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INTRODUCTION |
Prokaryotic DNA methyltransferases
(MTases)1 are usually
components of restriction-modification (R-M) systems that enable cells to resist propagation of foreign genomes that would otherwise kill them
(1). DNA methylation is catalyzed by
S-adenosyl-L-methionine (AdoMet)-dependent DNA methyltransferases. Based on the
position of methyl group transfer on bases in DNA, DNA MTases are
classified into two groups, exocyclic or amino methyltransferases and
endocyclic or ring methyltransferases (2). The amino methyltransferases methylate exocyclic amino nitrogen and form either
N6 methyladenine or N4
methylcytosine, whereas ring methyltransferases methylate ring carbon
and form C5 methylcytosine. All exocyclic MTases
share a common architectural plan. They have nine highly conserved
motifs, and these can be arranged in different combinations (3). The
amino methyltransferases are further subdivided into three groups,
namely
,
, and
, which are characterized by a distinct linear
order of the AdoMet binding region (FXGXG),
target recognition domain, and catalytic (DPPY) motif. Target
recognition domains are long sequences containing no conserved amino
acids. The low amino acid sequence similarity between target
recognition domains explains the specificity of a particular
methyltransferase for a specific sequence.
Several type I and type II R-M systems have been isolated from strains
of Klebsiella pneumoniae. These include KpnI,
KpnK14I (an isoschizomer of KpnI),
Kpn2I (an isoschizomer of BspMII), Kpn49kI (an isoschizomer of EcoRI),
Kpn49kII (an isoschizomer of NciI), and various
yet unnamed enzymes that are isoschizomers of EcoRII,
BssHII, and PstI (4). Although the type I systems includes KpnAI (5) and KpnBI (6), KpnI
restriction-modification system belongs to the type II R-M systems. The
genes for KpnI endonuclease and methyltransferase are
present within close proximity to each other in K. pneumoniae genome (7). KpnI MTase recognizes the
recognition sequence 5'-GGTACC-3' (8) and modifies the DNA by
methylating the adenine residue at the N6
position (9). Based on the conserved motif arrangement, KpnI MTase belongs to the
-subgroup of MTases. Comparison of the amino acid sequence of KpnI MTase with the sequences of various
MTases revealed a significant homology with
N6-adenine MTases and partial homology with
N4-cytosine MTases but no sequence homology with
C5-cytosine MTases. Dot matrix comparison of
KpnI MTase with several N6-adenine
MTases and N4-cytosine MTases showed that it has
homology with the catalytic motif (DPPY) and AdoMet binding motif
(FXGXG) of most N6-adenine
MTases and only the FXGXG region of
N4-cytosine MTases. Interestingly, the amino
acid sequence of KpnI MTase is more closely related to those
of EcoP1 MTase and EcoP15I MTase, which belong to
type III R-M systems (7). Among all the MTases characterized so
far, most of them are monomers, whereas their cognate endonucleases are dimers.
Kinetic mechanisms of C5 MTases such as
HhaI (10-13), murine DNMT1 (14), MspI
(15), human DNMT1 (16), and mammalian DNMT3a (17) have been determined.
All known cytosine methyltransferases studied so far follow an ordered
bi bi mechanism with DNA as the leading substrate. Kinetic mechanisms
for members of the N4 cytosine MTase family such
as PvuII and N6-adenine MTases such
as EcoP15I (18), EcoRI (19, 20), RsrI (21), EcaI (22), CcrM (23), TaqI (24),
EcoRV (25), and T4 Dam (26) have also been elucidated.
Bacterial N6-adenine methyltransferases, in
general, exhibit different kinetic mechanisms.
Our aim is to study the interaction between DNA and KpnI
MTase by kinetic analysis of the enzymatic reaction. In the present study we report the oligomeric nature of KpnI MTase using
gel filtration, chemical cross-linking, and enzyme
concentration-dependent analysis. The kinetic parameters
for methylation reaction with DNA substrates of different length by
KpnI MTase have been determined. We also report the order of
substrate binding and product release using initial velocity dependence
analysis, product inhibition, substrate inhibition, and steady state
kinetic analysis. Our results demonstrate that KpnI MTase
exists as a dimer in solution and functions as a dimer during the
methylation reaction. The methylation reaction proceeds through a
steady state ordered bi bi kinetic mechanism in which AdoMet binds
first followed by DNA.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Plasmids Used--
Escherichia
coli stain DH10B [mcrA
(mrr hsd RMS
mcrBC) endA1 080 dlacZ
M15
lac X74 recA1 deoR
(ara,
leu) 7697ara D139 galU galK
nupG rpsL] was used as a host strain for
transformation of pACMK clones, which encode KpnI MTase
(27).
Enzymes and Chemicals--
Restriction and modifying enzymes
were purchased from New England Biolabs.
S-Adenosyl-L-[3H]methionine (78.9 Ci/mmol) was purchased from PerkinElmer Life Sciences, and AdoMet,
chloramphenicol, bovine serum albumin, HEPES, polyethyleneimine,
Coomassie Brilliant Blue, RNase A, glutaraldehyde, and
S-adenosyl-L-homocysteine (AdoHcy) were
purchased from Sigma. AdoHcy was prepared by dissolving 38.44 mg into 1 ml of 1 N HCl to give a 100 mM solution.
Dilutions were made fresh in distilled water. Phosphocellulose P11 and
DE81 anion-exchange filter papers were purchased from Whatman. All
other chemicals used were of highest grade purity. Centricon 10 microconcentrator units were purchased from Amicon. Oligonucleotides
for methylation assays were purchased from Microsynth GmbH, Switzerland
and Bangalore Genei Pvt. Ltd., India. Oligonucleotides used in this
study are listed in Fig. 1.
Double-stranded DNA concentration was measured spectrophotometrically,
assuming an A260 of 1.0 to correspond to 50 µg/ml for double-stranded DNA.

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Fig. 1.
Duplex substrates. Duplexes I-VII are
noncanonical oligonucleotides for KpnI MTase recognition
sequence. Duplex VIII (20-mer) and IX (38-mer) are oligonucleotides
containing canonical sequences, whereas duplex X is a hemimethylated
oligonucleotide sequence of KpnI MTase. A complementary
strand was used for all the oligonucleotides. In duplex XI,
B represents biotin labeled at the 5' end.
