not shown). The only exception was T250D for which no discrete band of the binary complex was observed under these conditions, most likely due to a fast decay of the complex (see below). As shown in Table I, the WT and mutant enzymes showed quite similar dissociation constants in the low nanomolar range (except for T250D). Overall, our analysis indicates that the size of the side chain of residue 250 has a very small effect on the stability of the binary M.HhaI·DNA complex.

                              
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Table I
Effects of Thr-250 mutations on interaction of M.HhaI with hemimethylated DNA

Similar experiments were performed to measure MTase-DNA interaction in the presence of the cofactor product AdoHcy (Fig. 2A). Previous studies of the WT enzyme indicated that addition of AdoHcy leads to the formation of the stable dead-end ternary M.HhaI·DNA·AdoHcy complex (5, 23). Indeed, we find that the WT protein and most mutants exhibit low picomolar affinities in the presence of 100 µM AdoHcy (Table I). Overall, this assay revealed a much wide range of DNA binding affinities among the mutants as compared with the respective binary complexes. Cofactor-dependent enhancement of binding ranges from ~2000-fold for the WT enzyme to only 20-fold observed for the T250H mutant. In the case of T250D, a discrete band was only observed for free DNA, whereas the complex band was smeared and could not be quantified. Therefore, the lower band corresponding to free DNA was used to estimate the binding equilibrium. Notably, the ternary complex involving the T250H and T250N mutants migrated slightly but consistently slower than the other ternary complexes (Fig. 2A) and co-migrated with the binary complex bands (not shown). One possibility is that a substantial structural change is induced by the mutations in the ternary complex. Alternatively, the lower compactness may arise from the loss of AdoHcy during electrophoresis (see below) leading to the binary M.HhaI·DNA complex. The latter appears to be dominated by the open catalytic loop conformers such as DNF·MT (Fig. 1B) in solution (23, 24).


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Fig. 1.   The catalytic and kinetic mechanism of the HhaI methyltransferase. A, major covalent transformations in the enzyme active site leading to C-5 methylation of cytosine. B, general mechanism of HhaI MTase based on known structural, biochemical, and kinetic information. Acronyms describing molecular complexes are: MT and MTL, open and closed (locked) forms of M.HhaI, respectively; DN and DNF, substrate DNA with paired and flipped-out target base, respectively; DM and DMF, methylated DNA with paired and flipped-out target base, respectively; AM, AdoMet; AH, AdoHcy; · and --- denote non-covalent and covalent bonding between molecules, respectively. Vertically aligned steps are as follows: 11-15, DNA binding/dissociation; 21-25, base flipping; 31-35, catalytic loop closing/opening; 41-45, covalent addition/elimination at C-6. Horizontally aligned steps are as follows: 61-65, AdoMet binding/dissociation; 71-75, AdoHcy binding/dissociation (4, 5, 8). Step 53 is the transfer of methyl group from AdoMet into the C-5 of target cytosine, the unnumbered step denotes a side reaction (catalytic exchange of H5 (5)). Questionable or disputed steps are indicated with ?.

The equilibrium binding studies were complemented with measurements of the decay rate in a displacement experiment. The dissociation rates of the binary complexes were too fast to be measured by this method (not shown), and only an estimate for the WT enzyme was determined (~0.04 s-1). In the presence of 100 µM AdoHcy, the decay profiles proved readily discernible in the minute or hour time scales (Fig. 2B). Dissociation profiles were best fit to a double exponential equation. The biphasic behavior of the ternary complexes is consistent with the presence of a certain fraction of binary MTase·DNA complexes or other intermediates at equilibrium. Decay of such an intermediate would occur faster and account for the faster phase, whereas the dominant slower phase would correspond to the dissociation of the ternary complex per se. The rates of the second phase for the Thr-250 mutants of MTase are presented in Table I.2 The cofactor AdoHcy slows down the dissociation of WT M.HhaI from hemimethylated 37-mer duplex by a factor of at least 1000. The relative decay velocities of the ternary complexes again followed a similar trend as observed in the binding studies as follows: WT, T250C, T250S, T250G < T250N < T250D < T250H. Changes in KD values are thus largely attributable to changes in dissociation rates koff.


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Fig. 2.   Electrophoretic analysis of ternary MTase·DNA·AdoHcy complexes involving WT and Thr-250 mutants of M.HhaI. A, equilibrium binding reactions contained 1 nM GCGC/GMGC duplex, 100 µM AdoHcy, and 3 nM MTase (if any). Samples were incubated for 30 min and analyzed on a 8% polyacrylamide gel. B, dissociation kinetics of MTase·DNA·AdoHcy complexes as determined by competition binding with excess unlabeled DNA. Decay curves for WT (), T250C (black-square), T250S (triangle ), T250G (), T250N (open circle ), T250D (down-triangle), or T250H (black-triangle) M.HhaI along with double-exponential fits are shown.

Single-turnover Kinetics of Cytosine Methylation-- To obtain kinetic insights into events underlying the enzymatic transfer of the methyl group from AdoMet onto the target cytosine, we measured the rate of the methylation reaction under single-turnover conditions. For single-turnover measurements, saturating concentrations of protein and 3H-labeled AdoMet were employed to methylate the hemimethylated 37-mer DNA duplex. The reactions were stopped after specified incubation times with 2 N HCl, and the DNA was separated from unreacted radiolabeled cofactor and analyzed for 3H-methyl groups content. The progress curves followed a single-exponential reaction (Fig. 3A). The measured methylation rate constant kchem = 0.26 s-1 for WT M.HhaI agrees well with that recently reported (10). Interestingly, the six Thr-250 mutants were nearly as efficient as the WT enzyme in the methylation step (Table II). The highest decrease by a factor of 4 was observed for the T250H variant. These results are consistent with our previous finding that kcat values determined with poly(dG-dC) are quite uniform for all the mutants examined (16).


