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• W1982188814 abstract "Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA andS-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA andS-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG·CCG)12, (m5CGG·CCG)12, and (CGG·CCG)73 but were not linear for (CGG·Cm5CG)12. Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at ≤20 μm AdoMet. Addition of the product (methylated DNA) to unmethylated plasmid DNA increased V max(app)resulting in mixed stimulation and inhibition. Velocity equations indicated a two-step mechanism as follows: first, activation of DNMT1 by methylated DNA that bound to an allosteric site, and second, the addition of AdoMet and DNA to the catalytic site. The preference of DNMT1 for hemimethylated DNA may be the result of positive cooperativity of AdoMet binding mediated by allosteric activation by the methylated CG steps. We propose that this activation plays a rolein vivo in the regulation of maintenance methylation. Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA andS-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA andS-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG·CCG)12, (m5CGG·CCG)12, and (CGG·CCG)73 but were not linear for (CGG·Cm5CG)12. Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at ≤20 μm AdoMet. Addition of the product (methylated DNA) to unmethylated plasmid DNA increased V max(app)resulting in mixed stimulation and inhibition. Velocity equations indicated a two-step mechanism as follows: first, activation of DNMT1 by methylated DNA that bound to an allosteric site, and second, the addition of AdoMet and DNA to the catalytic site. The preference of DNMT1 for hemimethylated DNA may be the result of positive cooperativity of AdoMet binding mediated by allosteric activation by the methylated CG steps. We propose that this activation plays a rolein vivo in the regulation of maintenance methylation. C 5-methylcytosine base pair(s) DNA (cytosine-5) methyltransferase S-adenosyl-l-methionine S-adenosyl-l-homocysteine duplex oligonucleotide (75 bp) corresponding to exon-1 of the small nuclear ribonucleoprotein-associated polypeptide N, which is part of an imprinting center on human chromosome 15q11–13 5-fluorocytosine double-stranded 40-mer of random sequence containing methylated cytosine (m5C) at all CG dinucleotide steps duplex oligonucleotide of composition CGG(F5CGG)11·(Cm5CG)12, containing the CGG triplet repeat sequence whose expansion is associated with the fragile-X mental retardation syndrome (the duplex oligonucleotide contains non-methylatable CG steps, F5C on the top strand and m5C on the bottom strand) The genome of most organisms contains modified nucleotides including N 6-methyladenine,N 4-methylcytosine, andC 5-methylcytosine (m5C)1 (1Landry D. Barsomian J.M. Feeherey G.R. Wilson G.G. Methods Enzymol. 1992; 216: 244-259Crossref PubMed Scopus (10) Google Scholar, 2Wilson G.G. Methods Enzymol. 1992; 216: 259-279Crossref PubMed Scopus (56) Google Scholar, 3Cheng X. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 293-318Crossref PubMed Scopus (283) Google Scholar). However, both the biological significance and the types of DNA methylation differ greatly between prokaryotes and eukaryotes. In prokaryotes, most modified bases participate in restriction-modification, a defense mechanism that protects the host from heterologous phage infection (4Roberts R.J. Halford S.S. Linn S.M. Lloyd R.S. Roberts R.J. Nucleases. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 35-88Google Scholar). In addition,N 6-methyladenine plays a role in the initiation of DNA replication (5Noyer-Weidener M. Trautner T.A. Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Significance. Birkhauser Verlag, Basel1993: 39-108Crossref Scopus (98) Google Scholar) and in post-replicative methyl-directed mismatch repair (6Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar).In higher eukaryotes, DNA methylation is confined to m5C and is implicated in the regulation of development, genomic imprinting (7Li E. Beard C. Forster A.C. Bestor T.H. Jaenisch R. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 297-305Crossref PubMed Scopus (87) Google Scholar, 8Li E. Beard C. Jaenisch R. Nature. 