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Expression and Purification of KpnI MTase--
KpnI
methyltransferase was expressed in E. coli DH10B cells
containing plasmid pACMK (27). E. coli cells harboring
KpnI MTase clone pACMK were grown for 12-16 h at 37 °C
in LB medium containing 20 µg/ml chloramphenicol, and cells were
harvested by centrifugation for 10 min at 10,000 rpm. Harvested cells
(10 g) were suspended in 20 ml of buffer A (10 mM potassium
phosphate, pH 7.0, 1 mM EDTA, 100 mM KCl, and 7 mM
mercaptoethanol) and sonicated. Care was taken that
the temperature during sonication was maintained at 4 °C. Unless
indicated otherwise, all steps were carried out at 0-4 °C. The
sonicated cell suspension was centrifuged at 100,000 × g for 2 h. The crude extract was treated with 1% (v/v)
polyethyleneimine in the presence of 250 mM KCl. The sample
was centrifuged at 10,000 rpm, and the supernatant was subjected to
0-30% ammonium sulfate fractionation. The pellet was dissolved in
buffer A, dialyzed, and loaded onto a phosphocellulose column
previously equilibrated with buffer A. The enzyme was eluted with a
linear gradient of 0.1-1 M KCl. Fractions
containing the enzyme were pooled and dialyzed against buffer B (10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 100 mM KCl, 7 mM
-mercaptoethanol, and 10% glycerol). Dialyzed sample was loaded onto a heparin-Sepharose column
equilibrated with buffer B and eluted with a linear gradient of
0.1-1 M KCl. Fractions containing enzyme were
pooled and dialyzed against buffer B containing 50% glycerol, and the
enzyme was stored at
20 °C. The purity of the protein was judged
as being greater than 99% on SDS-PAGE, and the yield of purified
KpnI MTase from 10 g of cells was ~5 mg. Purified
KpnI MTase was stable for at least 3 months at
20 °C.
Protein concentration was estimated by the method of Bradford (28)
using bovine serum albumin as standard.
Methylation Assay--
All methylation assays monitored
incorporation of tritiated methyl groups into DNA by using a modified
ion-exchange filter assay (29). Reactions contained 100 mM
HEPES, pH 8.0, 0.25 mM EDTA, and 11 mM
-mercaptoethanol. Enzyme and substrate concentrations (DNA and
AdoMet) were used as noted in each experiment. After the incubation of
enzyme and substrates at 37 °C, reactions were stopped by
transferring aliquots onto small Whatman DE81 filter paper discs.
Filters were washed 3 times (5 min each) with 0.2 M
ammonium bicarbonate solution equilibrated at 4 °C, once with 95%
(v/v) ethanol, and once with diethyl ether. Filters were air-dried, and
the tritium content was determined in 3 ml of scintillation fluid
(0.5% 2,5-diphenyloxazole and 0.05% 1,4-bis[2-(5-phenyl-oxazoyl)] benzene in 1:1 (v/v) 2-methoxyethanol and toluene) using an LKB Rack
BETA model II liquid scintillation counter. All data are corrected for
nonspecific binding of [3H]AdoMet to the washed filter.
Background counts were measured at zero-time incubation, the incubation
in the absence of enzyme was subtracted (less than 100 cpm), and data
were analyzed. The amount of enzyme that transferred 1 nmol of
[3H]methyl group to the substrate DNA was, thus,
obtained. One unit of KpnI MTase activity was defined as the
amount of enzyme that incorporated 1 nmol of [3H]methyl
group into the DNA/min at saturating concentrations of substrates at
37 °C. The specific activity of KpnI MTase is referred to
as units/mol of protein; that is, nmol of
[3H]methyl groups/min/mol of protein.
Gel Filtration Analysis of KpnI MTase--
Native molecular mass
of KpnI MTase was determined by gel filtration analysis and
performed on a Superdex 200 column in buffer B. To determine the
molecular mass of KpnI MTase, the column was calibrated with
suitable molecular weight markers ranging from 12 to 150 kDa. The void
volume (Vo) of the column was found to be 40 ml, and
the bed volume was 120 ml. The elution volumes (Ve)
of marker proteins and KpnI MTase were determined. The
molecular mass of KpnI MTase was calculated from the plot of
Ve/Vo versus log
molecular weight.
Chemical Cross-linking of KpnI MTase--
KpnI MTase
(4.44 µM) was incubated on ice for 15 min. Increasing
amounts of glutaraldehyde were then added to the protein solution to a
final concentration range of 0.05-0.5%, and the mixture was further
incubated on ice for 1 h. Reaction products were separated by
electrophoresis on a denaturing polyacrylamide gel (0.1% (w/v) SDS,
10% (w/v) polyacrylamide) and visualized by silver-staining.
Preincubation Studies--
Preincubation experiments were
carried out by incubating 1 µM KpnI MTase with
either 1 µM DNA or 2 µM
[3H-methyl]AdoMet at 37 °C for 5 min. The reaction was
initiated by adding AdoMet or DNA, respectively. At 10-, 20-, 30-, 40-, 50-, and 60-s time intervals, 20-µl aliquots were removed and analyzed for product formation using the DE81 filter binding assay. In
a control experiment, AdoMet and DNA were preincubated at 37 °C for
5 min, and the reaction was started with KpnI MTase.
Determination of Kinetic Parameters--
Kinetic studies were
done using pUC18 supercoiled plasmid DNA. Methylation assays were
carried out as described earlier for 2.5 min to determine initial
velocity dependence. In a series of similar reactions containing
KpnI MTase (1 µM) and [3H]AdoMet
(2 µM), the concentration of DNA was varied in the range of 50 nM-1 µM. A double reciprocal plot of
the initial velocity versus DNA concentration allowed the
determination of KmDNA and
Vmax. Similarly, initial velocity experiments
were carried out by varying the concentration of
[3H]AdoMet in the range of 50 nM-2
µM while keeping the DNA concentration fixed at 1 µM and keeping other reaction conditions identical. The
double reciprocal plot of initial velocity versus
[3H]AdoMet concentration allowed the determination of
KmAdoMet and
Vmax. The turnover number
(kcat) was calculated as the ratio of
Vmax to the enzyme concentration used. The
kinetic constants were also calculated for the
ScaI-linearized pUC18 DNA, unmethylated oligonucleotide
(38-mer), and hemimethylated oligonucleotide (38-mer). Data obtained
were plotted by regression analysis using Sigma Plot. The equations
used to obtain the kinetic constants, Vmax, KmDNA
KmAdoMet, and
kcat were as described (30, 31). Unless
otherwise indicated, all enzyme activity data were the average of at
least triplicate determinations.