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Fig. 3.   Single-turnover kinetic analysis of M.HhaI. A, time course of 3H-methyl group incorporation into hemimethylated DNA at 37 °C. 100 nM GCGC/GMGC duplex and 200 nM M.HhaI were premixed and then rapidly mixed with 1.3 µM [methyl-3H]AdoMet (final concentrations). Reaction were then quenched in a timely manner with 2 N HCl, spotted on DE-81 filters, and processed as described under "Experimental Procedures." Data were analyzed by fitting to a single exponential equation. B, analysis of the extent of enzymatic methylation by restriction endonuclease cleavage. Rapid-quench reactions contained 100 nM 32P-labeled GCGC/GMGC duplex and 200 nM M.HhaI and 670 nM AdoMet (final concentrations) and were quenched after a specified period. The MTase-treated DNA was re-annealed with a 170-fold excess of the unlabeled lower strand oligonucleotide GCGC, and the resulting labeled duplex was subjected to cleavage with R.Hin6I and analyzed by electrophoresis on a 10% polyacrylamide gel.

                              
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Table II
Single-turnover kinetic parameters of cytosine and 5-fluorocytosine methylation by mutant (Mut) and WT M.HhaI

The amount of 3H-methylated DNA formed in the single-turnover experiments approximately equaled that of the starting hemimethylated duplex, although variations in different measurements by as much as 50% were observed (not shown). By taking into account that the methylation reaction by M.HhaI is irreversible (5), this would imply that the enzyme binds most hemimethylated sites in a productive orientation for catalysis. Randomly bound enzyme would have half of the molecules oriented in the alternative unproductive orientation and thus the amount of product obtained in the reaction cycle would be only 0.5 of the starting hemimethylated DNA. Discrimination between the two mechanisms might in principle be achieved by comparing the theoretical and experimental reaction amplitudes (10). We realized, however, that measurement of absolute 3H counts, due to significant quenching of low energy beta -particles on paper filters (25), may not be sufficiently accurate for the required 2-fold discrimination of reaction amplitudes. To exclude this sort of error, we determined the extent of methylation of the target strand by an independent method that is based on the ability of restriction endonucleases to discriminate between unmethylated and hemimethylated DNA (26). The hemimethylated duplex that was labeled on the target strand (32P-GCGC/GMGC) was subjected to single-turnover methylation reaction with M.HhaI and unlabeled AdoMet. The methylation products, which may contain either the original hemimethylated sites or the fully methylated product (32P-GMGC/GMGC), were reannealed with a large excess of the unlabeled unmethylated lower strand oligonucleotide GCGC. This leads to the displacement of the lower GMGC strand with GCGC yielding a labeled unmodified duplex (32P-GCGC/GCGC) or a labeled hemimethylated duplex (32P-GMGC/GCGC), respectively. Since the restriction endonuclease Hin6I does not fragment hemimethylated DNA, but is fully active on the unmodified GCGC sites,3 gel electrophoretic analysis of the labeled cleavage products thus affords reliable determination of the relative amounts of methylated and unmethylated GCGC sites produced during the reaction. The methylation time course, which included a zero time control and a reaction mid-point of 3 s (Fig. 3B), followed within error the exponential profile observed in the tritium experiment above (Fig. 3A). Less than 4% fragmentation was detected after methylation for 20 s or longer, indicating that nearly full modification of the target strand is achieved during the first turnover. This result directly demonstrates that M.HhaI binds the hemimethylated target sites in the productive orientation for catalysis with high selectivity.

Cofactor Exchange Analysis in the Binary MTase·AdoMet Complex-- Recent isotope partitioning studies of the MTase·[3H]AdoMet complex revealed no radiolabeled product in the presence of excess unlabeled AdoMet, which was interpreted by the authors (10) as lack of catalytic competence for this binary intermediate. However, the reported concentration of labeled AdoMet in the reaction mixture (400 nM) was much below the KD value of the binary complex (6 µM); only a small fraction of enzyme (6%) was bound in the binary complex at the moment when other reaction ingredients were added. Under these conditions, the radioactive product could hardly be observed, in particular at the 5-fold reduction of the specific radioactivity of [3H]AdoMet in the chase. Therefore, we performed a similar experiment in the presence of 1 µM enzyme and 20 µM labeled AdoMet (final concentrations in reaction), which was expected to give 76% saturation of enzyme. The reactions were allowed to proceed for 30 s (10 × t1/2) in the presence of 1 µM hemimethylated DNA duplex and a 125-fold excess of unlabeled AdoMet. Control reaction B contained no unlabeled cofactor in the chase; control C contained cofactor that was pre-diluted with unlabeled AdoMet at the 1:125 ratio in both pulse and chase (see legend to Fig. 4). The results presented in Fig. 4 for the hemimethylated 37-mer duplex indicate that incorporation of 3H label into the product under conditions A is discernibly higher than that in the control reaction C. The experiment was repeated several times with the 37-mer duplex or with poly(dG-dC) DNA using both hand-mixing and rapid mixing in a rapid-quench device, with similar results. This observation indicates that ~4-5% of the original binary complex (determined as (A - C)/(B - C)) is converted into product without going through steps of dissociation and rebinding of AdoMet.


