From the Department of Chemistry and Program in Biochemistry and
Molecular Biology University of California, Santa Barbara,
California 93106-6081 and the Department of Molecular
Physiology and Biophysics, Vanderbilt University Medical Center,
Nashville, Tennessee 37232-0615
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA methyltransferases are excellent prototypes for investigating DNA distortion and enzyme specificity because catalysis requires the extrahelical stabilization of the target base within the enzyme active site. The energetics and kinetics of base flipping by the EcoRI DNA methyltransferase were investigated by two methods. First, equilibrium dissociation constants (KDDNA) were determined for the binding of the methyltransferase to DNA containing abasic sites or base analogs incorporated at the target base. Consistent with a base flipping mechanism, tighter binding to oligonucleotides containing destabilized target base pairs was observed. Second, total intensity stopped flow fluorescence measurements of DNA containing 2-aminopurine allowed presteady-state real time observation of the base flipping transition. Following the rapid formation of an enzyme-DNA collision complex, a biphasic increase in total intensity was observed. The fast phase dominated the total intensity increase with a rate nearly identical to kmethylation determined by rapid chemical quench-flow techniques (Reich, N. O., and Mashoon, N. (1993) J. Biol. Chem. 268, 9191-9193). The restacking of the extrahelical base also revealed biphasic kinetics with the recovered amplitudes from these off-rate experiments matching very closely to those observed during the base unstacking process. These results provide the first direct and continuous observation of base flipping and show that at least two distinct conformational transitions occurred at the flipped base subsequent to complex formation. Furthermore, our results suggest that the commitment to catalysis during the methylation of the target site is not determined at the level of the chemistry step but rather is mediated by prior intramolecular isomerization within the enzyme-DNA complex.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein-DNA complexes reveal diverse mechanisms leading to sequence-specific interaction. Direct readout of DNA base functionalities within the major groove and the indirect readout of sequence-dependent phosphate backbone geometry are thought to contribute binding discrimination (1, 2). For DNA modification and repair enzymes the correct assembly of active site residues frequently demands the insertion of protein side chains into and rotating of a base completely out of the DNA helix (3, 4). The stabilization of an extrahelical base is often coupled to sequence-dependent DNA base pair rearrangement (5) and DNA bending (6). However, the energetic cost of the enzyme-mediated DNA deformations integrating site-specific recognition and catalysis are only now being elucidated.
The mechanism leading to the stabilization of an extrahelical base is thought to involve a multi-step binding process with discrete conformational intermediates (4, 7). Enzyme-mediated weakening or breakage of Watson-Crick hydrogen bonds at the target base pair and intercalation of amino acid side chains into the DNA helix are likely to be critical to the initiation of the base flipping process (8). The enhanced discrimination provided by the major groove readout of DNA base functional groups (9) appears for DNA-modifying enzymes to require the sterically encumbered process of extruding a base via the DNA minor groove (3, 5). In contrast, DNA repair enzymes use minor groove readout and extrude the base via the DNA major groove (4, 6). Tighter binding to DNA containing mismatches or otherwise modified target base pairs is observed for DNA-modifying enzymes (10-12). Similarly, enhanced specificity (kcat/Km) with substrates that reduce the investment of binding energy required to "flip out" a base has been detected for DNA repair enzymes (13, 14). These studies suggest that the energetic cost of base flipping substantially affects the equilibrium for enzyme-DNA complex formation. However, the elusive nature of structural transitions such as nucleotide flipping, base pair rearrangement, and DNA bending combined with the lack of suitable detection methodologies leaves obscure the underlying kinetics and catalytic consequences.
EcoRI DNA methyltransferase (M.EcoRI)1 catalyzes methyl-transfer from S-adenosyl-L-methionine (AdoMet) to adenine N6 within double-stranded DNA (15). The methyl-transfer step (kmethylation) of this essentially irreversible reaction is significantly faster than kcat, showing that product release or prior conformational change limits turnover (16). The chemical mechanism proceeds by direct attack of adenine N6 upon the methylsulfonium moiety of AdoMet (17), inducing an inversion in configuration of a chirally labeled methyl group (18). The conserved active site residues between the N6 adenine and N4 cytosine DNA methyltransferases indicates that these enzymes are likely to share a common mechanism of exocyclic amino modification (19).