Initial Velocity Dependence on DNA and AdoMet--
Methylation
assays were carried out to determine the initial velocity dependence of
DNA and AdoMet as described earlier. In a series of identical reactions
containing 1 µM KpnI MTase and 250 nM AdoMet, the concentration of the substrate DNA was
varied in the range of 50 nM-1 µM. Initial
velocities were obtained at different fixed concentrations of
[3H]AdoMet (500 nM and 1, 1.5, and 2 µM). The reaction mixture was incubated for 2.5 min at
37 °C. Similarly, by varying the concentration of
[3H]AdoMet in the range of 50 nM-2
µM, other reaction conditions remaining identical,
methylation assays were carried out to determine initial velocities.
Initial velocities were obtained at various fixed concentration of DNA
(250, 500, and 750 nM and 1 µM). The double
reciprocal plot allowed determination of the dependence of DNA on
AdoMet and vice versa. While determining initial velocities, the
product formed was measured under such conditions that overall inhibition of the reaction by AdoHcy was less than 5%.
Product Inhibition Studies--
Methylated DNA was obtained for
inhibition studies as follows. pUC18 supercoiled plasmid DNA was
incubated with KpnI MTase (2 µM) and AdoMet (5 µM) at 37 °C for 1 h. Methylated DNA was extracted twice with phenol-chloroform, precipitated with absolute ethanol, and dried. The concentration of DNA estimated, and the DNA was
used. Methylation of DNA was further confirmed by restriction digestion
with KpnI endonuclease.
Product inhibition studies were done under identical conditions as
described for initial velocity dependence experiments. Inhibition by
AdoHcy was studied using 1 µM pUC18 supercoiled plasmid
DNA (fixed concentration) while keeping the AdoHcy concentrations fixed
(0, 1, 5, and 10 µM) and varying the concentration of
[3H]AdoMet from 50 nM to 2 µM
for each of the fixed concentrations of AdoHcy. Similarly, another
series of identical reactions included 2 µM
[3H]AdoMet (fixed concentration); AdoHcy concentrations
fixed at 0, 1, 5, and 10 µM and DNA concentration varied
in the range of 50 nM-1 µM for each of the
fixed concentration of AdoHcy. Double reciprocal plots of initial
velocity versus [3H]AdoMet or DNA
concentrations were obtained at each concentration of the AdoHcy.
Ki AdoHcy was determined from these double reciprocal plots of initial velocity versus AdoMet
concentration followed by secondary plots with their intercepts (data
not shown). For inhibition by methylated DNA, the unmethylated DNA
concentration was kept constant at 1 µM, and AdoMet
concentration was varied in the range of 50 nM-2
µM against each of the chosen methylated DNA
concentrations (0, 0.5, 1, and 2 µM). Similarly, another
series of identical reactions was performed at a fixed concentration of
[3H]AdoMet (2 µM) and varying
concentrations of unmethylated DNA ranging from 50 nM to
1 µM against each of the fixed concentrations of methylated DNA (0, 0.5, 1, and 2 µM). Double
reciprocal plots were generated from these data, and
Ki of methylated DNA was estimated using secondary plots.
Fluorescence Spectroscopy Analysis of KpnI MTase-AdoMet
Interaction--
Fluorescence emission spectra and fluorescence
intensities were measured for KpnI MTase on a Shimadzu, RF
5000 spectrofluorimeter using a 1-cm stirred quartz cuvette at
37 °C. The emission spectra were recorded over a wavelength of
300-400 nm with an excitation wavelength of 280 nm. KpnI
MTase was allowed to equilibrate for 2 min in methylation buffer before
measurements were made. Small aliquots of cofactor (final concentration
1 µM-100 µM) were added to KpnI
MTase (5 µM), and spectra were recorded. The binding of AdoMet to KpnI MTase resulted in quenching of fluorescence.
Each spectra recorded was an average of three scans. The fluorescence intensities were plotted against the total AdoMet concentration, and
the data were analyzed according to Stern-Volmer and the modified Stern-Volmer equation (32). The Stern-Volmer relationship is represented by,
|
(Eq. 1)
|
where F0 and F are fluorescent
intensities in the absence and presence of cofactor respectively,
KSV is the collision Stern-Volmer constant, and
Q is the quencher (AdoMet) concentration. In the case where there is a
heterogeneous population of fluorophores, the modified Stern-Volmer
relationship is used,
|
(Eq. 2)
|
where fa is the fractional number of fluorophores
accessible to the quencher, and KQ is the quenching
constant. The dissociation constants were calculated graphically using
the modified Stern-Volmer plot (a plot of
F0/(F0
F)
versus 1/[Q]), where KQ = 1/Kd (33, 34).
Isotope Partitioning Analysis--
KpnI MTase (1 µM) was preincubated with [3H]AdoMet (4 µM) at 37 °C for 5 min. The preincubated reaction mix
was brought to a final volume of 150 µl with methylation buffer
containing 4 µM
[methyl-3H]AdoMet and 600 nM DNA.
Aliquots of 20 µl each were removed at 10-, 20-, 30-, 40-, 50-, and
60-s time intervals, and the reaction was stopped by snap-chilling the
samples in liquid nitrogen. Samples were then analyzed for radiolabeled
product formation using a DE81 filter binding assay. In a parallel
reaction, the above-mentioned preincubated mix was brought to 150 µl
with methylation buffer containing 4 µM unlabeled AdoMet
and 600 nM DNA, and the reaction was carried out as
described earlier.
DNA Binding Studies; Surface Plasmon Resonance Analysis--
The
binding kinetics of purified KpnI MTase with DNA was
determined by surface plasmon resonance spectroscopy using the BIACORE 2000 optical biosensor (Amersham Biosciences). The 24-mer (duplex XI)
with one KpnI recognition sequence containing a
5'-biotinylated oligonucleotide was immobilized on a
streptavidin-coated chip (Amersham Biosciences) as per the
manufacturer's recommendations. KpnI MTase titration
(concentration range, 75-150 nM) was performed in buffer
containing 10 mM HEPES, pH 7.4, 25 mM NaCl, 1 mM EDTA, and 0.05% surfactant P-20. The surface was
regenerated by passing 5 µl of 0.05% SDS followed by 10 µl of 1 M NaCl for further binding reactions. The binding data was
analyzed using BIA evaluation software (version 3.1). One of the four
surfaces was used as a negative control for the interaction.