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Fig. 4.   Isotope partitioning analysis of the binary M.HhaAdoMet complex. 3H Radioactivity incorporation into hemimethylated 37-mer duplex after 1 µM M.HhaI preincubated with 20 µM high specific activity [methyl-3H]AdoMet and then mixed with 1 µM DNA and 2.5 mM unlabeled AdoMet (A); 1 µM M.HhaI preincubated with 20 µM high specific activity [methyl-3H]AdoMet and then mixed with 1 µM DNA (B); 1 µM M.HhaI preincubated with 20 µM diluted (1:125) [methyl-3H]AdoMet and then mixed with 1 µM DNA and 2.5 mM diluted [methyl-3H]AdoMet (C). Reactions were incubated at 37 °C for 30 s and quenched with 0.5% SDS.

Fluorescence Spectroscopy and Kinetic Analysis of MTase-AdoMet Interaction in Solution-- M.HhaI contains a unique tryptophan residue (Trp-41), which is located in the cofactor binding pocket. It is thus not surprising that binding of cofactor AdoMet or product AdoHcy leads to a dramatic quenching of tryptophan fluorescence (Fig. 5A). In fact, the quenching is so strong that the 350 nm emission band is virtually eliminated at saturating concentrations of AdoMet or AdoHcy. Such a strong change in fluorescence intensity offered an opportunity to quantitatively follow MTase-cofactor interactions in solution (10, 27) at concentrations of fluorophore (M.HhaI) as low as 0.25-1 µM. The fluorescence titration curves with AdoMet (Fig. 5B) and AdoHcy (not shown) followed single-site binding isotherms reasonably well. The fitted binding constants are in the low micromolar range and are shown in Table III. Fitting to various two-step or two-site mechanisms (see below) did not give any improvement in the residuals. It thus can be concluded that in both cases the fluorescence binding experiments in solution are consistent with the simple binary interaction mechanism.


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Fig. 5.   Tryptophan fluorescence analysis of binary M.HhaI-AdoMet interaction in solution. A, emission spectra of M.HhaI (upper) and M.HhaI·AdoMet complex (lower) at lambda Ex = 290 nm. B, fluorescence titration of M.HhaI at 1 µM concentration with AdoMet (open circles). Data were fitted to a one-step single-site binding mechanism with DynaFit (line) to give a binding constant KD as shown in Table III. C, kinetic analysis of AdoMet binding. Fluorescence intensity progress curves at lambda Ex = 295 nm and lambda Em >320 nm were obtained by rapid mixing of 0.5 µM M.HhaI and 0, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 30, and 40 µM AdoMet (traces top to bottom) at 25 °C in a stopped-flow apparatus. Fits were obtained by global regression analysis of all 11 traces to the one-step reversible binding model. The fitted parameters are presented in Table III.

                              
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Table III
Kinetic and equilibrium constants for binary M.HhaI-cofactor interaction
The uncertainties are shown as 95% confidence intervals.

The fluorescence experiments were further extended to determine kinetics aspects of M.HhaI-cofactor interactions. Stopped-flow fluorescence measurements were performed with constant fluorophore and varied cofactor concentrations (Fig. 5C). Preliminary analysis showed that the progress curves could be fit quite well to the single-exponential equation. With both cofactors, a linear concentration dependence was observed (not shown) for experimental points corresponding to pseudo-first order reaction conditions ([cofactor] >=  5 × [M.HhaI]), suggesting a simple bimolecular binding mechanism. A more rigorous analysis was achieved by global fitting of the whole data set to a particular molecular mechanism. In this case, all experimental points can be used, and both the reaction rates and the amplitudes are utilized in the fitting process (20). As in the preliminary analysis above, the one-step mechanism gave a satisfactory fit (Fig. 5C). The obtained kinetic parameters (Table III) are internally consistent with the titration data.

In light of the reported two types of M.HhaI·AdoMet complexes in crystals (8, 9) and also in solution (10), we considered an alternative mechanism with two different binary complexes (two binding orientations of cofactor molecule) at the same binding site: MTase + AdoMet left-right-arrow complex I + complex II. The refined squared residual was slightly lower (7.9 · 10-6 versus 13.4 · 10-6) with this model. Given the higher number of parameters refined, the observed improvement of fit appears marginal for unequivocal discrimination between the two mechanisms. The KD values for complexes I and II were refined at 8 and 170 µM, respectively. This means that at submicromolar or lower concentrations, only one type of MTase·AdoMet complex is observed (complex I), since the contribution from the second binding mode is negligible. However, even at high micromolar and millimolar AdoMet, the occupancy of the second state (complex II) would not exceed 5%. Almost identical results were obtained from analogous analysis of AdoHcy binding (not shown). Therefore, even if the dual binding mode exists, it is not cofactor-specific. This makes unlikely that the observed effect is due to impurities present in AdoMet preparations, because AdoHcy, which is usually quite easy to purify and preserve from decomposition, also showed similar behavior. The most plausible explanation is that a small fraction of the protein exists in a different conformation with respect to cofactor binding, or perhaps is even inactive in our preparations. However, the dominant species (>95%) observed in solution is a fast-reversible single-orientation binary complex.