2-Aminopurine (2AP) is a strongly fluorescent adenine isomer that is highly quenched within duplex DNA due largely to intrastrand base stacking interactions (20). The sensitivity of the 2AP probe to localized DNA conformation and dynamics has been exploited to monitor insertion and excision kinetics by DNA polymerases (21, 22), RNA polymerases (23, 24), helicase activity (25, 26), and conformational changes within the hammerhead ribozyme (27). 2AP-substituted oligonucleotides retain B-form helical parameters and are cleaved by the EcoRI endonuclease (28-30). Using a steady-state 2AP-based base flipping assay, we recently demonstrated that M.EcoRI stabilizes the targeted base extrahelically in a low dielectric environment (7). Herein we extend this assay to the presteady-state, providing assessment of enzyme-assisted base-flipping dynamics continuously in real time. Our data suggest that the rate determining step for methyl-transfer is a first-order isomerization within the enzyme-DNA complex.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Enzyme Expression and Purification--
M.EcoRI was
overexpressed from pXRI (7) and purified essentially as described (31)
with the addition of another anion exchange column (Bio-Rex), yielding
an enzyme of >99% homogeneity. The purified enzyme was dialyzed
extensively in a buffer containing 100 mM NaCl, 10 mM potassium phosphate, pH 7.0, 1 mM EDTA, 7 mM -mercaptoethanol, 1 mM sodium azide at
4 °C. The enzyme preparation was confirmed to be free of detectable
contaminating AdoMet by restriction analysis of pBR322 DNA (contains an
EcoRI site) following incubation of the DNA and the enzyme
with and without added AdoMet. In the absence of added AdoMet, the
plasmid DNA was linearized following challenge with the
EcoRI endonuclease (data not shown). M.EcoRI
concentration (108 µM) was determined
spectrophotometrically at 280 nm utilizing the published extinction
coefficient (15).
Oligonucleotide Synthesis and Purification--
Oligonucleotides
(14-mers) were synthesized on an Biosearch 3810 DNA synthesizer using
-cyanoethyl phosphoramidites. Modified base analogs
(N6-methyladenosine-1-
-D-2
-deoxyriboside,
2-aminopurine-1-
-D-2
-deoxyriboside, nebularine(purine-1-
-D-2
-deoxyriboside),
2,6-diaminopurine-1-
-D-2
-deoxyriboside, and abasic)
were purchased from Glenn Research (Sterling, VA), and standard
phosphoramidites were from Pharmacia Biotech Inc. Oligonucleotides were
purified on a Dynamax C18 reversed-phase PureDNA column (Rainin
Instrument Co.). DNA purity was assessed by 32P
radiolabeling and visualization by overexposure of 20% denaturing polyacrylamide gel electrophoresis and confirmed to be >99% pure by
densitometric analysis utilizing a UVP (San Gabriel, CA) imaging system. Concentrations were determined spectrophotometrically (32).
Complementary strands were annealed in 10 mM Tris, 1 mM EDTA, 100 mM NaCl utilizing an MJ Research
(Watertown, MA) programmable thermocycler with a slight excess of the
unmodified DNA strand. M.EcoRI does not bind single-stranded
DNA with detectable affinity (33). The following 14-mers were used: top
strands, d(GGCGGAXTTCGCGG) (X = 6-aminopurine(adenine), 2-aminopurine, 2,6-diaminopurine, nebularine
(purine), or a stable abasic site (spacer)); bottom strands,
d(CCGCGAATTCCGCC), d(CCGCGAATTCCGCC), and
d(CCGCGZATTCCGCC) (A = N6-methyladenosine, Z = 2-aminopurine).
Hemi-methylated substrates were used to facilitate the formation of
unique binding orientations.
