 |
INTRODUCTION |
The mammalian genome is epigenetically modified by the transfer of
methyl groups from S-adenosyl-L-methionine
(AdoMet)1 to acceptor bases
in double-stranded DNA, mostly at the carbon 5 of cytosine when the
base is part of a CG dinucleotide (1). Several DNA methyltransferases
(Dnmt) have been identified, including Dnmt1 (2), Dnmt2 (3), Dnmt3A,
Dnmt3B (4), a splice variant of Dnmt1 (Dnmt1b) (5), and an
oocyte-specific isoform of Dnmt1 that lacks the first 118 N-terminal
amino acids (6). Targeted mutations of Dnmt1 (7),
Dnmt3A, and Dnmt3B genes are recessive lethals
(8) in mice attesting to their essential role in development. In
humans, mutations in the DNMT3B gene have been associated
with the (immunodeficiency, centromere
instability, and facial abnormalities syndrome (ICF
syndrome) (9), a recessive disorder characterized biochemically by
hypomethylation of satellite 2 and 3 DNA from the juxtacentromeric
heterochromatin of chromosomes 1 and 16 (10-12) as well as other
common non-satellite repeats (13).
Genetic studies suggest that Dnmt1 and Dnmt3 carry out two types of
modifications as follows: both Dnmt3A and -B establish patterns of
methylation early in embryogenesis by de novo methylation of
cytosine residues, whereas Dnmt1 maintains such patterns throughout life by copying them onto newly synthesized DNA (4, 14). Methylation
studies in vitro do not reveal such distinction of functions. In fact, whereas kinetic determinations show that Dnmt1 utilizes hemimethylated DNA 5-10-fold better than unmethylated DNA
(15-19), they also show that this enzyme methylates unmethylated DNA
more efficiently than Dnmt3A and Dnmt3B (4). Therefore, it is unclear
whether the de novo activity of Dnmt1 is repressed in
vivo.
Most cases of fragile X syndrome, a relatively frequent (1:4000 births)
hereditary neurological disease that causes mental retardation (20),
are associated with the expansion of a CGG·CCG repeat in the
5'-untranslated region of the FMR1 gene (21). The repeat is
polymorphic in the general population, varying from 6 to 60 units.
Occasional expansions from 61 to 200 units, in the so-called
pre-mutation range, trigger further instability such that the tract
expands up to thousands of repeats (full mutation) when transmitted to
offspring (22). In individuals with full mutation, the disease state
also requires the de novo methylation of the CGG·CCG tract
and the proximal CpG island (23-25), which leads to silencing of
FMR1 transcription. However, two important unknowns include
the following: first, what mechanisms trigger this de novo
activity, and second, which methyltransferase(s) are involved.
Herein, we present steady-state kinetic investigations on human
recombinant DNMT1 (19) using DNA substrates of random sequence or
substrates composed of CGG·CCG repeats. The data show that DNMT1 is
under allosteric repression, a control mechanism that reconciles the
results in vitro with the genetic studies in
vivo. The binding of AdoMet and the DNA to the active site occurs
by a random mechanism; however, the preferred pathway involves binding of AdoMet followed by DNA. The enzymatic activity depends on the topological state of the DNA and increases with negative supercoiling. These results indicate that although the methylation reaction is under
tight control, factors such as CGG·CCG expansion and the accumulation
of high levels of supercoiling stimulate de novo activity,
which are consistent with the possibility that DNMT1 may be involved in
the fragile X syndrome.
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EXPERIMENTAL PROCEDURES |
Human (Cytosine-5) DNA Methyltransferase Transfer Vector,
Viral Transfection, and Recombinant Protein
Expression--
Full-length human DNMT1 expression
constructs were derived from pBKSHMT5.0(b) (gift of Prof. S. Baylin,
The Johns Hopkins University). This plasmid had the full-length human
DNMT1 cDNA based on the previously published sequence
(26). Baculovirus expression of the full-length DNMT1 was
from clone HMT8. Transfer vector pVICHMT (19) has three
BamHI sites, one before the DNMT1 translation
initiation (in the vector) and the other two in the DNMT1
open reading frame at nucleotide positions 1704 and 2784 (GenBankTM accession number X63692). For the preparation of
the N-terminal 501 amino acid deletion mutant,
DNMT1
501, partial digestion of pVICHMT was
done with BamHI. A 14.5-kilobase pair band corresponding to
the DNA fragment lacking the first 1704 nucleotides of the
DNMT1 cDNA was excised from the gel, religated, and
transformed into Escherichia coli. The ligated junctions in the final pVICD501HMT construct were verified by DNA sequencing. The
enzyme was purified from clone 21 (HMT21). A pupal ovarian cell line
(SF9) from the worm Spodoptera frugiperda was used for co-transfection and expression of the human DNMT1 and
DNMT1
501 as described previously (27) with
the following modifications. SF9 cells were maintained as a suspension
culture in TNM-FH media (JRH Biosciences, Lenexa, KS)
supplemented with 10% v/v fetal calf serum and an
antibiotic/antimycotic solution at a final concentration of 5 units of
penicillin, 50 µg/ml streptomycin, and 0.125 µg/ml amphotericin B
at 27 °C on a Bellco steering platform at 70-80 rpm.
