From the Institute of Biotechnology, Laboratory of Biological DNA
Modification, LT-2028 Vilnius, Lithuania and Institut
für Organische Chemie der RWTH Aachen,
D-52056 Aachen, Germany
Received for publication, February 14, 2001, and in revised form, March 28, 2001
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ABSTRACT |
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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 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 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.
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 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
(
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 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.
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).
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 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
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
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 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.
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]
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 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
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),
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 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
The results presented in Fig. 4 demonstrate that, despite the fast
exchange rate, a certain fraction of the binary M.HhaI· [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
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 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
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
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
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
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 K1, KD = 6 µM) is partially converted into products during
isotope-partitioning experiments, suggesting that it is catalytically
competent. Chemical formation of the product
M.HhaI·MeDNA·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
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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ex) of 280 or 290 nm (slitwidths 1 and 2 mm) and
emission wavelength (
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).
ES) or
one-step dual-mode mechanism (E + S
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
Effects of Thr-250 mutations on interaction of M.HhaI with
hemimethylated DNA
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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 ?.
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 (
), T250S (
), T250G (
), T250N
(
), T250D (
), or T250H (
) M.HhaI along with
double-exponential fits are shown.
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.
Single-turnover kinetic parameters of cytosine and 5-fluorocytosine
methylation by mutant (Mut) and WT M.HhaI
-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.
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.HhaI·AdoMet 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.
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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 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
Ex = 295 nm and
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.
Kinetic and equilibrium constants for binary M.HhaI-cofactor
interaction
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.
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.
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
1.
(Eq. 1)
where the burst amplitude
= (kchem/(kchem + koff))2, burst rate
= 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.
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 (
), T250S (
), T250G (
), T250N (
), T250D
(
), or T250H (
). The data were fitted to the single-exponential
equation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
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.
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.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. R. Goody and MBI Fermentas for continuous support.
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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. Merkien and S. Klima
auskas, unpublished observations.
6
S. Serva, G. Vilkaitis, and S. Klimaauskas,
unpublished observations.
5
E. Merkiene· and S. Klimaauskas, manuscript in preparation.
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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.
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REFERENCES |
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