Data Analysis--
Unless otherwise indicated, all enzyme
activity data are the average of at least triplicate determinations.
Data points were collected in duplicate, plotted as Lineweaver-Burk
double reciprocal plots, and fit to weighted linear regressions. The
Michaelis-Menten equation was used for all studies described here. The
following equations were used.
|
(Eq. 3)
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Ordered mechanism,
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(Eq. 4)
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(Eq. 5)
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Kia is the dissociation constant for the
EA complex, Ka is the Michaelis constant for A, and
Kb is the Michaelis constant for B. Other mechanisms
were eliminated by demonstrating unacceptable fits of the data to the
appropriate equations.
|
(Eq. 6)
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(Eq. 7)
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The parameters are as follows: v, initial velocity;
Vmax, maximum velocity; Km,
Michaelis constant; Ki, inhibition constant.
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RESULTS AND DISCUSSION |
Preliminary Characterization of KpnI MTase--
In the present
work, we sought to obtain kinetic insights into events underlying the
interaction of enzyme with substrates and their relationship to
catalysis. KpnI MTase was purified to homogeneity and purity
of the protein judged as being greater than 99% on SDS-polyacrylamide
gel electrophoresis. Enzyme preparations used for the kinetic analysis
showed an optimal activity at a pH 8.0 and 37 °C in the presence of
100 mM KCl. No product inhibition was observed under
initial velocity conditions. Thermal stability experiments showed that
KpnI MTase was completely inactive when incubated at
37 °C for 30 min in the absence of substrates.
Molecular Mass Determination--
Gel filtration analysis was
performed to determine size and subunit structure of KpnI
MTase in solution. Superdex 200 column was calibrated with proteins of
known size (12-150 kDa), and different concentrations of
KpnI MTase were loaded (11.1-222.2 µM).
KpnI MTase eluted as a symmetric peak at a position
corresponding to a globular protein of ~90 kDa (Fig.
2), suggesting that the enzyme exists as
a dimer under native conditions.

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Fig. 2.
Determination of the molecular mass of
KpnI MTase by gel filtration chromatography under
nondenaturing conditions. The standard curve
Ve/Vo versus the log
of the molecular weight was derived from the elution profiles of the
standard molecular weight markers with Ve
corresponding to the peak elution volume of the protein and
Vo representing the void volume of the column
determined using blue dextran (2,000,000). The peak position of
KpnI MTase is indicated by a line. 1, lysozyme
(12.4 kDa); 2, carbonic anhydrase (29 kDa); 3,
ovalbumin (45 kDa); 4, bovine serum albumin (66 kDa);
5, alcohol dehydrogenase (150 kDa). Inset,
elution profile of KpnI MTase.
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Glutaraldehyde is a homobifunctional cross-linking reagent that
cross-links N-terminal primary amines of lysine residues, resulting in
the formation of amidine cross-links between protein subunits.
KpnI MTase contains 36 lysine residues/molecule. Chemical cross-linking of KpnI MTase with glutaraldehyde was carried
out to determine the oligomeric nature of the enzyme.
Glutaraldehyde-treated KpnI MTase migrated with a relative
molecular mass of 90 kDa (Fig. 3). It was
observed that increasing the concentration of glutaraldehyde in the
cross-linking reaction mixture resulted in an increase in the
cross-linked KpnI MTase. These results demonstrate that KpnI MTase exists as a dimer in solution.

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Fig. 3.
Glutaraldehyde cross-linking of
KpnI MTase. KpnI MTase (4.44 µM) was incubated with 0.05%-0.5% of glutaraldehyde
(final concentration) at 4 °C for 60 min. Reactions were stopped by
adding SDS-loading buffer and boiled for 3 min at 100 °C. The
reaction mixtures were analyzed on a 10% polyacrylamide gel containing
0.1% SDS. The gel was silver-stained to visualize the protein. The
control lane is KpnI MTase without glutaraldehyde
treatment.
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Based on the amino acid sequence, the molecular mass of KpnI
MTase was calculated to be ~47 kDa. The molecular mass of purified protein determined by SDS-polyacrylamide gel electrophoresis was ~45
kDa. However, size-exclusion chromatography analysis (Fig. 2) and
chemical cross-linking by glutaraldehyde (Fig. 3) showed that
KpnI MTase exists as a dimer in solution in the
concentration range of 11.1-222.2 µM.
Most of the characterized MTases such as HhaI MTase (10),
EcoRI MTase (29), and EcoDam (35) MTase exist as
monomers in solution. However, MTases such as CcrM MTase
(36), adenine MTases from DpnII R-M systems (37),
EcoP15I MTase (38), human placental DNA (cytosine-5)
methyltransferase (39), and HaeIV MTase (40) exist as dimers
in solution. RsrI MTase (41) and MspI MTase have
been shown to dimerize at high protein concentrations (42).
Rate of Methylation Versus Enzyme Concentration--
To establish
the relationship between the initial velocity of the reaction and
enzyme concentration, the rate of DNA methylation catalyzed at
different KpnI MTase concentrations was determined. The
substrate used for this analysis was pUC18 plasmid DNA with a single
KpnI (GGTACC) site. Varying concentrations (0.1-1
µM final concentration) of KpnI MTase was
added to the reaction mixture containing DNA and AdoMet and incubated
at 37 °C. The progress of methylation was monitored by withdrawal of
20-µl samples at timed intervals. All time courses displayed a rapid
burst in product formation followed by a slower rate of product
formation at various KpnI MTase concentrations (Fig.
4A). This is consistent with
an initial burst in product formation followed by a rate-limiting step
after methyl transfer. These curves were further used to calculate the
initial velocity (vo=
d[P]/dt) of each reaction catalyzed by a given
KpnI MTase concentration. When initial velocities were
plotted against the corresponding enzyme concentration a nonlinear plot
was obtained (Fig. 4B). This indicated that, at low
concentrations of KpnI MTase, the initial velocity of the reaction is not directly proportional to the enzyme concentration, suggesting that KpnI MTase-catalyzed reaction is higher than
first order with respect to enzyme concentration. The nonlinear
relationship of enzyme concentration on rate of DNA methylation
strongly suggests cooperative binding of two molecules of
KpnI MTase to DNA. Furthermore, replotting the initial
velocity of the reaction against the square of KpnI MTase
concentration yielded a linear plot (Fig. 4C). These results
indicate that the cooperative binding of two molecules of
KpnI MTase is required to methylate DNA.