Co-transfection of a monolayer of SF9 insect cells was carried out
using BaculoGold DNA, a modified, linearized Autographa
californica nuclear polyhedrosis virus DNA (PharMingen, San Diego,
CA), and the transfer vector pVICD501HMT. Screening for recombinant
clones and plaque purification was described previously (28). For
routine protein expression, SF9 cells were grown in spinner culture
flasks. SF9 cells at a density of 1.2-1.5 × 106
ml
1 were infected at a multiplicity of
infection between 7 and 10 with HMT8/HMT21. The cells were kept at
27 °C and stirred at 60 rpm. Cells were harvested 48 h
post-infection, pelleted, and washed with phosphate-buffered saline.
Protein purification and quantitation were described previously (19).
Enzyme preparations were >95% pure (19).
Determination of the Kinetic Constants--
We previously
developed a velocity equation for DNMT1 (29), based on the King-Altman
and Cleland methods (30), that considered the enzyme species bound to
the AdoMet and unmethylated DNA substrates but not to the AdoHcy and
methylated DNA products (initial forward velocity equation). These
conditions are met experimentally when the amount of substrate utilized
is negligible compared with its total concentration. The velocity
equation in the presence of products would be quite complex. In our
velocity equation, the term associated with Kia was
arbitrarily chosen to be AdoMet because the order for substrate
addition in the methyl transfer reaction was unknown. Because evidence
is now presented that the reaction order for DNMT1 is random, we
re-write the velocity equation in the form shown in Equation 1:
|
(Eq. 1)
|
where both [A] and [B] are
the concentrations of AdoMet and DNA, in CG (or CI, where CI is the CpI
dinucleotide in the poly(dI-dC)·poly(dI-dC template) steps, and
Kia(app) indicates the apparent dissociation constant for both AdoMet and the DNA
(K
and
K
). Accordingly,
K
= K
(1 + KDNA/[DNA]),
K
= K
(1 + KDNA/[DNA]),
K
= k4/k3,
K
= k6/k5,
KDNA = k2/k1, K
= K
(1 + KDNA/[DNA]),
K
= K
(1 + KDNA/[DNA]) as outlined in
Fig. 1. This modification of the velocity
equation does not alter the estimation of the Michaelis constants and
kcat; however, it enables the calculation of the dissociation constants for AdoMet and DNA as follows. In Equation 1
when [A] is the variable substrate the reciprocal of the
velocity is as shown in Equation 2.
|
(Eq. 2)
|
When the slopes in Equation 2, which are obtained by varying the
fixed concentrations of [B], are replotted as a function of 1/[B], they yield the following Equation 3,
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(Eq. 3)
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Fig. 1.
Reaction order for recombinant human
DNMT1. *D, hemimethylated or fully methylated DNA that
binds to the N-terminal regulatory site in DNMT1; E, DNMT1;
Am, S-(5'-adenosyl)-L-methionine
(AdoMet); CG, unmethylated CG step in unmethylated or
hemimethylated DNA that binds to the catalytic center; Ah,
S-(5'-adenosyl)-L-homocysteine;
m5CG, methylated CG step.
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|
Alternatively, when [B] is the variable substrate
in Equation 1, the reciprocal of the velocity is given in Equation 4.
|
(Eq. 4)
|
Similarly, when the slopes in Equation 4, which are obtained by
varying the fixed concentrations of [A], are replotted as a function of 1/[A], they yield the following Equation 5,
|
(Eq. 5)
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Because Equations 3 and 5 have the identical slope of
Kia(app)K
/Vmax,
where Kia(app) is the apparent
dissociation constant for substrate A and
K
is the Michaelis constant for
substrate B,
K
may be found by
setting K
= K
, whereas
K
may be found by
setting K
= K
in the replots of the
slopes of the double-reciprocal plots obtained from Equations 2 and 4.
A comparable method, which we did not utilize, would be to analyze the
vertical coordinate of the crossover point for the same
double-reciprocal plots (31).
The reaction conditions for the methyl transfer reaction with
recombinant human DNMT1
501 and DNMT1 with
all of the DNA substrates used and the calculation of the steady-state
kinetic constants were as described (29). The concentrations of the
reactants and those of the products analyzed were always converted to
nanomoles or micromoles per liter and therefore are expressed in
nM or µM, respectively. Also, the given
concentrations are always those in the final reaction mixture.
DNA Substrates and Products--
The DNA substrates used for
calculating the kinetic constants were the polymer
poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech, average length
7000 bp); the oligonucleotides (CGG·CCG)12, (m5CGG·CCG)12,
(CGG·Cm5CG)12, and (CGG·CCG)73;
SNRPN (exon 1 of the small nucleoriboprotein N
gene); SNRPN methylated on the upper strand;
SNRPN methylated on the lower strand; and plasmid DNA that
included linear, relaxed circular, and negatively supercoiled pRW3602.
The synthesis and characterization of these substrates were reported
(19, 29). Negatively supercoiled pRW3602 (32) was isolated from
E. coli strain HB101 and purified by CsCl banding (33). Its
average negative superhelical density (
<
> = 0.045) was
determined from a series of agarose gel electrophoreses performed in
the presence of chloroquine using known populations of topoisomers as
reference (32). Two oligonucleotides were employed for product
inhibition studies as follows: MeCG20, a
chemically synthesized 40-bp duplex oligonucleotide containing 5-methylcytosine at all 20 CG steps, and
F/MeCG12, a chemically synthesized 36-bp duplex
oligonucleotide containing the FMR1-associated CGG·CCG
triplet repeat substituted with 5-fluorocytosines on the upper strand
and 5-methylcytosines on the lower strand at the CG steps as described
(29).