Pre-steady-state and Steady-state Kinetics-- Multiple-turnover kinetic analysis of WT HhaI MTase with the multisite substrate poly(dG-dC)·poly(dG-dC) suggested that the enzyme operates by an ordered Bi Bi mechanism where DNA binds first (5). Comparison of the Thr-250 mutants under similar conditions (16) indicated that their kcat values are very similar, whereas certain mutations lead to substantial increases in K<UP><SUB>m</SUB><SUP>DNA</SUP></UP> and K<UP><SUB>m</SUB><SUP>AdoMet</SUP></UP>. In the present work we determined the steady-state parameters of WT M.HhaI using a 37-mer DNA duplex with a unique hemimethylated GCG(C/G)MGC site as a substrate. The multiple turnover rate kcat = 0.03 s-1 with the hemimethylated DNA agrees with that previously reported with the same DNA (28), is 2-fold higher than that with unmethylated 37-mer duplex (24), and is similar to kcat = 0.02 s-1 observed with the unmethylated copolymer DNA (5, 16). The K<UP><SUB>m</SUB><SUP>AdoMet</SUP></UP> = 45 nM obtained with the hemimethylated duplex is 3-fold higher than K<UP><SUB>m</SUB><SUP>AdoMet</SUP></UP> = 15 nM with poly(dG-dC). Overall, we find that the macroscopic kinetic parameters are fairly similar with both types of DNA. In the recent kinetic study of M.HhaI with 30-mer hemimethylated DNA (10), somewhat higher values were reported, K<UP><SUB>m</SUB><SUP>AdoMet</SUP></UP> = 280 nM and kcat = 0.08 s-1.

Comparison of kchem and kcat is important for understanding the partitioning of intermediates along the reaction pathway and assigning the rate-limiting step. However, direct comparison of these two rate constants may be problematic because kchem is measured under single-turnover conditions and is derived from the time dependence of product formation and is thus independent of absolute tritium counts. In contrast, kcat was obtained by determination of the absolute amount of tritiated methyl groups transferred under steady-state conditions. As discussed above, this type of measurement may lead to relatively large errors due to quenching of tritium counts. To measure both rate constants in a single experiment, reaction progress must be followed during the first and subsequent turnovers. The Equation 1 describing product formation for an irreversible reaction includes both exponential and linear terms (20),
[<SUP><UP>Me</UP></SUP><UP>DNA</UP>]=[<UP>MTase</UP>] · (&pgr; · (1−e<SUP><UP>−</UP>&lgr;t</SUP>)+k<SUB><UP>cat</UP></SUB> · t)=A<SUB>0</SUB>{(k<SUB><UP>chem</UP></SUB>/(k<SUB><UP>chem</UP></SUB>+k<SUB><UP>off</UP></SUB>))<SUP>2·</SUP> (Eq. 1)

(1−<UP>exp</UP>(−(k<SUB><UP>chem</UP></SUB>+k<SUB><UP>off</UP></SUB>) · t)+k<SUB><UP>chem</UP></SUB> · k<SUB><UP>off</UP></SUB>/(k<SUB><UP>chem</UP></SUB>+k<SUB><UP>off</UP></SUB>) · t}
where the burst amplitude pi  = (kchem/(kchem + koff))2, burst rate lambda  = kchem + koff, and the linear slope kcat = kchemkoff/ (kchem + koff). Since all terms contain contributions from both kchem and koff, it is thus theoretically possible to extract both rates constants by shape analysis of progress curves. Such analysis is less dependent on the absolute counts or enzyme concentration, etc., which are defined by the scale factor A0. Indeed, the obtained pre-steady-state reaction profile (Fig. 6) shows a clear initial burst followed by a linear phase of product formation. The progress data were analyzed by fitting to the expanded form of Equation 1 yielding the two rate constants. The values of kchem from the single-turnover experiment and the pre-steady-state burst are identical within error. The values for koff = 0.045 s-1 and kcat = 0.04 s-1 inferred from the latter experiment also agree well with kcat obtained under steady-state conditions (see above). Our data demonstrate in a quantitative manner that product release (koff) is substantially slower than all preceding steps and is thus the major contributor to the multiple turnover rate of the HhaI MTase. At both 25 °C (not shown) and 37 °C, we find that the ratio kchem/kcat is about 6-8 which is slightly higher than that (3-fold) reported by Reich and co-workers (10).


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Fig. 6.   Pre-steady-state kinetic analysis of M.HhaI. Time course of 3H-methyl group incorporation into hemimethylated DNA at 37 °C was obtained with 200 nM GCGC/GMGC duplex, 15 nM M.HhaI, and 1.3 µM [methyl-3H]AdoMet (final concentrations). Reactions were quenched at specified times with 2 N HCl and processed in quadruplet as described in Fig. 3. Experimental data were fitted to the full burst equation for irreversible reaction (see Equation 1). The refined parameters are as follows: kchem = 0.26 s-1, koff = 0.045 s-1, and A0 = 1200 cpm.