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Fig. 4.
Rate of DNA methylation at different
KpnI MTase concentrations. Methylation assays
were carried out in a reaction mixture containing 1 µM
supercoiled pUC18 plasmid DNA, 2 µM
[methyl-3H]AdoMet, and 100 nM to 1 µM KpnI MTase in standard reaction buffer at
37 °C. At regular time intervals, the samples were removed and
analyzed as described under "Experimental Procedures."
A, curves obtained from time course methylation assays at
different concentrations of KpnI MTase. B,
initial velocity versus KpnI MTase concentration.
C, initial velocity versus square of
KpnI MTase concentration. D, initial velocity
versus KpnI MTase concentration with unmethylated
oligonucleotide (duplex IX). E, initial velocity
versus KpnI MTase concentration with
hemimethylated oligonucleotide (duplex X).
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Similar experiments were carried out using both unmethylated (duplex
IX) and hemimethylated oligonucleotides (duplex X) to determine whether
KpnI MTase exists as a dimer on these short oligonucleotides. Dependence of initial velocity on enzyme
concentration with these oligonucleotides was higher than first order
(Fig. 4, D and E). But the nonlinear relationship
of enzyme concentration on the rate of DNA methylation is much less
pronounced with hemimethylated oligonucleotide than what was observed
when plasmid DNA and unmethylated oligonucleotide were used.
It is clear from the data shown above that KpnI MTase exists
as a dimer in solution and during methylation of its recognition sequence, unlike almost all other MTases belonging to type II R-M
system. The significance of the dimeric nature of KpnI MTase in solution and upon interaction with DNA is not clear at the present
time. We have considered the possibility that dimeric enzymes have
higher affinity for DNA and may be more proficient. But the results
presented below do not reflect this behavior in the case of
KpnI MTase, which is clear from the kinetic
parameters determined (Table I and see
Fig. 12). An alternative possibility is that KpnI MTase
originated from an ancestral dimeric MTase. The amino acid sequence of
KpnI MTase has maximum homology with the amino acid sequence
of the members of type III R-M systems (7). It is tempting to speculate
that KpnI MTase might have derived from MTases belonging to
type III R-M systems.
Progress Curve Analysis of Methylation Reaction--
Time courses
of methylation by KpnI MTase at various concentrations of
AdoMet were carried out to determine the period during which the rate
of product formation remains linear. A plot of methyl groups
transferred versus time was obtained (Fig.
5A). KpnI
MTase-catalyzed rate of methylation decreased progressively with
increasing AdoMet concentrations. The decrease in rate was inversely
proportional to the AdoMet concentration. The inhibition appears to be
competitive with respect to AdoMet due to the formation of AdoHcy or
methylated DNA, and the nonlinear kinetics are consistent with
competition by AdoHcy generated in the reaction (see later in this
section of text). This behavior was not because of enzyme degradation
under these conditions (data not shown).

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Fig. 5.
Progress curve analysis of the methylation
reaction of KpnI MTase. Time courses of
methylation assays were carried out in reactions containing 1 µM pUC18 supercoiled DNA, the indicated concentration of
[methyl-3H]AdoMet, and 1 µM
KpnI MTase under standard conditions. Samples were withdrawn
at the time intervals indicated and analyzed as described under
"Experimental Procedures." The data shown in A were
fitted to Equation 8, and the data obtained were plotted as shown in
B.
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The progress curves shown in Fig. 5A fit in Equation 8,
which describes a reaction wherein the product, AdoHcy in this case, competes with substrate, AdoMet,
|
(Eq. 8)
|
where P is the amount of AdoHcy formed at time
t, S0 is the initial AdoMet
concentration, and Ki is the dissociation constant
of AdoHcy. The amount of AdoHcy formed corresponds to the amount of
methylated DNA, the product that is measured in these experiments. A
plot of P/t versus ln
S0/(S0
P)/t provides a series of lines with positive
slopes converging on the negative side on the ordinate (Fig.
5B).
Preincubation Studies with KpnI MTase--
For the catalytic cycle
of DNA MTases, binding of substrates could occur in a random or
sequential order. To determine this, KpnI MTase was
preincubated with [methyl-3H]AdoMet or with
pUC18 DNA for 5 min, and the reaction was initiated by adding DNA or
[methyl-3H]AdoMet, respectively. Under
saturating substrate conditions, the order of preincubation of
KpnI MTase with AdoMet and DNA had significant influence on
the rate of product formation. As seen in Fig.
6, the preformed enzyme-DNA complex is
slightly less efficient than the preformed enzyme-AdoMet complex in
methylating DNA. Preincubation studies with unmethylated
oligonucleotides (duplex IX) produced similar results (data not
shown).

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Fig. 6.
Preincubation analysis of
KpnI MTase. Methylation reactions were carried
out in methylation buffer containing 1 µM KpnI
MTase, 1 µM pUC18 supercoiled DNA, and 2 µM
[methyl-3H]AdoMet. Methylation reactions were
started by the addition of DNA to a solution containing enzyme and
AdoMet ( ) or by the addition of AdoMet to a solution containing
enzyme and DNA ( ).
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From preincubation experiments it appears that the enzyme may have a
random order of substrate binding; the route via the enzyme-AdoMet
complex is kinetically more efficient than via the enzyme-DNA complex.
One explanation for this would be that the preformed enzyme-DNA complex
is not catalytically competent and that it must dissociate, bind
AdoMet, and then rebind DNA, which could slow down productive complex
formation. Therefore, although preincubation studies suggest that
either substrate could bind enzyme, it is evident from initial velocity
experiments and product inhibition studies (see below in this section)
that KpnI MTase obeys an ordered mechanism in which AdoMet
binds first, followed by DNA. A similar study performed with
EcoRV MTase clearly showed differences in product formation.
Under the preincubation conditions, EcoRV MTase-DNA complex
was not active, whereas enzyme-AdoMet complex was active (25). In the
case of T4 Dam, the preformed T4 Dam-AdoMet complex was catalytically
more efficient than the preformed T4 Dam-DNA complex during the first
round of catalysis, and this is consistent with the order of binding
where AdoMet binds first (43).
