Recombinant Human DNA (Cytosine-5) Methyltransferase

III. ALLOSTERIC CONTROL, REACTION ORDER, AND INFLUENCE OF PLASMID TOPOLOGY AND TRIPLET REPEAT LENGTH ON METHYLATION OF THE FRAGILE X CGG·CCG SEQUENCE*

Albino Bacolla, Sriharsa PradhanDagger , Jacquelynn E. Larson, Richard J. RobertsDagger , and Robert D. Wells§

From the Institute of Biosciences and Technology, Center for Genome Research, Texas A & M University System Health Science Center, Texas Medical Center, Houston, Texas 77030-3303 and Dagger  New England Biolabs, Beverly, Massachusetts 01915

Received for publication, January 16, 2001, and in revised form, February 26, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Steady-state kinetic analyses revealed that the methylation reaction of the human DNA (cytosine-5) methyltransferase 1 (DNMT1) is repressed by the N-terminal domain comprising the first 501 amino acids, and that repression is relieved when methylated DNA binds to this region. DNMT1 lacking the first 501 amino acids retains its preference for hemimethylated DNA. The methylation reaction proceeds by a sequential mechanism, and either substrate (S-adenosyl-L-methionine and unmethylated DNA) may be the first to bind to the active site. However, initial binding of S-adenosyl-L-methionine is preferred. The binding affinities of DNA for both the regulatory and the catalytic sites increase in the presence of methylated CpG dinucleotides and vary considerably (more than one hundred times) according to DNA sequence. DNA topology strongly influences the reaction rates, which increased with increasing negative superhelical tension. These kinetic data are consistent with the role of DNMT1 in maintaining the methylation patterns throughout development and suggest that the enzyme may be involved in the etiology of fragile X, a syndrome characterized by de novo methylation of a greatly expanded CGG·CCG triplet repeat sequence.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, DNMT1Delta 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 DNMT1Delta 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:


v=<FR><NU>V<SUB><UP>max</UP></SUB>[A][B]</NU><DE>K<SUB>ia(<UP>app</UP>)</SUB>K<SUP>B</SUP><SUB>m</SUB>+K<SUP>B</SUP><SUB>m</SUB>[A]+K<SUP>A</SUP><SUB>m(<UP>app</UP>)</SUB>[B]+[A][B]</DE></FR> (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<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> and K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP>). Accordingly, K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> K<UP><SUB><IT>i</IT></SUB><SUP>AdoMet</SUP></UP> (1 + KDNA/[DNA]), K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> = K<UP><SUB><IT>i</IT></SUB><SUP>DNA</SUP></UP> (1 + KDNA/[DNA]), K<UP><SUB><IT>i</IT></SUB><SUP>AdoMet</SUP></UP> k4/k3, K<UP><SUB><IT>i</IT></SUB><SUP>DNA</SUP></UP> = k6/k5, KDNA = k2/k1, K<UP><SUB><IT>m</IT>(app)</SUB><SUP>AdoMet</SUP></UP> K<UP><SUB><IT>m</IT></SUB><SUP>AdoMet</SUP></UP> (1 + KDNA/[DNA]), K<UP><SUB><IT>m</IT>(app)</SUB><SUP>DNA</SUP></UP> = K<UP><SUB><IT>m</IT></SUB><SUP>DNA</SUP></UP> (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.
<FR><NU>1</NU><DE>v</DE></FR>=<FR><NU>1</NU><DE>V<SUB><UP>max</UP></SUB></DE></FR><FENCE>1+<FR><NU>K<SUP>B</SUP><SUB>m</SUB></NU><DE>[B]</DE></FR></FENCE>+<FR><NU>K<SUP>A</SUP><SUB>m(<UP>app</UP>)</SUB></NU><DE>V<SUB><UP>max</UP></SUB></DE></FR><FENCE>1+<FR><NU>K<SUB>ia</SUB>K<SUP>B</SUP><SUB>m</SUB></NU><DE>K<SUP>A</SUP><SUB>m</SUB>[B]</DE></FR></FENCE> · <FR><NU>1</NU><DE>[A]</DE></FR> (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,
<UP>Slope</UP><SUB>1/A</SUB>=<FR><NU>K<SUP>A</SUP><SUB>m(<UP>app</UP>)</SUB></NU><DE>V<SUB><UP>max</UP></SUB></DE></FR>+<FR><NU>K<SUB>ia(<UP>app</UP>)</SUB>K<SUP>B</SUP><SUB>m</SUB></NU><DE>V<SUB><UP>max</UP></SUB></DE></FR> · <FR><NU>1</NU><DE>[B]</DE></FR> (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.