Methylation Reaction on Topological Isomers of
pRW3691--
Sixteen µg of CsCl-purified pRW3691 (29, 34) were
treated with chicken erythrocyte topoisomerase I in the presence of 0, 0.5, 1.0, 1.5, 2.0, and 3.0 µg/ml ethidium bromide, purified, and
analyzed for their average number of superhelical turns as described
(32). The concentration of CG steps for each of the six purified
topoisomer populations was obtained from the DNA concentration measured
spectrophometrically at 260 and 280 nm. For each topoisomer population,
an amount of pRW3691 corresponding to 25 µM CG steps in
the final reaction mixture was incubated with 40 nM DNMT1
and 10 µM
S-(5'-adenosyl)-L-[methyl-3H]methionine
([3H]AdoMet) (specific activity 15.0 Ci/mmol (1 Ci = 37 GBq), Amersham Pharmacia Biotech) in a total volume of 25 µl in
buffer A (29) at 37 °C for 30 min. Samples were frozen, heated to
65 °C for 20 min to inactivate DNMT1, supplemented with 5 µl of
10× buffer B (1 M NaCl, 100 mM
MgCl2, 10 mM dithiothreitol, 1 mg/ml bovine serum albumin), 2 µl each of HindIII and BamHI,
16 µl of H2O, and incubated at 37 °C for 4 h to
release the (CGG·CCG)73-containing fragment and the
vector DNA. This restriction splits the total number of CG steps of
pRW3691 into two nearly equal parts, 170 CG steps in the vector and 164 in the (CGG·CCG)73-containing fragment. The two
restriction fragments were isolated on a preparative 1.2% agarose gel,
stained with ethidium bromide, cut from the gel, transferred to
scintillation vials containing 2 ml of distilled H2O,
melted, immediately diluted with 15 ml of Ultima-Gold liquid scintillation mixture (Packard Instrument Co.), and counted on a liquid
scintillation analyzer (Packard Instrument Co.). Control vials
containing free [3H]AdoMet, but no melted gels slices, or
free [3H]AdoMet plus a melted gel slice of comparable
weight (~0.8 g), but no DNA, indicated that the melted gel matrix did
not quench the radioactive signals. The concentration of
[3H]CH3 incorporated in each DNA fragment was
obtained from the ratio of the counts/min of the DNA samples minus the
counts/min of the blank, divided by the counts/min given by 2 µl of
free [3H]AdoMet. The blank consisted of an average
counts/min value obtained from four melted gel slices of 0.8 g
each that were excised from the DNA lanes of the agarose gels used to
separate the restriction fragments. This value was 590 cpm. The values
for the DNA samples ranged from 3,788 to 2,306,472 cpm.
 |
RESULTS |
An N-terminal Peptide Regulates the Reaction Rate through DNA
Binding--
Previous studies with full-length recombinant human DNMT1
revealed the linear and intersecting velocity patterns for the majority of the DNA substrates used (19, 29). However, complex patterns were
observed with two DNA substrates, poly(dI-dC)·poly(dI-dC) (35) and
hemimethylated (CGG·Cm5CG)12, which revealed
both inhibition and activation of the reaction. Furthermore, inhibition
studies with fully methylated random sequence DNA (29) showed that this
product stimulated methylation. These data suggested that the reaction
was normally repressed and that de-repression could be achieved once
the DNA substrate was bound to an allosteric site.
To test this hypothesis, deletions were made in the N-terminal portion
of DNMT1, which was believed not to be involved in catalysis (2), and
the velocity curves were re-evaluated at various concentrations of
AdoMet and DNA (poly(dI-dC)·poly(dI-dC) and hemimethylated
(CGG·Cm5CG)12). Herein, we report the results
with DNMT1
501, a catalytically active enzyme
that lacks the first 501 N-terminal amino acids (19) (Fig.
2).

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Fig. 2.
Schematic diagram of full-length recombinant
human DNMT1 and DNMT1 501.
Functionally mapped regions of the full-length DNMT1 are illustrated
along with the C-terminal catalytic domain. DMAP1,
DNMT1-associated protein binding region, amino acids 1-126 (40);
PCNA, proliferative cell nuclear antigen-binding motif,
amino acids 162-174 (41); NLS, nuclear localization signal,
amino acids 194-213 (2); RF, replication fork targeting
peptide sequence, amino acids 320-567; Zn, zinc-binding
region. Repressor domain shows homology to the
tri-thorax-related proteins HRX (69) and ALL1 (70). HDAC,
histone deacetylase binding region, amino acid 653-812, located in the
repressor domain (71). Rb-1 and Rb-2,
retinoblastoma gene product binding region 1, amino acids 416-913
(61), and 2, amino acids 1-3362 (42). *, phosphorylated
serine at amino acid 509 (72). B, BamHI site at
nucleotide position 1704. Human DNMT1 501
shows the translation of the first four amino acids along with the
initiator methionine.