Determination of Kinetic Parameters--
To determine
initial velocities, product formation was measured under conditions
such that maximal final concentration of AdoHcy generated in the
reactions was less than 1 nM. These constraints ensured
that the overall inhibition of the reaction by AdoHcy was less than
5%.
Using double reciprocal plots of initial velocity versus
substrate concentration, the kinetic parameters
(KmDNA,
KmAdoMet, and
Vmax) were calculated. The catalytic constant,
kcat, was obtained as the ratio of maximal
velocity to enzyme concentration. The specificity constant was
determined as
kcat/KmDNA. In
all these assays, the substrate used was pUC18 plasmid DNA with a
single KpnI recognition sequence. Values for all the kinetic parameters for different DNA substrates and AdoMet have been listed in
Table I. The kcat of KpnI MTase with
plasmid DNA obtained is 3.3 × 10
4 s
1,
whereas the measured kcat values for other
MTases were 5.3 × 10
3 s
1 for
EcoP15I (44), 3.4 × 10
3 s
1
for EcaI (22), 0.175 s
1 for BamHI
(45), 2.2 × 10
2 s
1 for
HhaI (10), 0.124 s
1 for EcoRI (19),
4.2 × 10
5 s
1 for DNMT1, and 7.2 × 10
6 s
1 for DNMT3a (17). The
kcat value obtained with duplex X
(hemimethylated) (5.6 × 10
3 s
1) was
2.5-fold more than the kcat value obtained with
duplex IX (unmethylated) (1.1 × 10
3
s
1). This is consistent with hemimethylated DNA being a
preferable substrate for DNA MTases in vivo. DNMT1 has been
shown to have a 7-21-fold preference for hemimethylated DNA than
unmethylated DNA. However, DNMT3a preferred unmethylated DNA more than
3-fold over hemimethylated DNA (17). RsrI MTase has a
2-3-fold higher affinity for hemimethylated DNA than unmethylated DNA
(21), unlike EcoRI MTase, which displays no binding
preference between unmethylated and hemimethylated DNA (46).
KpnI MTase showed higher affinity to linear plasmid DNA than
to supercoiled DNA. KpnI MTase showed lesser affinity toward
oligonucleotide compared with longer DNA substrates (Table I).
KpnI MTase methylated hemimethylated oligonucleotide (duplex
X) more efficiently with higher affinity than unmethylated
oligonucleotide (duplex IX). The specificity constant for plasmid DNA
(2.2 × 103 m
1s
1) was
3.6-fold lesser than linear DNA (8.0 × 103
m
1s
1). In the case of hemimethylated
oligonucleotide, kcat/Km was
4-fold higher than unmethylated oligonucleotide. KpnI MTase did not methylate single-stranded oligonucleotides (data not shown). All of the DNA MTases that have been characterized kinetically display
very slow turnover or rate of methylation. The low turnover coupled
with strong binding to their DNA target sequence, as determined by
Km, means that
kcat/Km is high and that the MTases show fair to high specificity for methylation of their target sequence.
Initial Velocity Studies with DNA and AdoMet--
The effect of
different concentrations of DNA and AdoMet on the initial velocity was
determined by the initial velocity dependence studies (Fig.
7). Initial velocity experiments provide
clues to differentiate between ordered and ping-pong mechanisms.
These experiments were carried out at various
concentrations of [3H]AdoMet and pUC18 plasmid DNA. The
double reciprocal plots of 1/v versus 1/[DNA]
(Fig. 7A) and 1/v versus 1/[AdoMet]
(Fig. 7B) gave a series of lines intersecting on the left
side of the 1/v axis that are characteristic patterns for a
ternary complex formation. The transformed data were best fitted by
lines intersecting at quadrant IV. These results suggest that
methylation proceeds by a random or ordered bi bi mechanism. The fact
that double reciprocal plots (1/v versus 1/[S]) show
linear dependence imply that a random mechanism can be ruled out. A
ping-pong mechanism is also ruled out, because the lines are not
parallel.

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Fig. 7.
Double reciprocal plots of the initial
velocity versus substrate concentration.
Reactions contained the indicated concentrations of pUC18 plasmid DNA
and [methyl-3H]AdoMet in standard buffer at
37 °C. KpnI MTase (1 µM final
concentration) was added to start the reaction. The samples were
withdrawn at 2.5-min intervals and assayed as described under
"Experimental Procedures." A, double reciprocal plots of
the rates of methylation versus AdoMet concentrations at
different fixed concentrations of DNA. B, double reciprocal
plots of the rates of methylation versus DNA concentrations
at different fixed concentrations of AdoMet.
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Product Inhibition Kinetics--
Product inhibition studies with
AdoHcy and methylated DNA were carried out to distinguish between
ordered and random mechanisms. Product inhibition is examined by the
slopes and y axis intercepts from families of double
reciprocal plots. One substrate is held constant, whereas one product
and the other substrate being studied are varied in a concentration
range around its Michaelis constant. Inhibition profiles are
characterized as competitive, noncompetitive, or uncompetitive. The
inhibition constants are derived from analysis of the intercept and
slope effects in secondary plots. Ordered mechanisms give competitive
patterns with the first substrate that binds to the enzyme
versus the last product that leaves the enzyme and
noncompetitive patterns with any other combinations. AdoHcy was a
competitive inhibitor of AdoMet (Fig.
8A). The
competitive nature of AdoMet with respect to AdoMet binding
(Ki = 0.62 µM) suggested that AdoMet
and AdoHcy bind to the same form of the enzyme. The AdoHcy inhibition
with respect to varying DNA concentrations yielded a series of lines
intersecting on the left side of the x axis and is,
therefore, a noncompetitive inhibitor with respect to DNA (Fig.
8B).

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Fig. 8.
Product inhibition analysis of methylation
catalyzed by KpnI MTase. The reactions contained
the indicated concentrations of pUC18 DNA,
[methyl-3H]AdoMet, AdoHcy, and methylated DNA
in standard reaction buffer at 37 °C. KpnI MTase was
added to a final concentration of 1 µM. The samples were
withdrawn at regular time intervals and assayed as described under
"Experimental Procedures." A, double reciprocal plots of
the rates of methylation versus AdoMet concentrations at
different fixed concentrations of AdoHcy. B, double
reciprocal plots of rates of methylation versus DNA
concentrations at different fixed concentrations of AdoHcy.