Equilibrium Dissociation Constants-- KDDNA values were determined in the presence of the cofactor analog sinefungin essentially as described (34) with minor variations; 100 mM NaCl was used in binding mixtures, all preincubations were done at 20 ± 2 °C for 30 min prior to sample loading onto a prerunning 12% polyacrylamide gel, and electrophoresis was at 4 °C for 1 h at 200 V. These variations minimize dissociation during electrophoresis (data not shown). Following electrophoresis, the relative amounts of free and bound DNA were determined by densitometric analysis of autoradiogram band intensities using a UVP (San Gabriel, CA) imaging system. The percentage of complex was plotted versus enzyme concentration, and the KDDNA was determined by fitting the data to a standard hyperbolic binding expression using KaleidaGraph 2.1.2 (Adelbeck Software).
Stopped Flow System and Fluorescence Detection--
An SFM-3
stopped flow unit containing three stepper-motor driven syringes
(Molecular Kinetics, Pullman, WA) with an FC.15 cuvette (50-µl
volume) and a hard stop shutter was used for stopped flow reactions.
Fluorescence detection for the stopped flow studies utilized a
home-built single photon detector consisting of the following: a
Hamamatsu R928 photomultiplier, a 5 × 300-MHz amplifier (Stanford
Research SR445, Sunnyvale, CA), a discriminator (Stanford Research
SR400) and a multichannel scaler (Tennelec Model MCS-II, Oak Ridge,
TN), interfaced to an 80486 microcomputer. The detection system was
activated by an external synch-out pulse from a Molecular Kinetics
stepper motor controlling unit. Data acquisition began at least 100 ms
before sample mixing. Data were collected using 1-15-ms dwell times in
8000 total channels. A 250-W xenon arc lamp (SPEX Fluorolog model 1681)
with fiber optic output directed into the 50-µl cell was used for
excitation at 310 nm. Fluorescence emission was collected through a
360-nm cut-on filter (Hoya Optics type L36). Dead time values were
calibrated as described (21). Solutions of enzyme (diluted to 2.4 µM in a degassed buffer containing 100 mM
Tris, 10 mM EDTA, 100 mM NaCl, 1 mM
DTT at pH 7.5), double-stranded 2AP-containing duplex DNA (diluted to
0.8 µM), and buffer alone were loaded into three separate
syringes. Reactions were initiated by mixing equal volumes (100 µl)
of enzyme and DNA solutions at flow rates between 4-10 ml
s1. Multiple runs (typically 15) were summed to increase
the signal to noise ratio. Background measurements were made by
measuring the fluorescence emission of "pre-shots" of buffer and
2AP-containing duplex DNA alone prior to mixing reactants.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
M.EcoRI DNA Binding Affinity Is Strongly Influenced by the
Stability of the Target Base Pair--
DNA containing destabilized
base pairs (abasic, mismatched, or modified bases) are bound tightly by
DNA methyltransferases (10-12). This was first observed for the
C5 cytosine-specific M.HhaI (10) following the
determination of the enzyme-DNA cocrystal structure, which clearly
shows that the cytosine to be methylated is stabilized in an
extrahelical conformation (3). To further test the hypothesis that the
N6 adenine-specific M.EcoRI utilizes a similar
base flipping mechanism and confirm our previous conclusions (7),
equilibrium dissociation constants
(KDDNA) were
determined for M.EcoRI binding to a series of
oligonucleotides that are modified at the target base pair. A binding
isotherm derived from the gel mobility shift data for the
2AP-substituted DNA (inset) is shown in Fig.
1. The dissociation constants
(KDDNA), free energy
differences relative to the unmodified site (G°), and structures of the modified base pairs are summarized in Fig. 2.
|
|
Real Time Analysis of Base Flipping by Stopped Flow Fluorescence of
2AP--
Steady-state fluorescence of 2AP-substituted DNA showed a
14-fold increase in fluorescence emission intensity following titration of double-stranded DNA containing 2AP at the target base with saturating M.EcoRI (7). Although enzyme-assisted
stabilization of an extrahelical base can only be confirmed by an x-ray
diffraction structure of an enzyme complexed with double-stranded DNA,
the large increase in 2AP fluorescence intensity clearly indicates an
unstacking of the probe within the complex. Total intensity stopped
flow fluorescence time courses of M.EcoRI interaction with
2AP-substituted duplex DNA are shown in Fig.