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Fig. 3 shows the velocity patterns for
(CGG·CCG)12, as control, and for
(CGG·Cm5CG)12 (note the changes of scale on
the horizontal axes). The reciprocal transfer of
[3H]CH3 from AdoMet to the DNA is indicated
on the ordinate as a function of DNA concentration at
various fixed concentrations of AdoMet (A and C)
or as a function of AdoMet at various fixed concentrations of DNA
(B and D). The patterns for
(CGG·CCG)12 with the truncated enzyme are identical to
those obtained with the full-length enzyme (29), i.e. they
are linear and intersect to the left of the vertical axis. This result
indicates that the truncation did not alter the bi-reactant sequential
mode of substrate binding characteristic of DNMT1 and gives confidence
that kinetic studies carried out with
DNMT1
501 contribute to elucidating the
properties of the full-length enzyme. The patterns for the
hemimethylated (CGG·Cm5CG)12 are also linear
and intersecting (C and D), contrary to those
observed with DNMT1 (29), which were curved and not intersecting. This
result indicates that a region within the 501 N-terminal amino acids
was involved in generating the complex kinetic pattern (29) for
(CGG·Cm5CG)12 with DNMT1.

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Fig. 3.
Double-reciprocal plots for the methylation
of FMR1 locus by
DNMT1 501. The Lineweaver-Burk
plots present 1/v (vertical axis) expressed as
the reciprocal of nM [3H]CH3
contained in the DNA following the transfer from AdoMet by 1 nM DNMT1 501 in 1 min
versus the reciprocal concentration of the variable
substrate (horizontal axis). The
DNMT1 501 concentration was 40 nM; the symbols in Figs. 3 and 4 for the changing fixed
concentrations of substrate are in the following order: filled
circles, open circles, filled squares,
open squares, filled diamonds, and open
diamonds. A, reciprocal of
[3H]CH3 concentration as a function of
varying the DNA at various fixed concentrations of AdoMet for
(CGG·CCG)12; AdoMet concentrations were 50.0, 15.9, 9.26, 5.02, 4.50, and 4.02 µM. B, reciprocal of
[3H]CH3 concentration as a function of
varying AdoMet at various fixed concentrations of CG for
(CGG·CCG)12; CG concentrations were 5.0, 2.5, 1.25, 0.83, 0.62, and 0.50 µM. C, reciprocal of
[3H]CH3 concentration as a function of
varying the DNA at various fixed concentrations of AdoMet for
(CGG·Cm5CG)12; AdoMet concentrations were
10.0, 5.02, 2.56, 1.74, 1.43, and 1.02 µM. D,
reciprocal of [3H]CH3 concentration as a
function of varying AdoMet at various fixed-concentrations of CG for
(CGG·Cm5CG)12; CG concentrations were 1.0, 0.5, 0.25, 0.16, 0.12, and 0.10 µM.
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The velocity curves with full-length DNMT1 showed substrate inhibition
with poly(dI-dC)·poly(dI-dC), particularly at low AdoMet concentrations (19). The patterns obtained with
DNMT1
501 are shown in Fig.
4. At the identical AdoMet
concentrations, substrate inhibition is no longer present
(A), confirming a role for the N-terminal domain in
regulating the turnover rate. When AdoMet is the variable substrate
(B) the velocity responses are linear from 1.74 to 20 µM (1/AdoMet of 0.05-0.57). The loss of linearity at
1.02 µM (1/AdoMet = 0.98) probably reflects a
departure from steady-state conditions.

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Fig. 4.
Double-reciprocal plots for the methylation
of poly(dI-dC)·poly(dI-dC) by
DNMT1 501.
DNMT1 501 concentration was 8 nM.
A, reciprocal of [3H]CH3
concentration as a function of varying DNA at various fixed
concentrations of AdoMet; AdoMet concentrations were 20.0, 10.0, 5.02, 2.56, 1.74, and 1.02 µM. B, reciprocal of
[3H]CH3 concentration as a function of
varying AdoMet at various fixed concentrations of CI (CpI
dinucleotide); CI concentrations were 1.0, 0.5, 0.25, 0.16, 0.12, and
0.10 µM.
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|
In summary, this transformation from curved velocity patterns with
DNMT1 (19, 29) into linear and intersecting patterns with
DNMT1
501 (Figs. 3 and 4) shows that the
N-terminal domain of the enzyme is involved in the regulation of the
reaction rate through binding of the DNA.
DNMT1
501 Maintains the Preference for Hemimethylated
DNA While Acquiring Increased Catalytic Activity--
Replots of
slopes and intercepts of the double-reciprocal plots shown in Figs. 3
and 4 indicate linear relationships with respect to the various fixed
concentrations of substrate (Fig. 5).
These results enable the evaluation of the Michaelis constants for DNA
and AdoMet as well as the turnover numbers (Table
I) (see "Experimental Procedures" and
Ref. 29). A comparison of the kcat and
kcat/K
values for the unmethylated and the hemimethylated FMR1
sequence indicates that preference for the hemimethylated template is
retained in DNMT1
501, showing that the
residues involved are downstream of the N-terminal regulatory
domain.

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Fig. 5.
Replots of slopes and intercepts. Replot
of slopes and vertical axis intercepts of the
double-reciprocal plots from Figs. 3 and 4 as a function of the
changing fixed concentrations of substrate. A, data from
Fig. 3A. B, data from Fig. 3B.