C, double reciprocal plots of rates of methylation
versus AdoMet concentrations at different fixed
concentrations of methylated DNA. D, double reciprocal plots
of rates of methylation versus DNA concentrations at
different fixed concentrations of methylated DNA.
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To distinguish between ordered and random mechanisms and to
characterize the order of substrate binding, we determined the inhibition pattern with methylated DNA and DNA at saturating AdoMet concentrations. The double reciprocal plot of initial velocity versus varying concentrations of AdoMet with respect to
different fixed concentrations of methylated DNA yielded a series of
lines intersecting just above the left side of the x axis
(Fig. 8C). Therefore, the methylated DNA inhibition is
noncompetitive with respect to varying AdoMet as a substrate. Because
the double reciprocal plot of initial velocity versus
concentrations of DNA yielded a series of lines intersecting at
x axis, methylated DNA was noncompetitive with DNA when
AdoMet was held constant (Fig. 8D). The observed noncompetitive pattern is inconsistent with any ordered mechanism in
which DNA is required to bind first. A random bi-bi order of substrate
addition and product release should produce a series of four product
inhibition plots that are all noncompetitive. Hence, the patterns in
Fig. 8 are inconsistent with a random bi-bi mechanism. Consistent with
the observed pattern is an AdoMet binding first, ordered steady state
mechanism. The data obtained from KpnI MTase product
inhibition studies are summarized in Table II.
In the case of EcoRI MTase (19-20), EcoRV MTase
(25), and T4 Dam (26), the reaction mechanism is ordered with AdoMet
binding first to the enzyme, which is similar to the proposed reaction mechanism for KpnI MTase. In the case of TaqI
MTase (24), EcaI MTase (22), human DNMT1 (47), and
EcoP15I MTase (18), the order of substrate binding is
random, and in the case of HhaI MTase (10), MspI
MTase (15), murine C5 cytosine methyltransferase
(14), and mammalian DNMT3a (17), the order of substrate binding is just
the reverse, i.e. DNA binds first followed by AdoMet. It is
noteworthy to mention that DNA MTases in which DNA has been shown to
bind first act processively during methylation of specific DNA
sequences (10, 47, 23), whereas other DNA MTases in which AdoMet has
been shown to bind first methylate in a distributive manner (25,
48).
Substrate Inhibition Studies--
To further confirm that the
binding of substrates follows an ordered mechanism, studies at high
substrate concentrations were carried out. This is also known as
substrate inhibition. This analysis has been successfully used to
determine the order of binding of substrates in the case of
EcoP15I MTase (18), T4 Dam (26), and mammalian DNMT3a (17).
In a compulsory ordered mechanism, if the initial concentration of the
second substrate is sufficiently high, an inhibitory effect on the
enzyme would be observed due to the formation of nonproductive binary
and/or dead-end ternary complexes. On the contrary, the first substrate to bind does not have an inhibitory effect even at higher
concentrations, because the binary complex is catalytically active, and
a dead-end complex is not formed. No substrate inhibition is observed
in a random mechanism unless a dead end ternary complex is formed. This
is because neither of the two binary complexes (Enzyme-AdoMet or
Enzyme-DNA) can prevent a productive ternary complex formation. When
substrate inhibition studies were carried out with KpnI
MTase, no substrate inhibition was observed at AdoMet concentration of up to 3.5 µM (Fig.
9A). On the other hand, an
inhibition of KpnI MTase was observed by DNA at
high concentrations, above 1 µM (Fig. 9B).
When this experiment was carried out at a lower enzyme concentration, a
similar pattern was observed (data not shown). These results suggest
that KpnI MTase follows an ordered bi bi mechanism, where AdoMet is the first substrate to bind followed by the binding of DNA.
These data are consistent with the results obtained from product
inhibition studies.

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Fig. 9.
Substrate inhibition analysis of
KpnI MTase. A, each reaction contained
1 µM KpnI MTase, 1 µM pUC18
supercoiled DNA, and varying concentrations of AdoMet (0.05-3.5
µM). B, KpnI MTase concentration
was fixed at 1 µM, and AdoMet concentration was fixed at
2 µM. DNA concentration was varied from 0.05 to 7.5 µM. Reaction mixtures were incubated for 2.5 min, and
product was estimated as described under "Experimental
Procedures."
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Our assignment of the kinetic mechanism rests on the following lines of
evidence. (a) Double reciprocal plots of initial velocity data with AdoMet as the variable substrate and DNA as the fixed substrate and vice versa gave lines of intersecting pattern, which is
characteristic of ternary complex formation. These results rule out the
random as well as the ping-pong mechanism. (b) Product inhibition studies demonstrate that AdoHcy is a competitive inhibitor with respect to AdoMet in methylation reaction (Ki = 0.62 µM) and, therefore, binds to the free enzyme form.
(c) Inhibition studies with methylated DNA demonstrate that
it binds to the enzyme-AdoMet binary complex. Noncompetitive inhibition
demonstrates the formation of both an enzyme-DNA and enzyme-AdoMet
complex. (d) Substrate inhibition studies showed that at
higher concentrations of DNA, the inhibition was severe, but AdoMet did
not show inhibition even at higher concentrations.
Isotope Partitioning Studies--
Isotope partitioning analysis of
KpnI MTase was carried out to examine the competency of
KpnI MTase-AdoMet complex. First, we determined the
KD for AdoMet by fluorescence-quenching experiments.
The intrinsic fluorescent properties of KpnI MTase were
exploited in a fluorescence-quenching assay to determine the
dissociation constant for AdoMet. The Stern-Volmer plot (49) and
modified Stern-Volmer plot (32) were used to analyze the quenching
data. The Stern-Volmer plot for AdoMet showed a negative deviation from
linearity, a result expected only when a fraction of the
tryptophans are accessible to quenching by ligand binding (data not
shown). Therefore, a modified Stern-Volmer plot was used to analyze the
quenching. The data in Fig. 10 gave a
linear plot. This linearity suggests that cofactor binding is the
dominant fluorescence-quenching phenomenon over the range of
concentrations checked (1-100 µM). The
KD value calculated from modified Stern-Volmer
relationship (Equation 2) was 4 µM.