Kinetics of 5-Fluorocytosine Methylation-- 5-Fluorocytosine (5FC) is an isosteric analogue of cytosine and is widely used for studying the mechanisms of enzymes performing covalent transformation of pyrimidine nucleotides. It was demonstrated that C5-MTases undergo AdoMet-dependent inactivation by forming stable covalent protein-DNA complexes (13, 14, 29). The reaction with 5FC proceeds through the steps of covalent activation and methyl transfer (Fig. 1A, steps 42 and 53), but the dihydrocytosine product containing methyl and fluorine substituents at C-5 cannot resolve in the usual manner with release of enzyme (steps 44 and 45 are blocked). In this work, we compared quantitatively the efficiency of the Thr-250 mutants in this single-turnover methylation reaction. The rate of covalent trapping of DNA containing 5FC corresponds to kchem determined for the native substrate in the rapid-quench experiment above. The formation of such trapped complexes of M.HhaI with the hemimethylated 37-mer GFGC/GMGC was analyzed by polyacrylamide gel electrophoresis in the presence of 0.1% SDS (Fig. 7). Concentrations of the substrates in this experiment were held well above the known nanomolar Ki and Km values determined for reaction with poly(FdC-dG) (13). However, there was a theoretical possibility that Km for AdoMet was significantly higher in the case of mutants, and the observed reaction rates would then underestimate the intrinsic rate constant due to incomplete saturation of enzyme. To exclude this possibility, we performed control reactions with WT M.HhaI and the T250N mutant in which the concentration of AdoMet was varied in the range from 40 to 1000 µM. No variations in the covalent trapping rate were detectable (not shown) indicating that full saturation of the enzyme active site was attained. The progress data were fitted to a single exponential equation to give the single-turnover rate constant for methyl group transfer onto 5-fluorocytosine k<UP><SUB>chem</SUB><SUP>5FC</SUP></UP>. The observed methylation rate for WT M.HhaI in the hemimethylated 37-mer duplex (0.04 min-1) is in excellent agreement with that measured for poly(FdC-dG) (0.05 min-1) (13) and is about 400-fold slower than kchem for cytosine. Remarkably, this reaction proved highly discriminative with respect to the Thr-250 mutants (Table II). A small decrease in methylation rate (~4-fold) was observed even for the T250S and T250G mutants, and a substantial drop (~20-fold) in the case of T250N and T250D. The activity of the T250H mutant was barely detectable, <5% conversion in 30 h which gives an estimated rate constant of 3·10-5 min-1 (~1300-fold slower than WT). Similarly, loss of activity toward 5FC has been observed in the T237V mutant of MspI MTase (30).


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Fig. 7.   Kinetics of covalent trapping of Thr-250 mutants of M.HhaI with 5-fluorocytosine. Reactions containing 2.7 µM of MTase, 400 µM AdoMet, and 7 µM 37-mer hemimethylated duplex (GFGC/GMGC) were incubated at 37 °C. Aliquots were withdrawn at time intervals from 0 to 25 h and quenched by heating with 0.5% SDS at 100 °C for 5 min. Samples were analyzed by electrophoresis on 10% polyacrylamide gel in the presence of SDS and processed as described. Progress curves of covalent complex formation for WT (), T250C (black-square), T250S (triangle ), T250G (), T250N (open circle ), T250D (down-triangle), or T250H (black-triangle). The data were fitted to the single-exponential equation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enzymatic cytosine-5 methylation in DNA proceeds by a series of molecular events that involve binding of substrates, dramatic conformational rearrangements in both the DNA and protein, covalent transformations in the catalytic site of enzyme, and release of products (see Introduction). We attempted to integrate the vast biochemical, kinetic, and structural information available on M.HhaI into a general mechanistic scheme (Fig. 1B). This extensive scheme of interactions and transformations includes theoretically possible kinetic routes that connect multiple intermediates and serve as a working framework for further studies of the reaction mechanism. It is the aim of this and subsequent studies to establish the reaction pathway by determining the thermodynamic and kinetic contributions of individual steps on the catalytic cycle. Therefore, discussions of the results are held in the context of the proposed scheme (Fig. 1B).

MTase-Cofactor Interactions and the Catalytic Competence of the MTase·AdoMet Complex-- Previous studies found that AdoMet can bind in two different orientations in the cofactor binding pocket as observed in two types of nearly isomorphous crystals obtained at different conditions. In crystals of the binary M.HhaI·AdoMet complex produced in the presence of a short nonspecific DNA duplex, AdoMet binds in a primed orientation (9), identical with that observed in all available structures of the ternary complexes involving enzyme, cognate DNA, and AdoHcy or AdoMet (4, 28, 31-33). This orientation differs from the previously observed unprimed orientation in the binary M.HhaI·AdoMet complex, where the methylsulfonium center of AdoMet is in contact with the aromatic ring of Trp-41 (34). Our fluorescence solution studies of M.HhaI (Fig. 5) indicate the dominance of one binding mode in solution (at least 95% of the population) for both AdoMet and AdoHcy. The high efficiency of fluorescence quenching observed in the M.HhaI·cofactor complexes (Fig. 5A), which show emission spectra nearly identical with that of the W41F mutant,4 would be more consistent with the primed orientation (9) in which the purine ring of cofactor stacks face-to-face with the aromatic ring of Trp-41 (35). Moreover, four analogues of AdoHcy that have deletions in various parts of the molecule but retain the adenosine moiety also exhibit strong quenching of Trp-41 fluorescence upon binding to enzyme (27). Therefore, we conclude that both AdoMet and AdoHcy bind to M.HhaI in a similar manner in solution, which is most probably the primed orientation.

A somewhat unexpected finding is that the binary M.HhaI·AdoMet complex dissociates and rebinds cofactor at a rate of ~20 s-1 (Fig. 5 and Table III). Such a rapid exchange is in apparent discord with previous reports that M.HhaI·AdoMet complex is unusually stable, since it is capable of surviving certain steps of chromatographic purification (7, 9). Consequently, preparation of AdoMet-free enzyme by the high salt back-extraction procedure (7) requires multiple rounds of dialysis (34). This apparent controversy could be explained by that fact that the concentration of protein during purification is usually near or above the KD value for the binary complex (in the order of 10-50 µM). Under these circumstances, considerable retention of AdoMet by the enzyme during dialysis could occur due to thermodynamic stability, which is independent of the exchange rate. This idea alone is insufficient to explain why AdoMet is retained during purification of M.HhaI on cation-exchange columns, such as Mono-S (7). It is possible, however, that the enzyme and AdoMet happen to exhibit comparable retention properties on the ion-exchange column, and both elute in partially overlapping fractions. Indirectly, this notion is supported by the apparent lack of bound AdoMet in M.HhaI preparations obtained by different procedures that involve two different chromatographic purification steps (10, 36).

