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INTRODUCTION |
Biological methylation of DNA plays an important role in the
expression of genetic information. In eukaryotes, this process is
essential for controlling transcription, genomic imprinting, developmental regulation, mutagenesis, DNA repair, and chromatin organization (1). In prokaryotes, Dam DNA methyltransferases (MTases)1 regulate a number
of cell functions (reviewed in Ref. 2). An essential biological role
for DNA adenine methylation in determining bacterial virulence was
discovered in Salmonella typhimurium, where
dam
mutants were found to be avirulent; such
strains were effective in producing live vaccines against murine
typhoid fever (3). In prokaryotes, other MTases are usually found as
components of restriction-modification systems (4). Three kinds of DNA
MTases are known to exist in prokaryotes, i.e. C5-cytosine
(Cyt), N4-Cyt, and
N6-adenine (Ade) MTases (1). The catalytic
mechanism of methyl group transfer from the donor,
S-adenosyl-L-methionine (AdoMet), in the case of
C5-Cyt MTases, involves an intermediate in which the enzyme is
covalently bound to the target Cyt (5). In contrast, N6-Ade and N4-Cyt MTases
transfer a methyl group to the exocyclic amino group without forming
such an intermediate. It appears, however, that all MTases access their
target base by flipping it out of the DNA double helix, as first
reported for HhaI MTase (6). In general, DNA MTases appear
to be functional monomers, and one of their natural substrates is
hemimethylated DNA, the product of semi-conservative DNA replication.
Hence, one methyl group transfer is sufficient to methylate completely
one specific (hemimethylated) site; this process is generally referred
to as "maintenance" methylation. In this regard, productive
interaction between a monomeric MTase and its hemimethylated target
site is accomplished only after the enzyme is correctly oriented to the
unmodified target-strand base.
Reich and Mashhoon (7), measuring single turnovers in the reaction
catalyzed by EcoRI MTase, showed that there was no
specificity in binding orientation; i.e. the enzyme
methylated only 50% of the substrate-hemimethylated duplexes. However,
a different result was obtained in pre-steady state kinetics of
methylation of 20-mer duplexes by the bacteriophage T4 Dam
(N6-Ade)-MTase. Equal bursts of product
formation were observed with both a symmetric site GATC/GATC and a
hemimethylated, asymmetric site, GATC/GMTC (where M represents
N6-methyl Ade) (8). This was contrary to
expectation for a monomeric enzyme binding randomly to its target site
with respect to an unmethylated (productive) versus
methylated (nonproductive) strand. We hypothesized that the T4
Dam-AdoMet complex is capable of undergoing a rapid reorientation
following binding to the nonproductive DNA strand. Analogous results
were obtained for the RsrI (9), HhaI (10), and
EcoDam (11) DNA MTases.
Earlier, we investigated the kinetic characteristics of relatively
short (20-mer) oligonucleotide substrates containing one specific site,
native 5'-GATC/5'-GATC or modified 5'-GATC/5'-GATC (hemimethylated, or
with substitutions/deletions of different structural elements in one of
the two strands) (8, 12). These studies permitted us to characterize
the interaction of T4 Dam with one specific site as well as the
influence of defined modifications in site structure on kinetic
parameters. In addition, we used fluorescence titration to investigate
the interaction of T4 Dam with these 20-mer oligonucleotide duplexes in
which one or two target Ade residues were substituted by 2-aminopurine
(N), and we showed that AdoMet plays a crucial role in T4 Dam
reorientation about the DNA duplex (13). The intensity of 2-aminopurine
(N) residue fluorescence is low inside the double-stranded DNA helix; however, it increases sharply if the fluorophor is out of stacking interaction, permitting one to study the base flipping process induced
by enzyme binding (14). The addition of T4 Dam at a saturating
concentration to an unmethylated target (N/A duplex) or its methylated
derivative (N/M duplex) resulted in an up to 50-fold increase in
fluorescence (13). This indicated that T4 Dam binding promotes or
stabilizes base (nucleoside) flipping out of the DNA helix. However,
the addition of AdoMet sharply reduced the Dam-induced fluorescence
with these complexes. In contrast, AdoMet had no effect on the
fluorescence increase produced with an N/N doubly substituted duplex.