3. M.EcoRI was preincubated
with the cofactor analog sinefungin (43) prior to mixing with the DNA.
The biphasic total intensity signal change (Fig. 3, upper
panel) indicated that at least two kinetic processes were
occurring. Approximately 75% of the observed signal increase was
associated with a rate constant of 21 ± 2 s1, and
the remaining 25% slowly increasing at 0.6 s
1. Stopped
flow total intensity data obtained with 0.6, 1.2, and 4.8 µM M.EcoRI each yielded nearly identical
fractional amplitude changes (75 ± 2%) and rate constants for
both phases. The fast phase of the 2AP fluorescence enhancement showed
a weak dependence on enzyme concentration. In contrast, the slow phase
was demonstrated to be concentration-independent. To directly determine
the kinetic coupling between DNA binding and base flipping,
spectroscopic binding anisotropy experiments were performed using
oligonucleotides extrinsically labeled with the fluorescent probe
Rhodamine-X. These stopped flow anisotropy experiments revealed that
the fast 2AP fluorescence increase occurred subsequent to a more rapid DNA binding event.3 The
combined results show that for the adenine-specific M.EcoRI, the base flipping process does not involve the capture of an
extrahelical base but rather occurs subsequent to the assembly of the
enzyme onto the target DNA site.
|
Base Restacking and Enzyme-DNA Complex Dissociation--
Further
support for the existence of two intramolecular kinetic phases
associated with the base flipping process was obtained by examination
of the off-rate kinetics. Rapid mixing of a preformed M.EcoRI-2AP-containing DNA complex with a 100-fold molar
excess of the tightly bound abasic DNA duplex (Fig. 2) resulted
in a biphasic decrease in the 2AP total intensity (Fig. 3, lower
panel). Double exponential solutions (30% exp.(0.011t) + 70% exp.(
0.0017t)) were statistically superior to monophasic fits at
a confidence level >99.99% using an F-statistic test criterion (44).
This fluorescence quenching clearly reflects the restacking of the extrahelical base within the DNA double helix. Interestingly the recovered off-rate amplitudes from this "flip off" experiment match
very closely with the observed amplitudes (75% fast, 25% slow)
associated with the 2AP "flip on" experiment (Fig. 3, upper panel). Although the coupling between base restacking and complex dissociation is not known, base restacking or a prior conformational change is probably the rate-limiting step for turnover. Comparison of
the KDDNA determined
for the 2AP-substituted DNA duplex (0.51 nM, Fig. 2) with
the two predicted dissociation constants (0.10 and 0.68 nM)
obtained from the ratio of the measured kinetic dissociation constants
(Fig. 3, lower panel) and association rate
constant3 shows that these results are internally
consistent. The two off-rate phases suggest that two DNA-bound enzyme
forms are in equilibrium on the DNA and that enzyme dissociation
requires the interconversion from a tightly bound form to one that
dissociates rapidly.
Discrimination at the Binding Orientation
Level--
Single-turnover experiments initiated from the
enzyme-AdoMet complex indicate that hemimethylated DNA is methylated to
only 50% of the level of unmethylated DNA (16). These data suggest that under presteady-state conditions the monomeric M.EcoRI
binds the asymmetric hemimethylated target site with equal probability in both orientations, leading to catalysis or the assembly of an
unproductive dead end product complex. To confirm and extend these
observations at the level of the base flipping step, stopped flow total
intensity data were obtained with DNA that has the 2AP probe
incorporated in place of the adenine adjacent to a methylated (N6-methyladenosine) target base (Fig.