The results presented in Fig. 4 demonstrate that, despite the fast exchange rate, a certain fraction of the binary M.Hha [3H]AdoMet complex survives during reaction in the presence of excess unlabeled AdoMet leading to the formation of labeled product. This observation has two important mechanistic implications. In quantitative terms, the amount of trapped label is defined by the ratio of rates for exchange/forward reactions (37). Given the dissociation rate constant of ~20 s-1 at 25 °C (Fig. 5 and Table III), which may be somewhat higher at 37 °C, the observed ~5% retention of the original label in products at 37 °C might suggest that cofactor is trapped into a committed complex with an apparent rate of >= 1 s-1. The only known irreversible forward step is methyl transfer, whose rate (kchem = 0.26 s-1) seems too low to account for the inferred trapping rate. Therefore, AdoMet is likely locked into a committed ternary complex (probably DNF·MT·AM or DNF·MTL·AM) that is observed before the methyl transfer step. This hypothesis is now undergoing thorough experimental examination.

The above considerations also imply that AdoMet does not necessarily need to dissociate and rebind after DNA binds as earlier suggested, but rather the binary M.HhaI·AdoMet complex can bind DNA productively (step 12). The following indirect evidence exits in favor of the catalytic competence of MTase·AdoMet complex. No inhibitory effect on the initial velocity (5, 10) and burst magnitude (10) was observed at concentrations of AdoMet as high as 1 mM. If the M.HhaI·AdoMet complex (MT·AM) was a dead-end complex as suggested, then all terms in the denominator of the rate equation representing free enzyme would be multiplied by (1 + [AdoMet]/K<UP><SUB>D</SUB><SUP>AdoMet</SUP></UP>) (38), resulting in detectable inhibition at concentrations above K<UP><SUB>D</SUB><SUP>AdoMet</SUP></UP>. Such an effect would be observable regardless of whether the complex is in fast exchange or not, since dead-end reactions are at equilibrium at steady-state in any mechanism (39). Similarly, there was no discernible inhibitory effect of AdoMet (at 0.3 mM) on the fast kinetics of DNA binding and base flipping as determined in stopped-flow fluorescence studies.4 In aggregate, the presented direct and indirect evidence strongly suggests that the MTase·AdoMet complex is catalytically competent, and thus a certain portion of the reaction is routed through this intermediate. From structural standpoint, this conclusion is consistent with the primed orientation of AdoMet in the binary complex (see above).

Strand Selectivity on Hemimethylated DNA-- Binding and recognition of the target GCGC site in DNA is a key early event in the catalytic cycle (Fig. 1B, steps 11 and 12). The most common state of the target site in vivo is hemimethylated DNA, which is produced after replication of a fully methylated duplex. It is therefore important to understand how MTases, which usually act as monomers (1) and should not a priori exert dyad symmetry on pseudo-symmetric sites, interact with hemimethylated DNA? In other words, are MTases able to distinguish between the unmethylated and methylated strand and bind in the productive orientation targeting the unmethylated strand. It turns out that there is no common rule, and particular classes or even individual enzymes appear to have different solutions to the problem. Kinetic studies of the adenine-N-6 MTases Ecodam (40), EcoRI (41), EcaI (42), and EcoRV (43) suggest that these enzymes show little, if any, orientation preference during catalysis, although some binding preference for hemimethylated versus unmethylated sites has been reported for M.EcoRV (44) and M.RsrI (45). Leaving aside the eukaryotic C5-MTases for which selectivity for hemimethylated DNA is crucial for their maintenance function (46, 47), the bacterial C5-MTases also show clear differences in how unmethylated and hemimethylated substrates are processed (48, 49). For M.HhaI, a high degree of strand discrimination in the presence of AdoHcy (10, 32, 49) has been demonstrated. Several lines of indirect evidence suggest that this may also be true at the level of binary complex. For instance, the DNA binding preference by M.HhaI in the absence of cofactor is hemimethylated > unmethylated > fully methylated (10, 23, 24, 32), suggesting that the enzyme can sense whether the target base is methylated (negative factor) and whether the cytosine on the opposite strand is methylated (positive factor). If these effects are additive, then the enzyme should be able to distinguish the two orientations based on a two positives versus two negatives rule. Recently, based on the relative amounts of 3H-methyl groups incorporated into unmethylated and hemimethylated substrates under single-turnover conditions, it has been suggested that half of the catalytic hemimethylated binding events by M.HhaI occurs in the unproductive orientation (10). The results of our restriction endonuclease analysis disagree with the latter interpretation. Simultaneous determination of the methylated DNA and the original substrate in reaction products clearly indicates that, at least under conditions of our experiment, the target cytosine in the hemimethylated substrate undergoes complete methylation during the initial burst phase (first turnover) (Fig. 3B). Therefore, we conclude that M.HhaI binds hemimethylated target sites in the productive orientation with high selectivity.