C, data from Fig. 3C. D, data from
Fig. 3D. E, data from Fig. 4A.
F, data from Fig. 4B. Filled circles, replot of
the slopes; open circles, replot of the vertical
axis intercepts. Fitting of the data and derivation of the
steady-state kinetic parameters K ,
K , and
kcat was performed as described (see Ref. 29 and
"Experimental Procedures").
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Table I
Steady-state kinetic parameters of recombinant human
DNMT1 501 using poly(dI-dC) · poly(dI-dC) and FMR1
locus as substrates
The standard deviations from two determinations ranged from 0.6 to 14%
of the mean values. The ratio between the hemimethylated and the
unmethylated FMR1 sequence is in parentheses.
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Table II shows the ratios of the
steady-state kinetic parameters between
DNMT1
501 and DNMT1. Both the values of
kcat and
kcat/K
are greater for the truncated enzyme, indicating that the N-terminal peptide represses the enzymatic activity. Inspection of the ratios for
the substrate dissociation constants
(K
and
K
) indicates that the
truncation increases the apparent affinity of
DNMT1
501 for the hemimethylated
FMR1 sequence. Furthermore, because the K
and
K
ratios are 0.1 and 0.3 for the unmethylated sequence, respectively, we conclude that the
truncation increases the apparent affinity for both AdoMet and the
FMR1 sequence. Thus, we propose that the N-terminal domain of DNMT1 interferes with the accessibility of the substrates to the
catalytic center, therefore reducing the overall reaction rate.
AdoMet and DNA Bind Randomly to the Active Site--
An aspect
that remained unresolved during the previous study (29) was the order
in which the two substrates bound to the active center, i.e.
whether either AdoMet or DNA can be the first to bind or whether their
addition must follow a defined order. The difficulty in establishing
this kinetic question was the presence of the N-terminal regulatory
site, which yielded stimulatory effects in the presence of fully
methylated DNA, thus obscuring the inhibition patterns. If the absence
of such a domain in DNMT1
501 confines
binding of methylated DNA to the active site and thereby eliminates
activation, this would reveal whether substrate addition is random or
ordered. For a bireactant ordered reaction, the inhibition pattern for
the first substrate that binds is competitive versus its
product, whereas that of the second substrate is non-competitive versus its product. Alternatively, the inhibition patterns
for both substrates are competitive versus their respective
products for a random reaction (36). Our previous experiments (29) showed that the inhibition pattern between AdoMet and its product AdoHcy was competitive.
The inhibition pattern of fully methylated DNA versus
unmethylated DNA for DNMT1
501 is shown in
Fig. 6. Fig. 6A shows the
reciprocal of the incorporation of [3H]CH3 by
supercoiled pRW3602 (vertical axis) as a function of DNA
(horizontal axis) at changing fixed concentrations of
methylated DNA and a constant AdoMet concentration of 6.67 µM. The convergence of the curves on the vertical axis
was based on the replot of their slopes and intercepts (Fig.
6B), which indicated that the slopes increased as a function
of methylated DNA, whereas the 1/Vmax(app)
values remained constant. This pattern shows competitive inhibition
between fully methylated and unmethylated DNA and, together with the
patterns obtained with AdoHcy, indicates that both AdoMet and DNA add
randomly to the catalytic center. Thus, we conclude that the methyl
transfer reaction for recombinant human DNMT1 occurs by a bireactant
sequential random mechanism.

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Fig. 6.
Product inhibition with
DNMT1 501 by fully methylated
(MeCG)20 duplex DNA. The sequence of
(MeCG)20 was reported previously (29).
A, double-reciprocal plot for supercoiled pRW3602 at varying
CG concentrations (horizontal axis) and changing fixed
concentrations of the (MeCG)20 inhibitor at the
constant AdoMet concentration of 6.67 µM.
m5CG concentrations in the (MeCG)20
duplex oligonucleotide were 0 (filled circles), 10 (open circles), 20 (filled squares), 30 (open squares), 40 (filled diamonds), 50 (open diamonds), and 60 µM (filled
triangles). B, replot of slopes and vertical
axis intercepts from A. Data points and error
bars are from the double-reciprocal plots before constraint to the
convergence point was applied.
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Negative Supercoiling Increases Reaction Rates--
The interest
in determining whether negative supercoiling influences the rate of
methylation was largely stimulated by the clinical observation that the
number of CGG·CCG repeats in the FMR1 locus expands to
more than 200 copies, from 6 to 60 of the general population, and
becomes de novo methylated in fragile X patients (22).
Because the bending restoring force, but not the torsional modulus, for
CGG·CCG DNA is ~40% lower than for random DNA (34), we
hypothesized that an expanded CGG·CCG tract would represent a genomic
site for the preferential partitioning of negative supercoiling (37),
which is known to unwind stretches of DNA (38). The free energy of
supercoiling associated with unwinding would consequently
generate alternative DNA structures such as slipped structures, which
could increase the rates of the methylation reaction. Thus, the CG
steps within an expanded CGG·CCG would become kinetically favorable
substrates for de novo methylation.
Six families of topological isomers of pRW3691, which contains an
insert with 73 consecutive CGG·CCG triplet repeats, with increasing
average negative supercoil densities were methylated with DNMT1 and
[3H]AdoMet under conditions where no more than 5-7% of
CG sites were modified. The insert harboring the triplet repeat tract, with a total of 164 CG steps (146 CG steps from the triplet repeat plus
18 CG steps from its flanking sequence), and the vector harboring 170 CG steps were separated by 1% agarose gel electrophoresis following
restriction enzyme cleavage, and the extent of methylation was
determined for the insert and the vector (Fig.