Alternatively, when [B] is the variable substrate in Equation 1, the reciprocal of the velocity is given in Equation 4.
<FR><NU>1</NU><DE>v</DE></FR>=<FR><NU>1</NU><DE>V<SUB><UP>max</UP></SUB></DE></FR><FENCE>1+<FR><NU>K<SUP>A</SUP><SUB>m(<UP>app</UP>)</SUB></NU><DE>[A]</DE></FR></FENCE>+<FR><NU>K<SUP>B</SUP><SUB>m</SUB></NU><DE>V<SUB><UP>max</UP></SUB></DE></FR><FENCE>1+<FR><NU>K<SUB>ia(<UP>app</UP>)</SUB></NU><DE>[A]</DE></FR></FENCE> · <FR><NU>1</NU><DE>[B]</DE></FR> (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,
<UP>Slope</UP><SUB>1/B</SUB>=<FR><NU>K<SUP>B</SUP><SUB>m</SUB></NU><DE>V<SUB><UP>max</UP></SUB></DE></FR>+<FR><NU>K<SUB>ia(<UP>app</UP>)</SUB>K<SUP>B</SUP><SUB>m</SUB></NU><DE>V<SUB><UP>max</UP></SUB></DE></FR> · <FR><NU>1</NU><DE>[A]</DE></FR> (Eq. 5)
Because Equations 3 and 5 have the identical slope of Kia(app)K<UP><SUB><IT>m</IT></SUB><SUP><IT>B</IT></SUP></UP>/Vmax, where Kia(app) is the apparent dissociation constant for substrate A and K<UP><SUB><IT>m</IT></SUB><SUP><IT>B</IT></SUP></UP> is the Michaelis constant for substrate B, K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> may be found by setting K<UP><SUB><IT>m</IT></SUB><SUP><IT>B</IT></SUP></UP> K<UP><SUB><IT>m</IT></SUB><SUP>DNA</SUP></UP>, whereas K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> may be found by setting K<UP><SUB><IT>m</IT></SUB><SUP><IT>B</IT></SUP></UP> K<UP><SUB><IT>m</IT></SUB><SUP>AdoMet</SUP></UP> 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 DNMT1Delta 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 (-<sigma > = 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 DNMT1Delta 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 DNMT1Delta 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 DNMT1Delta 501 shows the translation of the first four amino acids along with the initiator methionine.

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 DNMT1Delta 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 DNMT1Delta 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 DNMT1Delta 501 in 1 min versus the reciprocal concentration of the variable substrate (horizontal axis). The DNMT1Delta 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.

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 DNMT1Delta 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 DNMT1Delta 501. DNMT1Delta 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.

In summary, this transformation from curved velocity patterns with DNMT1 (19, 29) into linear and intersecting patterns with DNMT1Delta 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.

DNMT1Delta 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<UP><SUB><IT>m</IT></SUB><SUP>DNA</SUP></UP> values for the unmethylated and the hemimethylated FMR1 sequence indicates that preference for the hemimethylated template is retained in DNMT1Delta 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<UP><SUB><IT>m</IT></SUB><SUP>AdoMet</SUP></UP>, K<UP><SUB><IT>m</IT></SUB><SUP>CG</SUP></UP>, 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 DNMT1Delta 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.

Table II shows the ratios of the steady-state kinetic parameters between DNMT1Delta 501 and DNMT1. Both the values of kcat and kcat/K<UP><SUB><IT>m</IT></SUB><SUP>DNA</SUP></UP> 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> and K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP>) indicates that the truncation increases the apparent affinity of DNMT1Delta 501 for the hemimethylated FMR1 sequence. Furthermore, because the K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> and K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> 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.

                              
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Table II
Ratio of steady-state kinetic parameters between recombinant human DNMT1Delta 501 and DNMT1

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 DNMT1Delta 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 DNMT1Delta 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 DNMT1Delta 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.

