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From the Department of Chemistry and Program in Biochemistry and
Molecular Biology University of California, Santa Barbara,
California 93106-6081 and the 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.
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.
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 Oligonucleotide Synthesis and Purification--
Oligonucleotides
(14-mers) were synthesized on an Biosearch 3810 DNA synthesizer using
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
s 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 (
Direct Real Time Observation of Base Flipping by the
EcoRI DNA Methyltransferase*
, Department of Molecular
Physiology and Biophysics, Vanderbilt University Medical Center,
Nashville, Tennessee 37232-0615
ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-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).
-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.
1. 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
Top
Abstract
Introduction
Procedures
Results & Discussion
References
G°), and structures of the modified base pairs are summarized in Fig. 2.
View larger version (29K):
[in a new window]
Fig. 1.
Equilibrium binding of M.EcoRI to
oligonucleotides modified at the target base. Binding mixtures
containing 50 pM 32P-labeled DNA, 100 mM NaCl, 100 mM Tris, 10 µM
sinefungin, 10 mM EDTA, 1 mM DTT, pH 7.5, and
0-10 nM enzyme were preincubated at 20 ± 2 °C
prior to electrophoresis at 4 °C for 1 h at 200 V. The figure
shows the binding isotherm derived from gel shift data for the
2AP-substituted DNA duplex (inset). The other sequences were
analyzed similarly with the results summarized in Fig. 2.
View larger version (37K):
[in a new window]
Fig. 2.
KDDNA and
G° determinations for enzyme-DNA interactions and structures of
the modified target base pairs.
KDDNA and standard
error (shown in parentheses) were determined as described
under "Experimental Procedures." The binding free energy differences, relative to the unmodified DNA duplex, for the ternary (enzyme-DNA-sinefungin) complexes were calculated from G° = 
RT
ln(KDmodified)/(KDunmodified),
where R is the universal gas constant. a,
hemimethylated abasic duplex; b, unmethylated abasic
duplex.
G°) of
0.78 and 0.82 kcal mol1 for unmethylated and
hemimethylated abasic duplexes, respectively. Although the free energy
differences in the ternary (enzyme-DNA-sinefungin) complexes are not
extensive, these results are consistent with those reported for other
DNA methyltransferases (10-12) and correlate well with the thermal
stabilities determined when some of these single-analog substitutions
were incorporated into an octamer with an EcoRI site
(38).
The gain in binding energy observed upon removal of the exocyclic amino
moiety implies that unfavorable interactions are made at this position
within the ternary (enzyme-DNA-sinefungin) complex (Fig. 2).
Furthermore, replacing a hydrogen with an amino group at the adenine
C2 position did not lead to steric exclusion from the
enzyme active site.2 These
observations suggest that the stabilization of the extrahelical purine
is mediated by forces other than direct enzyme interaction with the DNA
base functional groups. Structural data and sequence comparisons for
the amino-specific DNA methyltransferases show that these enzymes
contain active sites rich in aromatic and hydrophobic amino acid
residues (19, 40-42). In addition, the large (~10 nm) blue shift in
the 2AP emission 
max following complex formation with
M.EcoRI (7) indicates placement of the target base in an
environment of significantly lower dielectric constant than the
interior of the DNA double helix (20). These observations suggest that
the extrahelical purine is likely to be stabilized largely by
hydrophobic interactions.
The overall inverse correlation between
KDDNA and the strength
of the base pair (Fig. 2) supports a base flipping mechanism for
M.EcoRI (10-12), allowing limits to be set regarding the
energetic cost of DNA distortion. Moreover, these results show that the disruption of the target base pair is a dominant energetic barrier to
the tightly bound complex. The lack of steric exclusion of the modified
target bases from the enzyme active site suggests that once the base is
positioned extrahelically, the primary determinant for methyl-transfer
is the presence of the N6 amino nucleophilic center. The
minimal substrate thus appears to be a flippable purine base with an
amino group at the N6 position. However, equilibrium
binding determinations reveal only the sum of numerous thermodynamic
effects and leave unresolved the rate of the base flipping step and the
relevance of extrahelical base stabilization to sequence specificity
and the catalytic cycle.
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 s1. 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.
|
. Upper panel, stopped flow reaction initiated at
20 °C by mixing equal volumes of 2.4 µM
M.EcoRI (preincubated with 200 µM sinefungin)
and 0.8 µM duplex DNA in a buffer containing 100 mM NaCl, 100 mM Tris, 10 mM EDTA, 1 mM DTT, pH 7.5. The inset shows total intensity
data collected during the initial 300 ms of the experiment. Lower
panel, kinetic off-rate data obtained following rapid mixing of a
preformed M.EcoRI-DNA ternary complex (1.2 µM
enzyme, 0.8 µM 2AP-containing DNA, 100 µM
sinefungin) with an equal volume of 100 µM abasic
double-stranded 14-mer. Data were fit using non-linear least squares
(1)
determined for hemimethylated DNA by rapid chemical quench-flow methods
(16). The slow (0.6 s1) intramolecular transition may
reflect a rearrangement of the extrahelical base within the enzyme
active site. The fact that the relative amplitudes of both phases are
concentration-independent is consistent with a sequential base flipping
mechanism. Thus, the biphasic nature of the total intensity increase
(Fig. 3, upper panel) may be the result of a sequential
process involving rapid targeted base stacking disruption, transition
of the unstacked base to the active site pocket, and finally
reorientation of the extrahelical base within the enzyme active
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 s1 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 s1). Because the rate of base flipping
is approximately 50-fold slower than the rate of spontaneous 2AP:T base
pair opening (1000 s1) (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.
|
-AAATTC-3),4
showing that a step prior to product release is rate-limiting. Furthermore, the kcat/Km
observed for the methylation of non-target sites is decreased up to
20,000-fold (48). Thus, the overall discrimination against non-target
site methylation is likely to be mediated at the level of the
precatalytic isomerization and the chemistry step. The formation and
interconversion among the conformationally distinct structural
intermediates would serve to enhance sequence specificity relative to
an enzyme that does not partition among multiple enzyme-DNA
intermediates. Ultimately, a quantitative understanding of enzyme
discrimination demands knowledge of the relative magnitude of both of
these energetic barriers toward the modification of target and
non-target sites.
The stabilization of an extrahelical base circumvents the problem of
catalyzing a stereo-specific reaction at sterically occluded DNA bases
or target lesions. For AdoMet-dependent enzymes, base flipping minimizes DNA-cofactor interaction by removing the bound AdoMet from the enzyme-DNA interface. This permits site-specific methylation at many different DNA sequences, which might otherwise not
be possible if sequence-specific recognition elements involved the
bound AdoMet molecule. This expansion of the recognition sequence diversity is not without energetic consequence. DNA methyltransferases typically bind DNA significantly less tightly than the related restriction endonucleases or repressor type proteins. The free energy
differences separating the closed and open forms of the base pair and
the large activation enthalpy (5-25 kcal mol1)
associated with spontaneous base pair opening (49) provide a rationale
for the differences in equilibrium binding affinities observed for
these distinct classes of proteins. In conclusion, direct real time
observation and rigorous solution-based characterization of the
nucleotide flipping process allows mechanistic investigation into the
origin of sequence specificity and enzyme discrimination derived from
this dramatic protein-nucleic acid interaction.
| |
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 s1 (B. W. Allan,
J. M. Beechem, and N. O. Reich, manuscript in
preparation).
4 N. O. Reich and K. Maegley, unpublished data.
| |
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Abstract
Introduction
Procedures
Results & Discussion
References
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