7).

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Fig. 7.
Supercoil-dependent methylation
of pRW3691 by recombinant human DMNT1. The graph shows the
nanomoles [3H]CH3 incorporated (left
ordinate) in the vector sequence (filled squares) or in
the insert containing the (CGG·CCG)73 triplet repeat
tract (filled circles) of pRW3691 at six different
superhelical densities. The right ordinate indicates the
fold increase in methylation where a value of 1 indicates the nanomoles
[3H]CH3 incorporated when the plasmid was in
the relaxed topological state. The data show the mean and standard
deviation from three separate experiments.
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The turnover rates increased with negative supercoiling, about 2-fold
for the vector and 7-fold for the triplet repeat tract, indicating that
the free energy of supercoiling plays a significant role in
methylation. The concentration of [3H]CH3
incorporated into the triplet repeat-containing fragment were
5-10-fold lower than those incorporated in the vector. These differences are not justified by the kcat values
for plasmid DNA (Table III) and the
(CGG·CCG)n triplet repeats (Table V). Instead, because these
two DNAs differ mostly in their
K
and
K
values (Table
IV), it is possible that DNA
sequence-specific interactions with DNMT1 and/or differences in DNA
binding at the catalytic versus regulatory domain dominate
the apparent turnover rates (i.e. at unsaturating substrate
concentrations). A second possibility is that the CGG·CCG repeats
display substrate inhibition [like poly(dI-dC)·poly(dI-dC)] at high
concentrations. In fact, because of the low
K
values, the concentrations
of CG steps ranged between 0.1 and 5 µM in the velocity
studies with (CGG·CCG)n, whereas they ranged between 3 and 40 µM for this plasmid DNA.
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Table III
Steady-state kinetic parameters of recombinant human DNMT1 using
pRW3602 as substrate
The standard deviations from two determinations ranged from 2 to 70%
of the mean values.
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Table IV
Dissociation constants for recombinant human DNMT1 and
DNMT1 501
The standard deviations ranged from 2 to 54% of the mean values.
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A second analysis was conducted by evaluating the steady-state kinetic
parameters with DNMT1 for pRW3602 under three different topological
conditions, supercoiled (
<
> = 0.045), relaxed circular, and
linear (Table III) (29). The kcat values were 5- and 3-fold higher for the supercoiled and linear conformers,
respectively, than for relaxed DNA. The most dramatic difference was
the 7.7-fold reduction in K
for relaxed DNA, suggesting that DNA topology influenced the rate
constants for substrate binding and/or product release. In summary,
these data show that the rates of methylation with the human
recombinant DNMT1 are accelerated by the free energy of supercoiling
and that such increases are sensitive to DNA sequence.
Analysis of the Dissociation Constants for AdoMet and DNA--
The
dissociation constants for AdoMet and DNA were obtained from the slope
replots (see "Experimental Procedures"). These equilibria
(K
and
K
) represent apparent values that contain an additional factor, i.e. the ratio
between the free enzyme and the enzyme bound to DNA at the allosteric site, Kia(app) = Kia(1 + [E]/[DE]). If the
binding affinity of the DNA for the regulatory site is high, the term in parentheses contributes little, whereas if binding is weak the value
increases significantly.
Table IV summarizes the data obtained with DNMT1 and
DNMT1
501 (19, 29). The dissociation
constants obtained with DNMT1
501 should not
contain the apparent term (1 + [E]/[DE])
since the regulatory site is no longer present. Hence, the range from 2 to 4 µM K
measured for poly(dI-dC)·poly(dI-dC), (CGG·CCG)12, and
(CGG·Cm5CG)12 should also represent the value
for DNMT1. The values for K
ranged between 0.1 and 1 µM for the same DNA substrates.
Contrary to K
, the
dissociation constant for the DNA is not expected to have a constant
value since sequences flanking the CG recognition step may contribute
to the binding affinity. Thus, the 10-fold variation between
(CGG·CCG)12 and (CGG·Cm5CG)12
shows that hemimethylated (CGG·Cm5CG)12 has a
greater affinity for the catalytic center than unmethylated (CGG·CCG)12, as expected (19).
The dissociation constants for both AdoMet and DNA with DNMT1 were
lowest with poly(dI-dC)·poly(dI-dC). These values were similar to
those obtained with DNMT1
501, indicating
that the (1 + [E]/[DE]) term did not
contribute substantially. Because these data show that this DNA has a
strong affinity for the catalytic site, the [DE] complex
may represent DNMT1 associated with DNA at the regulatory or at the
catalytic site. The K
values for the unmethylated (CGG·CCG)12 and the
SNRPN duplex DNA with DNMT1 were substantially higher than
for the corresponding hemimethylated substrates
(m5CGG·CCG)12,
(CGG·Cm5CG)12, SNRPN-methylated
upper strand, and SNRPN-methylated lower strand. This result
indicates that the (1 + [E]/[DE]) term
contributes significantly only for unmethylated DNA, confirming the
data from inhibition studies (29) that hemimethylation increases the
affinity of the DNA for the allosteric site. The same conclusion was
obtained from the comparison of the
K
values for
(CGG·CCG)12 and (CGG·Cm5CG)12
between DNMT1
501 and DNMT1, which shows an
8-fold increase for the unmethylated FMR1 duplex, but
identical values for the hemimethylated
(CGG·Cm5CG)12.