1993; 366: 362-365Crossref PubMed Scopus (1743) Google Scholar), X chromosome inactivation, gene expression (9Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Significance. Birkhauser Verlag, Basel1993Crossref Google Scholar), and retrotransposon inactivation (10Doerfler W. Biol. Chem. Hoppe-Seyler. 1991; 372: 557-564Crossref PubMed Scopus (125) Google Scholar, 11Woodcock D.M. Lawler C.B. Linsenmeyer M.E. Doherty J.P. Warren W.D. J. Biol. Chem. 1997; 272: 7810-7816Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 12Bestor T.H. Philos. Trans. R. Soc. Lond-Biol. Sci. 1990; 326: 179-187Crossref PubMed Scopus (178) Google Scholar). In mammals, the patterns of methylation are inherited from both parental genomes but are erased and reconstructed (de novo methylation) in somatic cells following implantation (13Razin A. Shemer R. Hum. Mol. Genet. 1995; 4: 1751-1755Crossref PubMed Scopus (239) Google Scholar, 14Trasler J.M. Hake L.E. Johnson P.A. Alcivar A.A. Millette C.F. Hecht N.B. Mol. Cell. Biol. 1990; 10: 1828-1834Crossref PubMed Scopus (67) Google Scholar). Such patterns are then copied and maintained by hemimethylation of the daughter strands during semiconservative DNA synthesis in S phase (maintenance methylation) (15Leonhardt H. Page A.W. Weier H.-U. Bestor T.H. Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (815) Google Scholar, 16Araujo F.D. Knox J.D. Szyf M. Price G.B. Zannis-Hadjopoulos A. Mol. Cell. Biol. 1998; 18: 3475-3482Crossref PubMed Scopus (74) Google Scholar). However, the mechanisms by which both de novo and maintenance methylation occur remain to be elucidated.Several DNA methyltransferases have been isolated from human and mouse (17Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1259) Google Scholar, 18Van den Wyngaert I. Sprengel J. Kass S.U. Luyten W.H.M.L. FEBS Lett. 1998; 426: 283-289Crossref PubMed Scopus (40) Google Scholar, 19Yoder J.A. Bestor T.H. Hum. Mol. Genet. 1998; 7: 279-284Crossref PubMed Scopus (224) Google Scholar, 20Xu G. Flynn J. Glickman J.F. Reich N.O. Biochem. Biophys. Res. Commun. 1995; 207: 544-551Crossref PubMed Scopus (25) Google Scholar), but it is not clear whether the de novo and maintenance methylations are carried out by separate proteins in vivo or whether both activities are shared by one or more enzymes (21Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3183) Google Scholar, 22Trasler J.M. Alcivar A.A. Hake L.E. Bestor T. Hecht N.B. Nucleic Acids Res. 1992; 20: 2541-2545Crossref PubMed Scopus (48) Google Scholar, 23Yoder J.A. Yen R.-W.C. Vertino P.M. Bestor T.H. Baylin S.B. J. Biol. Chem. 1996; 271: 31092-31097Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 24Tucker K.L. Talbot D. Lee M.A. Leonhardt H. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12920-12925Crossref PubMed Scopus (76) Google Scholar, 25Lei H. Oh S.P. Okano M. Jüttermann R. Goss K.A. Jaenisch R. Li E. Development. 1996; 122: 3195-3205Crossref PubMed Google Scholar, 26Okano M. Xie S. Li E. Nucleic Acids Res. 1998; 26: 2536-2540Crossref PubMed Scopus (336) Google Scholar). Nevertheless, a role for in vivo methylation has been established for isoforms of the human DNMT1 gene (27Deng J. Szyf M. J. Biol. Chem. 1998; 273: 22869-22872Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,28Gaudet F. Talbot D. Leonhardt H. Jaenisch R. J. Biol. Chem. 1998; 273: 32725-32729Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) whose product, DNMT1, methylates C at a CG dinucleotide step, both in single-stranded and double-stranded, unmethylated or hemimethylated, templates (29Wang R.Y.-H. Huang L.-H. Ehrlich M. Nucleic Acids Res. 1984; 12: 3473-3490Crossref PubMed Scopus (26) Google Scholar, 30Hitt M.M. Wu T.-L. Cohen G. Linn S. J. Biol. Chem. 1987; 263: 4392-4399Abstract Full Text PDF Google Scholar, 31Reale A. Lindsay H. Saluz H.P. Pradhan S. Adams R.L.P. Jost J.-P. Strom R. Biochem. J. 1995; 312: 855-861Crossref PubMed Scopus (7) Google Scholar, 32Tollefsbol T.O. Hutchison III, C.A. J. Biol. Chem. 1995; 270: 18543-18550Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 33Flynn J. Glickman J.F. Reich N.O. Biochemistry. 