Because the N/M duplex cannot be methylated, the AdoMet-induced
decrease in fluorescence could not be due to methylation per
se. We proposed that T4 Dam randomly binds to the asymmetric N/A
and N/M duplexes and that AdoMet induces an allosteric T4 Dam
conformational change, allowing a rapid reorientation of the enzyme to
the strand that contains the unmethylated Ade. This capability is
likely to be advantageous for more efficient maintenance methylation
in vivo.
Natural in vivo DNA substrates are much longer and contain
many potential methylation sites. Thus, in vitro methylation
of short single-site duplexes is not going to take into account
possible processive behavior of the DNA MTases; i.e.
movement of the enzyme along the DNA via one dimensional (or linear)
diffusion and carrying out multiple turnovers on the same substrate
molecule (11, 15-16). To initiate a bridge between studies using short
(20-mer) one-site DNA substrates and natural long DNA substrates, we
constructed two-site DNA duplexes to compare their substrate
characteristics with those of their one-site constituents. While this
work was in progress, Urig et al. (11) reported that the
EcoDam MTase acts in a processive manner on both synthetic
duplexes and polymeric phage
DNA.
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EXPERIMENTAL PROCEDURES |
Enzymes and
Chemicals--
[3H-CH3]AdoMet (15 Ci/mmol; 1 mCi/ml) was purchased from Amersham. Unlabeled AdoMet (Sigma) was
purified further by chromatography on a C18 reversed phase
column as described previously (17). Oligonucleotides (Table I) were
synthesized by Integrated DNA Technologies, Inc. (Coralville, IA);
their concentrations were determined spectrophotometrically. The
duplexes were obtained by heating and annealing complementary
oligonucleotide chains from 90 to 20 °C over 7-12 h. The 40-mer
duplexes were constructed from corresponding short component duplexes
using T4 polynucleotide kinase and T4 DNA ligase (SibEnzyme). T4 Dam
MTase was purified to homogeneity as described previously (17). The
protein concentration was determined by the Bradford method (18), which
yielded values in close agreement with those determined
spectrophotometrically at 280 nm from the known composition and molar
extinction coefficients of individual aromatic amino acid residues in
6.0 M guanidinium hydrochloride and 0.02 M
phosphate buffer, pH 6.5 (19).
DNA MTase Assay--
DNA MTase assays were similar to those
reported previously (20). T4 Dam reactions were carried out at 25 °C
in buffer containing 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and
0.2 mg/ml bovine serum albumin (17). The reactions were initiated by
the addition of prewarmed T4 Dam to preincubated mixtures of
[3H-CH3]AdoMet and substrate DNA. At
appropriate intervals, aliquots (15 µl) were withdrawn from the
mixture and spotted on a DE81 anion-exchange filter disc (Whatman, 1.5 cm). Filters were washed three times with 0.02 M
NH4HCO3, twice with water, once with 75% ethanol, and then dried. They were counted in a toluene liquid scintillator. The molar concentrations of
3H-CH3 groups incorporated into DNA were
quantified as described previously (21). The validity of the
quantification procedure was confirmed under complete methylation
conditions (~1 h at a 1:2 enzyme/substrate ratio) where the
calculated number of 3H-CH3 groups incorporated
into DNA coincided with the number of methylatable Ade residues. All
experiments were done at least twice. Kinetic data were analyzed using
the program Scientist 2.01 (MicroMath®) for regression
analysis. The burst values (B) and steady state rate constants
(kcat) were determined using the equation
[3H-DNA]/[enzyme] = B + kcatt.
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RESULTS AND DISCUSSION |
Rationale for Determining T4 Dam Processivity--
Investigation
of the kinetics and processivity of methylation of oligomeric and
concatemeric DNAs containing multiple GATC and (hemi) methylated GMTC
sites can extend our understanding of the mechanism of site/strand
selectivity. To approach these issues, we constructed substrates by
ligating short, defined synthetic duplexes containing complementary
single-stranded overhangs (Table II) and generated a series of 40-mer
duplexes with different combinations of GATC and GMTC sites (Table
III). For example, ligation of duplexes 1a/a and 2a/a (each containing
a single symmetric unmodified site GATC/GATC) created the control
prototype duplex A as illustrated below in Structure 1,
with further information provided in Tables
I and II. Consider the
methylation of the 40-mer duplex E
(1m/m*2m/a), which has only one GATC site available for methylation. At
high [DNA]/[enzyme] ratios, a T4 Dam monomer will bind the duplex,
as seen in Structure 2,
asymmetrically at one of four possible positions (denoted
"1"-"4"). If the enzyme is unable to reorient on the duplex
and does not act processively, then we would expect a maximum
"burst" value of 0.25 (number of 3H-CH3
groups transferred per bound T4 Dam in the pre-steady state phase)
because "4" is the only productive site, and appropriate orientation to it is necessary for methylation. However, if the enzyme
is capable of reorienting at position "3" to position "4," then
the burst could be as high as 0.5. Moreover, T4 Dam bound at position
"1" may be capable of moving (by linear diffusion) to "3" and
reorient to "4"; perhaps it might even reorient from "2" to
"1" and diffuse to "3," where it reorients. If some combination of these events occurs, then even higher burst values could be registered (with 1.0 as the theoretical maximum). Hence, by determining the burst value we can ascertain whether T4 Dam was able to adapt to a
substrate in which a productive target site-strand represented only
one-quarter of the initial binding orientations. This type of
experiment was carried out with several 40-mer duplexes containing differing combinations of variant sites.