The value of K
decreased 5-fold for the supercoiled pRW3602 compared with the relaxed
circular form showing that the free energy of supercoiling increases
the affinity of the DNA for the enzyme. The low value of
K
for the linear
pWR3602 was unexpected, and it is unclear what contributes to
the difference between relaxed circular and linear pRW3602; obviously,
these two substrates differ for the presence of free ends in the
linear molecule.
The range of K
values for DNMT1 varied by 140-fold. These variations likely reflect differences in the affinities of each DNA molecule for the catalytic and the regulatory sites and the competition for binding at both locations. The 10-fold decrease in
K
for
(CGG·Cm5CG)12) relative to
(CGG·CCG)12 with DNMT1
501
indicates that methylation increased the affinity of the DNA for the
catalytic site. Comparison of the increases in
K
obtained with DNMT1
relative to DNMT1
501 for
(poly(dI-dC)·poly(dI-dC), (CGG·CCG)12, and
(CGG·Cm5CG)12) with those for
K
indicates that
neither K
nor
K
increased for
poly(dI-dC)·poly(dI-dC) and (CGG·Cm5CG)12.
For (CGG·CCG)12, the
K
value increased
about 8-fold with DNMT1; however,
K
only increased 3-fold.
This difference suggests that if a duplex DNA has a low affinity for
the regulatory site but a high affinity for the catalytic site, then
this DNA may bind to the catalytic site prior to or without binding to
the regulatory site. This would then decrease the contribution of the
apparent term to K
but
not to K
. Thus, it is
possible that three pathways lead to the catalytically competent
central complex as follows: one in which DNA binds to the regulatory
site followed by AdoMet and then DNA to the catalytic site; a second in
which DNA binds to the regulatory site followed by DNA and AdoMet to
the catalytic site; and a third in which allosteric binding is bypassed
and DNA binds to the catalytic center followed by AdoMet (Fig. 1).
A comparison of the dissociation constants for the
(CGG·CCG)12 and (CGG·CCG)73 DNA substrates
reveals that although the
K
values did not
change, K
decreased
about 10-fold for the longer molecule. Comparison of the steady-state
kinetic parameters for the (CGG·CCG)12 and
(CGG·CCG)73 (Table V)
indicates that kcat values doubled for
(CGG·CCG)73 and that the
kcat/K
ratio increased five times. Together with the results from the
topoisomer distribution, these results show that both length and
negative supercoiling contribute to increasing the methylation rate of
the CGG·CCG triplet repeat sequence involved in the fragile X
syndrome.
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Table V
Steady-state kinetic parameters of recombinant human DNMT1 using
different lengths of the FMR1 locus as substrate
The standard deviations ranged from 5 to 45% of the mean value.
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Finally, the K
values for supercoiled, relaxed closed, and linear pRW3602 were the
highest of all the substrates evaluated. Together with the
K
values, these
results indicate that plasmid DNAs had a weak affinity for both the
regulatory and catalytic sites.
AdoMet Binding Is Preferred as the First
Substrate--
The results obtained with DNMT1 and
DNMT1
501 indicate that the turnover rates
were not limited by the chemical steps of the methyl transfer reaction
but rather by substrate binding or product release. Therefore, if one
of the three pathways shown in Fig. 1 dominates over the other two, a
correlation may unfold between the kcat and
Kia(app) values. Fig.
8 shows the relationship between kcat and
K
(open
circles) or between kcat and
K
(filled
circles) for the 11 DNA templates shown in Table IV with DNMT1. A
significant correlation (r2 = 0.862) was found
between log kcat and log
K
, as shown by the
95% confidence interval, whereas there was only poor correlation
(r2 = 0.250) between the log
kcat and log
K
. This result indicates
that, subsequent to DNA binding to the regulatory site, AdoMet binding
to the catalytic site followed by DNA was the most common pathway.

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Fig. 8.
Correlation between turnover number and
dissociation constants for recombinant human DNMT1. The log of the
dissociation constants for AdoMet and the DNA presented in Table IV for
DNMT1 were plotted versus the log of their respective
kcat values (19). Filled circles, log
K ; open
circles, log K and
log K . The shaded
area shows the 95% confidence interval for the correlation with
the K data.
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DISCUSSION |
Steady-state kinetics revealed that the methyl transfer
reaction with the recombinant human DNMT1 (19, 29) involves control mechanisms that act through DNA sequence, methyl groups, and DNA topology and that these properties are consistent with the maintenance methylation role of the enzyme in development.
Allosteric Repression and Activation--
The N-terminal region
comprising the first 501 amino acids of DNMT1 inhibits enzymatic
activity. This region also binds methylated DNA, which partially
relieves the inhibition. Therefore, methylated DNA is both an inhibitor
and an allosteric activator for the methyl transfer reaction. Previous
inhibition studies (29) indicated that two DNA molecules may bind
simultaneously to DNMT1, a methylated substrate at the regulatory site
and an unmethylated DNA at the catalytic site, suggesting that binding
at the regulatory site exposes the catalytic center, otherwise
occluded, to the AdoMet and DNA substrates. Because the N-terminal
region is also known to bind unmethylated DNA (39), activation requires
the presence of methyl groups.