1996; 35: 7308-7315Crossref PubMed Scopus (72) Google Scholar, 34Pradhan S. Talbot D. Sha M. Benner J. Hornstra L. Li E. Jaenisch R. Roberts R.J. Nucleic Acids Res. 1997; 25: 4666-4673Crossref PubMed Scopus (87) Google Scholar). Hemimethylated templates are the most effective for the reaction, and methylation rates increase in the neighborhood of pre-existing m5C residues (35Christman J.K. Sheikhnejad G. Marasco C.J. Sufrin J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7347-7351Crossref PubMed Scopus (72) Google Scholar, 36Lindsay H. Adams R.L.P. Biochem. J. 1996; 320: 473-478Crossref PubMed Scopus (26) Google Scholar, 37Tollefsbol T.O. Hutchison III, C.A. J. Mol. Biol. 1997; 269: 494-504Crossref PubMed Scopus (51) Google Scholar, 38Carotti D. Funiciello S. Palitti F. Strom R. Biochemistry. 1998; 37: 1101-1108Crossref PubMed Scopus (19) Google Scholar). However, little is known about the mechanisms responsible for this preference.DNMT1 has a bipartite structure with the C-terminal 570 amino acids containing the catalytic domain. This region shares sequence homologies with all prokaryotic type II cytosine-5 methyltransferases (39Bestor T. Biochem. Soc. Trans. 1988; 16: 944-947Crossref PubMed Scopus (16) Google Scholar, 40Bestor T. Laudano A. Mattaliano R. Ingram V. J. Mol. Biol. 1988; 203: 971-983Crossref PubMed Scopus (704) Google Scholar), including a PC dipeptide motif that is part of the catalytic center in the crystal structures of M.HhaI (41Klimasauskas S. Kumar S. Roberts R.J. Cheng X. Cell. 1994; 76: 357-369Abstract Full Text PDF PubMed Scopus (913) Google Scholar) and M.HaeIII (42Reinisch K.M. Chen L. Verdine G.I. Lipscomb W.N. Cell. 1995; 82: 143-153Abstract Full Text PDF PubMed Scopus (383) Google Scholar), and the binding site forS-adenosylmethionine (AdoMet), the methyl donor for methyltransferases (43Ramchandani S. Bigey P. Szyf M. Biol. Chem. Hoppe-Seyler. 1998; 379: 535-540Crossref PubMed Scopus (19) Google Scholar, 44Bestor T.H. EMBO J. 1992; 11: 2611-2617Crossref PubMed Scopus (386) Google Scholar). The remaining ∼1000 N-terminal amino acids, which are not present in the prokaryotic enzymes, contain a nuclear localization signal, a replication foci targeting sequence (15Leonhardt H. Page A.W. Weier H.-U. Bestor T.H. Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (815) Google Scholar), and are important in the discrimination between unmethylated and hemimethylated substrates (33Flynn J. Glickman J.F. Reich N.O. Biochemistry. 1996; 35: 7308-7315Crossref PubMed Scopus (72) Google Scholar).Herein, we report kinetic analyses with the human full-length, recombinant, DNMT1 on a variety of DNA substrates with the aim of learning about the mechanism of the methyl transfer reaction and the role of DNA in regulating the enzyme activity. The results confirm the preference of DNMT1 for pre-methylated DNA; however, the kinetics reveal a complex behavior with DNA substrates that are bound more tightly. The reaction follows a sequential mechanism whereby both substrates (DNA and AdoMet) must bind to the enzyme before any product (methylated DNA and AdoHcy) is released and is consistent with a two-step process. First, DNMT1 binds DNA at an allosteric site (probably in the N-terminal domain) and activates the catalytic center, and second, AdoMet and the DNA (which may either be the same molecule bound to the regulatory site or a new DNA molecule) occupy the catalytic site. Allosteric binding of pre-methylated CG is proposed to increase the accessibility of AdoMet to the catalytic center, which then results in an acceleration of the reaction rate.RESULTSInitial velocity experiments enable the evaluation of kinetic constants and provide insights into the mechanism of a reaction. A comprehension of the mechanism of this key enzyme, human DNMT1, is critical for understanding its role in developmental processes and in the etiology of fragile X syndrome and other diseases (7Li E. Beard C. Forster A.C. Bestor T.H. Jaenisch R. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 297-305Crossref PubMed Scopus (87) Google Scholar, 8Li E. Beard C. Jaenisch R. Nature. 1993; 366: 362-365Crossref PubMed Scopus (1743) Google Scholar, 9Jost J.P. Saluz H.P. DNA Methylation: Molecular Biology and Significance. Birkhauser Verlag, Basel1993Crossref Google Scholar, 10Doerfler W. Biol. Chem. Hoppe-Seyler. 