Dissection of the Chemistry Step-- To assess the relative contributions from individual events on kchem, we took advantage of the Thr-250 mutants of M.HhaI that introduce moderate structural perturbations in the catalytic center. The T250N, T250D, and T250H mutations lead to a decreased affinity of the MTase toward cofactor in the respective ternary MTase·DNA·cofactor complexes (elevated Km for AdoMet (16)) as well as weaker effect of AdoHcy on the stability MTase·DNA complex (higher KD and koff values in the presence of AdoHcy, Table I). In principle, such a behavior could be expected if the mutations were introduced in the cofactor binding pocket. However, Thr-250 is situated >13 Å away from the bound cofactor and thus can have only an indirect influence on the MTase-cofactor interactions. Indeed, we found that K<UP><SUB>D</SUB><SUP>AdoMet</SUP></UP>, determined in the absence of DNA, is very similar for WT, and the T250N and T250H mutants (not shown). Inspection of the crystal structures for M.HhaI and its T250G mutant indicates a fairly tight packing of atoms in the vicinity of Thr-250 (see Fig. 6 in Ref. 16), and therefore, introduction of larger side chains should inevitably lead to steric clashes with adjacent residues. The most likely atoms whose geometry would be altered are the side chains of Arg-165, Arg-163, and Lys-162, the O-4' atom in the sugar of the flipped out 2'-deoxycytidine, and several backbone atoms of the 5'-neighboring nucleotides on the target strand. These perturbations may propagate further to alter the positions of atoms in the flexible catalytic loop and/or even shift the conformational equilibrium of the loop toward the open state. Since the affinity of M.HhaI for cofactor is dramatically dependent on the formation of the closed ternary complex (see Table I and III)5 even slight conformational alterations in the catalytic loop and the target base would have a strong effect on M.HhaI-cofactor interactions. This notion is consistent with the lower overall compactness of the ternary complexes involving T250N and T250H (Fig. 2A).

Remarkably, neither kchem (Table I) nor kcat values (16) are substantially affected in the Thr-250 mutants as compared with the WT enzyme, which means that the conformational distortions in the reaction complex can be compensated by an appropriate increase of cofactor concentration. This implies that although the affinity of the bound cofactor is altered, this does not contribute to the rate of a rate-limiting step. Notably, a similar overall effect (higher K<UP><SUB>m</SUB><SUP>AdoMet</SUP></UP>, similar kcat) was observed in the EcoRII MTase when the geometry of the active site was perturbed by mutations of Pro-185 which is adjacent to the catalytic Cys-186 (50). From this perspective it is interesting to compare the effects of replacing the 4'O atom in the target nucleotide with sulfur on the behavior of M.HhaI (28). Due to its larger van der Waals radius, the sulfur atom appears to push the nearby Arg-165 residue with concomitant increase in separation between the terminal amino groups of Arg-165 and O-2 in the target cytosine. Thus the structural consequences of the latter substitution and the Thr-250 mutations may be similar. As observed with the Thr-250 mutants, the 4'-thiolated substrate shows essentially no effect on the binary interaction with DNA (K<UP><SUB>D</SUB><SUP>DNA</SUP></UP>); however, the stability of the ternary complex with AdoHcy is substantially decreased (10-fold increase in koff) (28).

A different picture emerges when the target cytosine is replaced with 5-fluorocytosine. The methylation rate kchem by the WT enzyme drops 400-fold upon introduction of a fluorine atom at C-5 of the cytosine ring. The mutants again lead to effects whose magnitudes are very similar to those discussed above. For instance, the relative change in K<UP><SUB>m</SUB><SUP>AdoMet</SUP></UP> observed with canonical DNA and the relative drop in the rate of covalent trapping with 5FC-DNA all increase in the order Asn < Asp < His and differ from that of WT enzyme by ~1, 2 and 3 orders of magnitude, respectively (Tables I and II). However, the major difference between cytosine and 5FC methylation is that in the latter case the reaction cannot be rescued by a mere increase of exogenous AdoMet concentration. Perturbations in the catalytic center induced by the mutations translate into the lower rates of methyl transfer onto 5FC, which means that in this case the velocity of a rate-limiting step is affected. Altogether, our findings allow us to conclude that different events determine the rate of methyl transfer (kchem) for cytosine and for 5-fluorocytosine.

As mentioned in the Introduction, the catalytic mechanism of cytosine C-5-methylation involves a step of covalent bond formation between Cys-81 and C-6 position in the ring, which is likely coupled with protonation of N-3 to yield an enamine intermediate (5, 11, 12). The C-5 can then attack the methyl sulfonium center on AdoMet resulting in the transfer of the methyl group onto the target base (51). Molecular calculations suggest a very favorable enthalpy change for the step of methyl transfer from AdoMet onto the covalently activated cytosine, which therefore should be relatively fast. The preceding formation of the covalent intermediate (step 42) is an endothermic reaction and is a likely candidate to limit the overall methylation rate of cytosine (52).

Replacement of the C-5-hydrogen with a much more electronegative fluorine atom in the cytosine ring is expected to lower the electronic density at C-5. Such an electron withdrawing effect is thought to increase the reactivity of the ring toward nucleophilic addition at C-6 (53), similar to the effects of N-3 protonation (11, 12, 54) or removal of the 4-amino group (30). Therefore, the formation of the covalent intermediate at C-6 (step 42) is unlikely to be adversely affected by the presence of fluorine at C-5. However, the negative inductive effect should reduce the nucleophilic character and reactivity of C-5 in the SN2 transmethylation reaction. By taking into account our kinetic results, we propose that methyl transfer (step 53) is rate-limiting in the case of 5-fluorocytosine and accounts for the 400-fold reduction of the overall methylation rate kchem. It is, however, not a major contributor to kchem in the case of cytosine; rather, the formation of the covalent intermediate or some other prior step must be rate-limiting for the single-turnover reaction.