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Table I
Oligodeoxyribonucleotides used in this study
Nucleotide residues included in recognition site, GATC, are underlined.
M is N6-methyl Ade.
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Table II
Synthetic single-site duplexes used to construct 40-mer substrates
containing two specific sites
Duplexes were produced by heating and annealing complementary
oligonucleotide chains (90-20 °C over 7-12 hours).
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Measurement of T4 Dam Processivity--
The pre-steady state burst
values (B) and steady state rate constants
(kcat) were determined for both the 40-mers and
the stoichiometric mixtures of the unligated component duplexes used to generate each 40-mer (Fig. 1 and Table
III). The kcat
values for 40-mer duplexes A-D (with two potential sites for
methylation) and the corresponding mixtures of constituents were all
comparable; they were close to the value of kcat = 0.015 s
1 observed for a single-site 20-mer duplex (20).
Hence, it appears that T4 Dam steady state methylation of 40-mer
duplexes is similar to the methylation of a shorter 20-mer duplex, and
both have the same rate-limiting step in the overall reaction. In this
regard, we recently showed that methylation of a 20-mer duplex is
consistent with a steady state-ordered bi-bi mechanism in which the
order of substrate binding and product release (methylated DNA,
DNAMe, and the S-adenosyl-L-homocysteine,
AdoHcy) is AdoMet
DNA
DNAMe
AdoHcy
(21). The
related EcoDam MTase has a steady state-ordered bi-bi
mechanism where the order of substrate binding is DNA
AdoMet
(11).
After the chemical step (methyl group transfer from AdoMet to DNA),
product DNAMe dissociates relatively rapidly
(koff = 1.7 s
1) from the complex.
In contrast, dissociation of product AdoHcy proceeds relatively slowly
(koff = 0.018 s
1), indicating that
its release is the rate-limiting step, consistent with the
kcat = 0.015 s
1 (20).

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Fig. 1.
Kinetics of T4 Dam methylation of 40-mer
duplexes A-F (triangles) and corresponding
stoichiometric mixtures of the unligated components
(circles). The concentrations of enzyme and
duplexes were 10 and 200 nM, respectively.
[3H-CH3]AdoMet was at 5 µM.
A, duplex A; B, duplex B; C, duplex C;
D, duplex D; E, duplex E; F, duplex
F.
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Table III
Kinetic characteristics of duplexes methylation
Pre-steady state and steady state kinetic analyses of T4 Dam
methylation were carried out on 40-mer duplexes with stoichiometric
amounts of their corresponding unligated component duplexes. Kinetic
data were analyzed using the program Scientist 2.01 (MicroMath®) for
regression analysis. The burst values (B, the number of
[3H-CH3] groups incorporated per T4 Dam during the
pre-steady state phase) and kcat values were
determined using the equation [3H-DNA]/[enzyme] = B + kcatt.
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In contrast to the results for steady state methylation, the burst
values differed for the 40-mer duplexes versus the
stoichiometric mixtures of their component-short duplexes. For example,
40-mer duplex A (1a/a*2a/a) had a burst of 1.86 compared with 1.01 for the corresponding mixture of short constituents "1a/a + 2a/a". The
latter burst value was also observed with a single-site 20-mer duplex
(8). It follows that, after methylation of one Ade in the palindromic
GATC/GATC site, T4 Dam dissociates from a short duplex prior to its
exchanging product AdoHcy for substrate AdoMet. In contrast, the burst
value of 1.8 with 40-mer duplex A suggests that, after methylation of
one site, T4 Dam is capable of linear diffusion, release of product
AdoHcy without dissociating from DNA, binding another AdoMet, and
methylation of a second site. This appears to contradict the sequence
of events observed during steady state cycles, viz.