4). The exponential increase in total
intensity (Fig. 4) shows that 2AP adjacent to the target base became
partially unstacked in the enzyme-DNA complex. The total intensity
increase was approximately 5-fold less than with 2AP substitutions at
the target base and only required a single exponential (5.7 s1) analysis. This lack of a biphasic fluorescence
enhancement further supports a sequential mechanism involving two
distinct conformational transitions at the flipped base. The observed
rate at which the methylated adenine became unstacked was significantly
slower than kmethylation. Interestingly, the
dynamics of spontaneous base pair opening measured by hydrogen exchange
are slowed down by adenine methylation (45, 51). The incorporation of
the 2AP probe adjacent or opposite to the target base might be a useful strategy to investigate the nucleotide flipping process for other DNA
modification or repair enzymes.
|
Implications of this Work--
Our model relating site-specific
DNA binding, complex isomerization, methyl-transfer, and product
release by M.EcoRI is depicted in Scheme
ins;2123s1}I. DNA binding is shown in a multi-step
mechanism involving the rapid formation of a "loosely" bound
site-specific encounter complex, followed by one or more conformational
changes within the enzyme-DNA complex. Because the majority of the base flipping transition occurred at a rate similar to
kmethylation (16), all of the conformational
changes required for catalysis must also be complete within this time
frame. In addition to stabilizing an extrahelical base,
M.EcoRI bends the DNA by 52 ° (39). DNA bending may occur
simultaneously (46) with complex formation and possibly strain the DNA
to destabilize the target base pair and induce base stacking
disruption. Alternatively, DNA bending may occur in a sequential
mechanism following site-specific complex formation or subsequent to
base extrusion. Because the extrahelical conformation of the target
base is clearly the preferred orientation at equilibrium (7), the rapid
methyl-transfer step during the modification of the target site
(5-GAATTC-3
) indicates that the chemistry step has a high degree of
commitment. Initial binding of the canonical site by M.EcoRI
leads to an active configuration of the enzyme-DNA complex where the
key catalytic residues converge into an alignment compatible with
transition state stabilization. A value of >200 s
1 is
tentatively assigned to the chemistry step because this step is likely
to be greater than 10-fold faster than the prior first-order isomerization (21 s
1). Because the rate of base flipping
is approximately 50-fold slower than the rate of spontaneous 2AP:T base
pair opening (1000 s
1) (35), enzyme-assisted and
spontaneous base pair opening most likely occur by different
pathways. Although the base flipping trajectory for M.EcoRI
is not known, the C5 cytosine-specific DNA
methyltransferases extrude the target base via the DNA minor groove (3,
5). This trajectory is distinct from the major groove route predicted
to mediate spontaneous base pair opening (47). Although base flipping
clearly occurs subsequent to complex formation, the ensuing events
remain obscure.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. Stanley M. Parsons and Asst. Prof. John Perona for enlightening discussion and critical review of the manuscript. We also thank Jason Sutin for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant MCB-9603567 (to N. O. R.).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.: 805-893-8368; Fax: 805-893-4120; E-mail: reich{at}sbmm1.ucsb.edu.
1 The abbreviations used are: M.EcoRI, EcoRI DNA N6 adenine methyltransferase; DTT, dithiothreitol; 2AP, 2-aminopurine; 2,6-DAP, 2,6-diaminopurine; AdoMet, S-adenosyl-L-methionine.
2 Although structural reorganization cannot be confirmed directly utilizing a gel mobility shift assay, several lines of evidence support the hypothesis that the M.EcoRI-DNA complexes have undergone structural isomerization. M.EcoRI-assisted extrahelical stabilization of the 2AP probe (7) and DNA bending (39) were both detected under similar conditions as these KDDNA measurements. In addition, M.EcoRI methylates 2,6-DAP-substituted DNA (30), which clearly requires the stabilization of the modified target base within the enzyme active site.
3
Rhodamine-X labeled oligonucleotides are the
preferred substrate to measure changes in anisotropy associated with
complex formation because data can be obtained at the picomolar to
nanomolar range and no changes in the fluorescence lifetime are
observed. These experiments revealed that the second-order collision
complex is formed with a rate constant of 1.6 × 107
M1 s
1 (B. W. Allan,
J. M. Beechem, and N. O. Reich, manuscript in
preparation).
4 N. O. Reich and K. Maegley, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|