1991; 372: 557-564Crossref PubMed Scopus (125) Google Scholar, 50Imbert I. Feng Y. Nelson D.L. Warren S.T. Mandel J.-L. Wells R.D. Warren S.T. Genetic Instabilities and Hereditary Neurological Diseases. Academic Press, San Diego, CA1998: 27-53Google Scholar). A variety of DNA templates (Table I) were methylated to less than 5–8% of the total CG steps by purified, recombinant DNMT1 with the aim of assessing the effect of sequences flanking the substrate CG on the kinetic constants, the role of negative supercoiling, and the mechanism of the reaction. Experimental data were analyzed by graphing the extent of methylation as a function of concentration of DNA or AdoMet, as variable substrates, on double-reciprocal Lineweaver-Burk plots. This report is the second of a series of three papers describing the purification and characterization of DNMT1 (45Pradhan S. Bacolla A. Wells R.D. Roberts R.J. J. Biol. Chem. 1999; 274: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar) and focuses on the mechanism of the methyltransferase reaction. The third paper 2A. Bacolla, S. Pradhan, J. E. Larson, R. J. Roberts, and R. D. Wells, manuscript in preparation. will describe the effect of DNA topology on the reaction rates at CG sites in random as well as CGG·CCG repeat tracts and compare the kinetic properties of DNMT1 with the bacterial M.SssI.Linear Velocity ResponsesFor bireactant enzymes (such as DNMT1), double-reciprocal plots generally give linear responses where 1/v is graphed as a function of 1/substrate. For most of the DNAs used, which included supercoiled pRW3602 as purified fromEscherichia coli (−ς̄ = 0.045), relaxed circular (−ς̄ = 0), or linear, as well as theSNRPN oligonucleotide (unmethylated or hemimethylated), (CGG·CCG)12, (m5CGG·CCG)12, and (CGG·CCG)73, the velocity curves were linear with respect to the variable substrate, whether this was the DNA or AdoMet. Fig.3 shows the data with supercoiled pRW3602. Fig. 3 A shows the concentration of [3H]CH3 groups incorporated when the DNA was the variable substrate (on the x axis) and AdoMet the fixed substrate. Conversely, Fig. 3 B shows the results with AdoMet as the variable substrate and DNA as the fixed substrate. For all of the DNA templates listed above, the velocity patterns were as in Fig.3, i.e. they converged to the left of the y axis and above or below the x axis for both substrates. Fig.4 shows the double-reciprocal plots for the triplet repeat sequences (CGG·CCG)12 (Fig. 4,A and B), (m5CGG·CCG)12 (Fig. 4, C andD), and (CGG·CCG)73 (Fig. 4, E andF). These patterns contrast with those obtained with M.HhaI (51Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar) and M.SssI2methylases, where the families of lines converge on the yaxis when AdoMet is the variable substrate. This result shows that AdoMet and DNA do not bind by an ordered and rapid equilibrium mechanism to DNMT1, as in the case with M.HhaI and M.SssI.Figure 4Initial forward velocities for the methylation of CGG·CCG triplet repeats. The Lineweaver-Burk double-reciprocal plots present 1/v (y axis) expressed as the reciprocal of nm[3H]CH3 contained in the DNA following the transfer from AdoMet by 1 nm DNMT1 in 1 minversus the reciprocal concentration of the variable substrate (x axis). The DNMT1 concentration was 40 nm. The concentrations of reactants for each are in the following order: filled circles, open circles,filled squares, open squares, filled diamonds, andopen diamonds. A, [3H]CH3 concentration as a function of variable DNA at changing-fixed AdoMet for (CGG·CCG)12; AdoMet concentrations were 50.0, 15.9, 9.26, 5.02, 4.50, and 4.08 μm. B, [3H]CH3concentration as a function of variable AdoMet at changing-fixed DNA for (CGG·CCG)12; CG concentrations were 5.00, 2.50, 1.25, 0.83, 0.62, and 0.50 μm. C, [3H]CH3 concentration as a function of variable DNA at changing-fixed AdoMet for (m5CGG·CCG)12; AdoMet concentrations were 10.0, 5.02, 3.38, 1.73, 1.23, and 1.02 μm. D, [3H]CH3 concentration as a function of variable AdoMet at changing-fixed DNA for (m5CGG·CCG)12; CG concentrations were 1.00, 0.50, 0.25, 0.17, 0.12, and 0.10 μm. E, [3H]CH3 concentration as a function of variable DNA at changing-fixed AdoMet for (CGG·CCG)73; AdoMet concentrations were 20.0, 9.00, 5.88, 4.00, 2.50, and 2.11 