AdoMet
DNA
DNAMe
AdoHcy
. To reconcile this
apparent discrepancy, we propose the following series of events during
the pre-steady state methylation phase of the two-site 40-mer duplex A.
First, T4 Dam binds substrate AdoMet (AdoMet
), this
complex binds randomly to the 40-mer duplex (DNA
), and methylation
of a target GATC ensues (GATC
GMTC). Next, T4 Dam bound with product AdoHcy leaves the methylated site (GMTC
); at a nonspecific DNA sequence, AdoHcy rapidly dissociates from the T4 Dam-DNA-AdoHcy complex
(AdoHcy
) and is exchanged with AdoMet (AdoMet
). Only this enzymic
form is capable of reorientation (8, 13) at a hemimethylated site (see
below). This sequence of the events does not contradict the scheme
AdoMet
DNA
DNAMe
AdoHcy
, but here the event
DNAMe
signifies departure from the GMTC site without
physically dissociating from the DNA duplex.
Reorientation of T4 Dam to the Methylation Target--
We proposed
earlier that AdoMet induces an allosteric T4 Dam conformational change,
allowing a rapid reorientation of the enzyme to the DNA strand that
contains the unmethylated GATC (8, 13). Thus, when comparing bursts for
different duplexes, it is important to keep in mind that, according to
our model, only T4 Dam-AdoMet is capable of reorientation at a
hemimethylated site. It follows that when a hemimethylated site is
generated enzymatically, the T4 Dam-product AdoHcy cannot reorient to
the other strand. Rather, it diffuses away from the newly created GMTC.
The 40-mer duplex B (Table III) is distinguished from duplex A by
having one hemimethylated target; however, the two bursts were almost
identical. This can be explained according to the following scenario.
T4 Dam bound oriented to the lower strand can methylate one or two A
residues (positions "2" and "4"), assuming for simplicity that
diffusion is unidirectional (e.g. only 5'
3'). (In
contrast, EcoDam appears to diffuse bi-directionally; i.e. does a random walk on the DNA; Ref. 11). Some T4 Dam
molecules may bind duplex B oriented to the top strand and methylate
"position 1." These then diffuse away from the product GMTC site,
and exchange AdoHcy for substrate AdoMet. Upon reaching methylated site
"3," the T4 Dam-AdoMet complex reorients to "4" and is
proficient for methyl transfer. This route allows for a successive
transfer of up to 3 methyl groups, at positions "1," "4," and
"2," respectively. Finally, some T4 Dam molecules may bind at
"3," where they can reorient to "4" and then transfer up to two
methyl groups to the bottom strand. Thus, with duplex B, the
theoretical maximum burst is somewhere between 2.0 and 3.0. However,
some T4 Dam molecules may catalyze a methyl transfer and diffuse off
the duplex before exchanging product AdoHcy for AdoMet. The observed
burst of 1.0 for both unligated A and B component 20-mer duplexes
(Table III) is consistent with this notion. It should be noted that our
assumption about unidirectional movement is not implausible. The
transfer of the methyl group from AdoMet to DNA can be considered
irreversible for the T4 Dam MTase (22), because the reverse reaction
was estimated to be at least 500-fold slower than the forward one. Hence, it follows, that DNA methylation is accompanied by the liberation of significant energy (
G0 =
3.7 kcal/mole).
This energy can be used in part for T4 Dam isomerization (22) and for
unidirectional 5'
3' movement of T4 Dam along the DNA.
We measured burst values for duplexes in which two methylated positions
are located in cis on one strand (duplex C) or in trans on the complementary strands (duplex D). As shown in Table III, their burst values were about 1.8. If no reorientation could occur, then we would expect a maximum burst of 0.75 with duplex C,
because only one or two methyl transfers would be catalyzed by T4 Dam
productively bound on the lower strand. T4 Dam bound unproductively
oriented on the top strand would not register any methyl transfers.
Furthermore, with duplex D, we'd expect a maximum of one methyl group
transferred to each strand, Thus, the observed bursts can best be
explained by T4 Dam being capable of rapid reorientation at
hemimethylated sites. In this regard, formation of T4 Dam dimers cannot
explain these results, because the calculated burst values would have
to be reduced 2-fold to account for the binding of two enzyme
molecules. Moreover, under the conditions similar to those used in
these experiments, we found no evidence for the formation of
appreciable amounts of T4 Dam dimers (13).