The protein domain involved in allosteric regulation also mediates the
interaction of DNMT1 with other cellular factors (Fig. 2) responsible
for targeting the enzyme to the nucleus (2) and to replication
foci2 (40-42). This domain
is also involved in transcriptional repression. Therefore, because of
the dual properties of catalytic repression and protein binding, the
"maintenance methylation" role of DNMT1, and therefore the
silencing of its de novo activity in vivo, may be
achieved by stable inhibition of the catalytic center through the
interaction of the N-terminal domain with binding proteins. This
interaction may either interfere with DNA binding at the regulatory
site or suppress its activation. The loss of DNMT1 activity following
truncations in the N-terminal domain (43, 44) or the binding with
specific antibodies (2) suggests that the catalytic center is in
proximity to N-terminal regions and that its function is sensitive to
their folding and/or dynamic properties.
Reaction Order--
Our inhibition studies show that the
reaction order is random. However, because the turnover numbers
correlate with the apparent dissociation constants for AdoMet, binding
of AdoMet as the first substrate occurs more frequently. DNA substrates
with high affinity for the catalytic center, such as
poly(dI-dC)·poly(dI-dC) and (CGG·Cm5CG)12,
may bind first without necessitating prior allosteric binding (18).
Preference for Hemimethylated DNA--
It has long been
recognized that hemimethylated DNA serves as a better substrate than
unmethylated DNA for mammalian Dnmt1 (16-18, 45). We find that
this property is independent of the presence of the first 501 N-terminal amino acids, which rules out a role for the allosteric
control region. Since the velocity patterns for
poly(dI-dC)·poly(dI-dC) and (CGG·Cm5CG)12
were curved and not intersecting with DNMT1, whereas they were linear
and intersecting with DNMT1
501, we propose
that the preference for hemimethylated DNA is an intrinsic property of
the catalytic center.
Role of Negative Supercoiling, Triplet Repeat Length, and
Hemimethylation in Methylation of the Fragile X (CGG·CCG)n
Sequence--
The methylation rates increased with increasing negative
supercoiling up to 2-fold for DNA of random sequence and up to 7-fold for the (CGG·CCG)73 triplet repeat, whereas the turnover
number was higher (10 h
1) for the supercoiled
form of the plasmid than for the relaxed and linear forms. These data
clearly show that DNA topology plays a significant role in methylation.
The finding is particularly relevant to the fragile X syndrome because
the (CGG·CCG)n repeat has a low persistence length (34) that
enables higher levels of superhelical stress to be present in the
repeat than in the flanking sequences (37). Negative supercoiling is
known to unwind and destabilize duplex DNA and promote alternative DNA structures (38, 46), which for (CGG·CCG)n may consist of
hairpin loops (47). Such hairpin loops are preferred substrates for
DNMT1 (48).
The turnover numbers and specificity constants
(kcat/K
)
also increased when the number of (CGG·CCG) repeats increased,
whereas the apparent dissociation constant decreased. Our
interpretation is that, due to the high affinity of the repeat for the
catalytic pocket, DNMT1 will "stick" in the vicinity of
(CGG·CCG)n molecules by non-elastic collisions (49) and will
associate with molecules with more triplet repeats for longer times
than with molecules with fewer repeats.
We also show that an m5CG step stimulates methylation both
at the complementary unmethylated CG dinucleotide as well as at flanking CG steps. Therefore, sporadic methylation in an otherwise unmethylated (CGG·CCG)n tract may act as a catalyst for further methylation and thereby elicit a cooperative methylation process.
Model for Involvement of DNMT1 in Fragile X Syndrome--
Although
the mechanism for the methylation of (CGG·CCG)n tracts that
lead to the fragile X syndrome is not understood, two consequences of
large repeat expansions are consistent with the involvement of DNMT1.
First, a delay in replication was observed. The timing of DNA
replication for the FMR1 locus and its flanking sequences
(~1 megabase pair total) is lengthened in fragile X males (50,
51), in concert with transcriptional silencing (52). A delay in
replication is also observed in chromosomes of carrier individuals
(53-55) where the CGG·CCG tract is only moderately expanded and
methylation is not observed (22). This suggests that repeat expansion
leads to delay in replication irrespective of its methylation status.
The compact, higher order structure of the transcriptionally silent
heterochromatin requires the interactions between heavily methylated
DNA (56), methylated DNA-binding proteins (57, 58), and deacetylated
histones (59, 60). It is possible that the late replication-specific
interaction of DNMT1 with deacetylated histones (40, 42, 61) activates the enzyme activity to sustain the heavy methylation patterns.
Second, DNA structure and topology may play a critical role in the
methylation process. Transient surges in negative supercoiling may
arise during replication due to the disassembly of nucleosomes (62-64)
and the helicase activity of the polymerase complex (65). An expanded
CGG·CCG tract may form hairpin structures (66) both under the
influence of negative supercoiling and the strand separation that
accompanies replication (67, 68). Therefore, negative supercoiling,
hairpins, and the high concentration of CG steps may trigger sporadic
methylation events that will catalyze further methylation in the
following rounds of replication, eventually leading to extensive methylation.