μm. F, [3H]CH3concentration as a function of variable AdoMet at changing-fixed DNA for (CGG·CCG)73; CG concentrations were 1.00, 0.40, 0.25, 0.18, 0.14, and 0.12 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5 shows the replots of the slope and y axis intercept (1/V max(app)) for each of the lines in Fig. 3 that were used to derive the kinetic constants. As shown in Equations 2 and 3, four replots are possible that give the following constants: (a) 1/V max on they axis intercept (Fig. 5 A) and 1/ KmAdoMet on thex-axis intercept by replotting the intercepts of Fig.3 A as a function of 1/AdoMet (Equation 3); (b) KmCG/V maxon the y axis intercept (Fig. 5 A) and 1/K ia on the x axis intercept by replotting the slopes of Fig. 3 A as a function of 1/AdoMet; (c) 1/V max on the y axis intercept (Fig. 5 B) and 1/ KmCG on the x axis intercept by replotting the intercepts from Fig. 3 B as a function of 1/CG (Equation 2); and (d) KmAdoMet/V maxon the y axis intercept (Fig. 5 B) and KmAdoMet/K ia KmCGon the x axis intercept by replotting the slopes from Fig.3 B as a function of 1/CG. Therefore, these four replots yield the maximum velocity and the Michaelis constants for AdoMet (at DNA = ∞) and DNA (at AdoMet = ∞). As pointed out previously, the dissociation constant K ia cannot be assigned to the DNA or AdoMet because the method does not distinguish the order of addition. The experimental values for the constants are reported in the companion papers2 (45Pradhan S. Bacolla A. Wells R.D. Roberts R.J. J. Biol. Chem. 1999; 274: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar).Figure 5Replots of intercepts and slopes of initial velocities for the methylation of supercoiled pRW3602. A, replot of slopes and y axis intercepts of the 1/v versus 1/CG data shown in Fig. 3 A. B, replot of slopes and y axis intercepts from the 1/v versus 1/AdoMet data shown in Fig. 3 B. Error bars are the standard error associated with the double-reciprocal plots before constraint to the convergence point was applied.Lines drawn through the experimental data points are from fitting of the data to the slopes and intercepts of Equation 3 forA and Equation 2 for B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Curved Velocity ResponsesUnexpectedly, two template DNAs (d(I-C·I-C)∼7000 and (CGG·Cm5CG)12) gave non-linear initial velocity curves. The results for d(I-C·I-C)∼7000 are described in the accompanying paper (45Pradhan S. Bacolla A. Wells R.D. Roberts R.J. J. Biol. Chem. 1999; 274: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar), whereas the double-reciprocal plots for (CGG·Cm5CG)12 are reported in Fig.6. Fig. 6 A shows that the methylation rate was linear when DNA was the variable substrate. The data also indicate that, contrary to Figs. 3 and 4, velocities were maximal at 1.74 μm AdoMet, such that further increases (up to 10.0 μm) did not result in a decrease in slope or intercept. Fig. 6 B shows that plots were not linear when AdoMet was the variable substrate. Velocities were unchanged, and plots were parallel to the x axis when AdoMet concentrations rose above ∼2 μm. The responses were still dependent on DNA concentration, since 1/V max(app) (yaxis intercepts) decreased with increasing CG content. A replot of 1/V max(app) versus 1/CG was linear (Fig. 7 A), whereas both intercept and slope replots from the data at fixed AdoMet were curved (Fig. 7 B).Figure 6Double-reciprocal plots for the methylation of (CGG·Cm5CG)12. A, [3H]CH3 concentration as a function of DNA at fixed AdoMet concentrations were as follows: open triangles,1.02 μm; filled triangles, 1.23 μm; open diamonds, 1.43 μm; filled diamonds, 1.74 μm; open squares, 2.56 μm; filled squares, 4.17 μm; open circles, 5.02 μm; andfilled circles, 10.0 μm. B, nm [3H]CH3 incorporated as a function of AdoMet at fixed DNA. CG concentrations were as follows:open diamonds, 0.10 μm; filled diamonds, 0.12 μm; open squares, 0.16 μm; filled squares, 0.25 μm;open circles, 0.50 μm; filled circles, 1.00 μm. DNMT1 concentration was 40 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Replots of slopes and intercepts for initial velocities with (CGG·Cm5CG)12. A, replot of y axis intercepts from the 1/v versus 1/AdoMet data shown in Fig. 6 B. B, replot ofy axis intercepts