The methylation results with duplexes E and F are distinct from those
obtained with the other 40-mer duplexes (Table III). First, duplexes E
and F have only one methylatable site (M/A and A/A, respectively) plus
one fully methylated site (M/M). Both 40-mers gave burst values of
about 1.0 (almost 2-fold lower compared with duplexes A-D). In the
case of duplex F, this indicates that T4 Dam was not able to reorient
following methylation at the A/A site, essentially what would be
predicted if T4 Dam-AdoHcy diffused away from an enzymatically created
GMTC site and could not reorient. Thus, the interaction of T4 Dam with
duplex F is functionally equivalent to that observed for a 20-mer
duplex with a single unmethylated site, because both give bursts of
1.0. The unligated duplex F component 20-mers, 1m/m and 2a/a, gave a
burst of 0.5 as expected, because one-half of the T4 Dam molecules
bound unproductively to the fully methylated 1m/m duplex. For duplex E,
T4 Dam binding oriented to the lower strand would result in the
transfer of zero or one methyl group, depending on the location of the
initial binding and assuming unidirectional diffusion. In contrast, T4 Dam binding oriented to the upper strand would not result in any methyl
group transfer to that strand. However, the enzyme could reorient at
the hemimethylated M/A site and catalyze methyl transfer. It follows
that a burst of one with duplex E is possible only if T4 Dam were able
to reorient at the hemimethylated site.
The mixtures of unligated, component 20-mers for duplexes E and F
contained stoichiometric amounts of molecules with a fully methylated
M/M site and a methylatable A/A or M/A site. These mixtures are similar
to those for duplexes A and C, except that the latter do not contain
any M/M duplexes. Because half of the 20-mer duplex E and F components
can not contribute to a burst, we'd predict that the burst values
should be ~50% of those obtained with the duplex A and duplex C
mixtures. As seen in Table III, this was indeed the case,
i.e. 1.01 versus 0.52 and 1.12 versus 0.69. Taken together, these results lend strong support to the model
for pre-steady state methylation described above.
Finally, it should be noted that the stoichiometric mixtures of the E
and F unligated component-short duplexes had about 2-fold lower
kcat and burst values compared with those for
duplexes A-D; this might be due to "product inhibition" by the
fully methylated, short duplex "1m/m." In contrast, judging from
the higher kcat values for the 40-mer duplexes E
and F, it would appear that a fully methylated site exerted little
inhibition. This suggests that T4 Dam is better able to move from one
site to another on longer 40-mer duplexes compared with
dissociation/reassociation with the shorter one-site duplexes.
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CONCLUSIONS |
Based on the results presented, we draw the following conclusions.
(i) T4 Dam MTase modifies the 40-mer two-site duplexes in a processive
fashion. (ii) During processive movement on DNA from one site to the
next, T4 Dam is capable of rapidly exchanging product AdoHcy (in the
ternary complex) for substrate AdoMet without dissociating from the DNA
duplex. (iii) The processive steps of T4 Dam action are consistent with
an ordered bi-bi mechanism
AdoMet
DNA
DNAMe
AdoHcy
. However, in contrast to
the steady state, here DNAMe
signifies departure from a
methylated site, GMTC
, without physically dissociating from the DNA
molecule. (iv) Following methyl transfer at one site and linear
diffusion to a hemimethylated site, T4 Dam-AdoMet is capable of rapidly
reorienting itself to the (productive) unmethylated strand. In
contrast, T4 Dam-AdoHcy is not capable of reorientation at an
enzymatically created GMTC site. (v) The inhibition potential of fully
methylated sites, 5'-GMTC/5'-GMTC, is much lower in a long DNA molecule
compared with short single-site duplexes.
The results of the present work are important for refining our
understanding of T4 Dam methylation and its relation to the mechanisms
of other processive DNA MTases. First, the ability of T4 Dam to
reorient to the methylation target, deduced earlier by us (8, 13) and
directly demonstrated in this work, may be a general property of
processive DNA MTases. Second, in long DNA molecules the inhibition
potential of fully methylated sites must be low for other DNA MTases.
If a processive DNA MTase performs a random walk on the DNA molecule,
as recently suggested for EcoDam (11), it may signify that
the enzyme passes over fully methylated sites without any kinetic retardation.