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Fig. 10.
Modified Stern-Volmer plot for AdoMet
quenching. Fluorescence experiments were performed at an enzyme
concentration of 1 µM at 37 °C in methylation buffer.
An excitation wavelength of 280 nm and emission spectra from 300-400
nm were recorded. Fluorescence changes were calculated from the
intensity of the maximal intensity of enzyme alone (before addition of
cofactor), F0, and the maximal intensity after the
addition of AdoMet, F (10-200 µM).
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AdoMet concentrations were chosen based on the
KDAdoMet determined from the
fluorescence-quenching experiments. In an isotope partitioning
experiment, a presaturated binary complex of KpnI MTase with
[3H]AdoMet was diluted into a mixture containing excess
DNA and unlabeled AdoMet to determine what fraction of the
bound-labeled AdoMet would react before exchanging with unlabeled
cofactor. The formation of radiolabeled product reflects the propensity of the enzyme-bound labeled substrate to undergo catalysis without dissociation (50). Preincubation of KpnI MTase with
[3H]AdoMet (4 µM) results in a burst of
product formation upon the addition of DNA and labeled AdoMet (Fig.
11,
). The burst was followed by a
constant rate of product formation. A decreased burst was observed when
unlabeled AdoMet was used in the chase (Fig. 11,
). However, this
burst was much smaller in size than that observed in the first case.
Burst magnitude is defined as the vertical axis intercept resulting
from extrapolation of the linear portion of the progress curve to zero
time. These results indicate that the KpnI MTase-AdoMet
complex formed is catalytically competent. This is so because a chase
including 600 nM DNA and 4 µM unlabeled
AdoMet produced detectable activity. The detection of a burst in the
isotope-partitioning assay also provides evidence that the prebound
AdoMet is catalytically competent and supports an ordered bi bi
mechanism wherein AdoMet binds first. If DNA were required to bind to
KpnI MTase before AdoMet for catalysis, all of the prebound
AdoMet would form catalytically nonproductive complex. These results
further support the kinetic mechanism as steady state rather than rapid
equilibrium.

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Fig. 11.
Isotope partitioning analysis of
KpnI MTase. Methylation assays were carried out
in reaction mixtures containing 1 µM KpnI
MTase, 600 nM DNA, and 4 µM AdoMet. Curve 1 ( ), product formation after enzyme was preincubated at 37 °C with
high specific activity [methyl-3H]AdoMet (78.9 Ci/mmol), and the reaction was started with DNA and labeled
[methyl-3H]AdoMet. Curve 2 ( ), product
formation after enzyme was preincubated with high specific activity
[methyl-3H]AdoMet, and the reaction was
started with DNA and unlabeled AdoMet.
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Isotope partitioning technique has been used successfully to determine
the catalytic competency of enzyme substrate complex and to decipher
the order of binding in EcoRI MTase (19), HhaI MTase (12, 13, 51) MspI MTase (15), the murine DNA
(cytosine) MTase (14), and RsrI MTase (21). The requirement
of DNA binding for the formation of competent complex was found in the
case of murine DNA (cytosine) MTase (14), whereas for EcoRI
MTase (19), HhaI MTase (13), and RsrI MTase (21)
the enzyme-AdoMet complex was found to be catalytically active.
Methylation Assays with Noncanonical Oligonucleotides--
The
recognition sequence of the KpnI R-M system is 5'-GGTACC-3',
and KpnI endonuclease cleaves within this sequence.
KpnI endonuclease is also known for its star activity as it
cleaves DNA at nonspecific
sequences.2 We were,
therefore, interested to find out if KpnI MTase would methylate oligonucleotides containing noncanonical sequences. Methylation assays were carried out with seven different
oligonucleotides containing noncanonical sequences (duplexes I-VII)
with one base change in the recognition sequence and an oligonucleotide
(duplex VIII) containing KpnI recognition sequence (data not
shown). These assays showed no significant methylation at nonspecific
sites by KpnI MTase even when enzyme was used in far excess
and the enzyme methylated only at its specific recognition sequence
(data not shown).
Kinetics of DNA Binding--
Surface plasmon resonance
spectroscopy was used to determine the kinetics of DNA binding for
KpnI MTase. Surface plasmon resonance measures change in the
refractive angle arising from a binding event. Experiments focusing on
DNA-protein interactions require the DNA to be immobilized on the
surface of the flow cell and the enzyme under study to be passed over
the surface in increasing concentrations to allow determination of
binding constants. The association and dissociation of the protein to
DNA was monitored by changes in the refractive index due to the binding
event on the sensor surface. To determine KpnI MTase-DNA
stoichiometry, a DNA substrate was synthesized that contained a
3'-biotin tag (duplex XI), which was immobilized through a
biotin-streptavidin interaction on the surface of a streptavidin chip.
The background nonspecific binding and bulk concentration of
KpnI MTase were experimentally determined by simultaneous
injection over a surface that lacked DNA. These results demonstrate
that KpnI MTase binds to oligonucleotide containing the
recognition sequence with high affinity (KD = 6.57 × 10
8 M) (Fig.
12). The Kd values
for other MTase-DNA binary complexes are 10 nM for
HhaI (13), 17.5 nM for RsrI (21), and
76.9 nM for CcrM (36). It has been shown that
the addition of AdoMet or its analogs increases the specificity of
several MTases to their recognition sequences (3, 52). However,
the addition of cofactor, AdoHcy, did not seem to influence the binding characteristics (data not shown). DNA binding studies and the interaction of cofactor have also been studied using surface plasmon resonance spectroscopy in the case of CcrM (36) and
EcoR124I (53).

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Fig. 12.
Interaction of KpnI MTase
at the recognition sequence by surface plasmon resonance.
Shown is a sensogram displaying the response of increasing
KpnI MTase concentration (75-150 nM).
KpnI MTase was injected for 300 s over streptavidin
chips containing immobilized cognate DNA at flow rate of 10 µl/min
followed by a dissociation phase of 300 s.
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The key finding of the present study is the identification of dimeric
nature of KpnI MTase in solution and that it functions as a
dimer during methylation reaction. Initial velocity dependence experiments, product inhibition, and substrate inhibition studies demonstrate an ordered mechanism for KpnI MTase where AdoMet
binds first followed by DNA. Isotope partitioning analysis showed that the preformed KpnI MTase-AdoMet complex is catalytically competent.