Kinetic Mechanism of HhaI Methyltransferase-- Pre-steady-state reaction profiles with an exponential burst followed by a phase of linear product build up have been recently demonstrated for M.HhaI (10) and for another C5-MTase, M.MspI (6). The parallel measurement of the rate of methylation kchem and product release koff in a single experiment (Fig. 6) provides firm quantitative evidence that a step following methyl transfer is the major contributor to the rate catalytic turnover kcat of M.HhaI. However, it is not clear which particular step (upwards from step 53) is rate-limiting. Interestingly, rate-limiting product release appears to be a feature characteristic also of some site-specific DNA adenine MTases (43, 55, 56), restriction endonucleases (57, 58), and DNA-repair enzymes (59).

Comparison of the rates on the sequential pathway of M.HhaI clearly shows that under steady-state conditions the enzyme is largely partitioned in reaction intermediates in the forward direction. The rate of decay of the binary MTase·DNA complex is in the range of 0.04-0.3 s-1 (5, 13),6 which is similar or slower than the rate of chemical methyl transfer (0.26 s-1). DNA partitioning experiments show that the starting MTase·DNA complex is largely converted to products in the presence of competitor DNA (10).6 Clearly, the rapid equilibrium assumption (koff kforward), which was originally established with a copolymer substrate, poly(dG-dC) (5), is not valid (step 11 is relatively slow) for DNA fragments containing hemimethylated target sites. On the other hand, our fluorescence solution studies (Fig. 5C and Table III) and isotope partitioning experiments (Fig. 4) indicate that AdoMet is exchanged rapidly (step 61), indicating a rapid equilibrium mechanism with respect to cofactor. This difference (rapid equilibrium for AdoMet versus steady-state for DNA) may account for the apparent kinetic inequality of the two substrates. By taking into account that AdoMet and AdoHcy can bind to enzyme in the absence of DNA, a compulsory binding order does not appear to be necessary. The key question for the formal assignment of the mechanism between compulsory order or random order appears to be the catalytic competency of the binary MTase·AdoMet complex. As discussed in the preceding sections, the binary M.HhaI·AdoMet complex can bind DNA productively (step 12 is possible) and thus may lie on the catalytic pathway. Therefore, on the basis of the data currently available, we suggest that the most likely mechanism of the HhaI MTase is a partial rapid equilibrium random Bi Bi with DNA at steady-state (60). The proposed mechanism is incompatible with any mechanism that implies a dead-end catalytically incompetent (10) MTase·AdoMet complex.

In retrospect, our conclusions should be regarded as further refinement of the kinetic mechanism determined by steady-state analysis of Santi and co-workers (5). One reason why the formation of the binary MTase·cofactor complex was not observed in those early studies is that K<UP><SUB>D</SUB><SUP>AdoMet</SUP></UP> = 6-11 µM turns out to be 3 orders of magnitude higher than K<UP><SUB>m</SUB><SUP>AdoMet</SUP></UP> = 15 nM (Table III). In contrast, K<UP><SUB>D</SUB><SUP>DNA(binary)</SUP></UP> is similar with K<UP><SUB>m</SUB><SUP>DNA</SUP></UP> (both within 1-15 nM) (Table I and see Refs. 5, 10, and 16). Therefore, the contribution of the binary MTase·cofactor complexes to the steady-state rate is rather small, and thus the compulsory binding order mechanism appears a plausible choice. On the other hand, although binary interactions with AdoMet are weaker than with DNA, the MTase·cofactor complexes may play an important role in vivo. For example, intracellular concentrations of AdoMet in Escherichia coli are reported to be on the order of 30-300 µM (61). These numbers are well above K<UP><SUB>D</SUB><SUP>AdoMet</SUP></UP>, suggesting that the enzyme predominantly exists in the cofactor-bound form (7). This way a molecule of AdoMet is used immediately or protected from decomposition in the binding cavity of enzyme.

    ACKNOWLEDGEMENTS

We thank Prof. R. Goody and MBI Fermentas for continuous support.

    FOOTNOTES

* This work was supported in part by a Volkswagen-Stiftung collaborative research grant (to S. K. and E. W.) and a Howard Hughes Medical Institute International Research scholarship (to S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 370 2 602114; Fax: 370 2 602116; E-mail: klimasau@ibt.lt.

Published, JBC Papers in Press, March 29, 2001, DOI 10.1074/jbc.M101429200

2 The decay rates obtained with single-exponential approximations are ~20% higher than the slower rates of the double-exponential fits.

3 Extensive digestion of a hemimethylated DNA duplex with excess R.Hin6I may lead to nicking of the unmethylated strand, with no detectable cleavage of the methylated strand (Z. Maneliene and A. Janulaitis, unpublished observations). No fragmentation of the hemimethylated 37-mer duplex with R.Hin6I was observed in our experiments.

4 E. Merkiene and S. Klimasauskas, unpublished observations.

6 S. Serva, G. Vilkaitis, and S. Klimasauskas, unpublished observations.

5 E. Merkiene· and S. Klimasauskas, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; C5-MTase, DNA cytosine-5-methyltransferase; 5mC, 5-methylcytosine; 5FC, 5-fluorocytosine; M.HhaI, HhaI DNA methyltransferase; MeDNA, methylated DNA; MTase, DNA methyltransferase; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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