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.

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<UP><SUB><IT>m</IT></SUB><SUP>DNA</SUP></UP> and K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> 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<UP><SUB><IT>m</IT></SUB><SUP>DNA</SUP></UP> 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 DNMT1Delta 501
The standard deviations ranged from 2 to 54% of the mean values.

A second analysis was conducted by evaluating the steady-state kinetic parameters with DNMT1 for pRW3602 under three different topological conditions, supercoiled (- <sigma > = 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<UP><SUB><IT>m</IT></SUB><SUP>DNA</SUP></UP> 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> and K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP>) 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 DNMT1Delta 501 (19, 29). The dissociation constants obtained with DNMT1Delta 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<UP><SUB><IT>i</IT></SUB><SUP>AdoMet</SUP></UP> 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<UP><SUB><IT>i</IT></SUB><SUP>DNA</SUP></UP> ranged between 0.1 and 1 µM for the same DNA substrates. Contrary to K<UP><SUB><IT>i</IT></SUB><SUP>AdoMet</SUP></UP>, 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 DNMT1Delta 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> values for (CGG·CCG)12 and (CGG·Cm5CG)12 between DNMT1Delta 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> 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<UP><SUB><IT>i</IT></SUB><SUP>DNA</SUP></UP> for (CGG·Cm5CG)12) relative to (CGG·CCG)12 with DNMT1Delta 501 indicates that methylation increased the affinity of the DNA for the catalytic site. Comparison of the increases in K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> obtained with DNMT1 relative to DNMT1Delta 501 for (poly(dI-dC)·poly(dI-dC), (CGG·CCG)12, and (CGG·Cm5CG)12) with those for K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> indicates that neither K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> nor K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> increased for poly(dI-dC)·poly(dI-dC) and (CGG·Cm5CG)12. For (CGG·CCG)12, the K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> value increased about 8-fold with DNMT1; however, K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> but not to K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP>. 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> values did not change, K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> 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<UP><SUB><IT>m</IT></SUB><SUP>DNA</SUP></UP> 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.

Finally, the K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> values for supercoiled, relaxed closed, and linear pRW3602 were the highest of all the substrates evaluated. Together with the K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> 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 DNMT1Delta 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP> (open circles) or between kcat and K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> (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<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP>, as shown by the 95% confidence interval, whereas there was only poor correlation (r2 = 0.250) between the log kcat and log K<UP><SUB><IT>i</IT>(app)</SUB><SUP>DNA</SUP></UP>. 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<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP>; open circles, log K<UP><SUB><IT>i</IT>(app)</SUB><SUP>CG</SUP></UP> and log K<UP><SUB><IT>i</IT>(app)</SUB><SUP>CI</SUP></UP>. The shaded area shows the 95% confidence interval for the correlation with the K<UP><SUB><IT>i</IT>(app)</SUB><SUP>AdoMet</SUP></UP> data.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 DNMT1Delta 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<UP><SUB><IT>m</IT></SUB><SUP>DNA</SUP></UP>) 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.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM46127 (to R. J. R.), GM52982, and NS37554 (to R. D. W.), the Robert A. Welch Foundation, and Polycystic Kidney Research Foundation Grant 99004 (to R. D. W.). This paper is the third in a series on human DNA (cytosine-5) methyltransferase. Papers I and II appeared in J. Biol. Chem. 274, 33002-33010 and 33011-33019.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: Institute of Biosciences and Technology, Center for Genome Research, Texas A & M University System Health Science Center, Texas Medical Center, 2121 Holcombe Blvd., Houston, TX 77030-3303. Tel.: 713-677-7651; Fax: 713-677-7689; E-mail: rwells@ibt.tamu.edu.

Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M100404200

2 S. Pradhan and G.-D. Kim, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; DNMT1, human DNA (cytosine-5) methyltransferase 1; DNMT1Delta 501, human DNA (cytosine-5) methyltransferase 1 lacking the first N-terminal 501 amino acids; Dnmt, mammalian DNA (cytosine-5) methyltransferases; SNRPN, exon 1 of the small nucleoriboprotein N gene; FMR1, fragile X mental retardation gene 1; bp, base pair.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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