and slopes from the 1/v versus1/CG shown in Fig. 6 A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This result indicates that when this particular DNA was bound to DNMT1, the velocity of the reaction was already maximal at a very low (∼2 μm) AdoMet concentration, contrary to the expectation that maximum velocity requires infinite amounts of AdoMet. The plots in Figs. 6 and 7 still enable the calculation of 1/V max, 1/ KmCG, and 1/ KmAdoMet; however,K ia cannot be derived.The most significant conclusion that can be drawn from these results is that the kinetic behavior of DNMT1 may be dramatically altered by both sequence of the DNA template and its pre-methylation status. Obviously, the scheme in Fig. 2 and its velocity equations are not adequate to describe the results with (CGG·Cm5CG)12 which implies that DNMT1 is capable of complex kinetics. Overall, this combination of linear plus non-linear responses indicates a steady-state mechanism, where the DNA can act simultaneously both as a substrate and as an activator for the reaction. The activation is proposed to occur through binding of a DNA molecule at a site distinct from the catalytic center, i.e. an allosteric, or regulatory, site. The precise sequence of the chemical steps that lead to such non-linear responses, however, is unknown. Nevertheless, it seems clear that the role of the DNA bound to the allosteric site is to increase the affinity of the DNA-DNMT1 complex for AdoMet since low levels of AdoMet are sufficient to maximally drive the reaction.It is noteworthy that the complex enzymatic behavior is associated with a DNA sequence, the (CGG·CCG) n triplet repeat, whose expansion in the chromosomal FRAX locus leads to aberrant methylation and to disease in humans.In summary, these studies indicate that the methylation reaction by DNMT1 may follow complex mechanisms, and both the sequence composition as well as the methylation status of the DNA substrate contribute to this complexity.Product Inhibition with AdoHcyProduct inhibition studies of bireactant enzymes provide a means to distinguish random from ordered sequential Bi Bi mechanisms. Ordered systems give competitive patterns with the first substrate that binds to the enzyme versus the last product that leaves the enzyme (the plots converge on they axis) and non-competitive patterns with the other combinations (the lines converge to the left of the y axis). On the contrary, random mechanisms give competitive inhibition between like substrates and products (with similar chemical structures) and non-competitive patterns between unlike reactants. In our case, determining whether the reaction is ordered or random would enable the assignment of the dissociation constant K ia to the DNA, to AdoMet, or to both in the case of a random system.In the first set of experiments (AdoHcy versus CG with fixed AdoMet) (unlike product and substrate), 2.5 to 25.0 μmAdoHcy were added to reactions where the concentration of CG with supercoiled pRW3602 was varied and AdoMet kept constant. In separate studies, the fixed concentration of AdoMet ranged from 2.0 to 40.0 μm. The pattern of inhibition was non-competitive from 2.0 to 10.0 μm AdoMet, rather uncompetitive at 15.0 and 20.0 μm AdoMet, and non-competitive again from 26.0 to 40.0 μm AdoMet (not shown).In double-reciprocal plots where AdoMet was the variable substrate, AdoHcy the changing-fixed inhibitor (like substrate and product), and CG the fixed co-substrate (4.0 to 25.0 μm), velocities increased, as expected, up to 20.0 μm AdoMet; however, higher concentrations caused strong inhibition by AdoHcy, a result that was not anticipated. In fact, the expectation was that AdoMet would progressively overcome the inhibition by AdoHcy, linearly increasing the reaction rates as its concentration rose. The slopes obtained from the 1/v versus 1/CG plots (which were linear) at various AdoHcy concentrations were graphed as a function of AdoMet. Increasing AdoMet up to 50 μm progressively reduced the slopes (higher velocities) to plateau levels in the absence of AdoHcy, as expected. AdoHcy increased all of the slopes as a result of inhibition, and increasing AdoMet progressively reduced such an inhibition. However, at concentrations of AdoMet >20 μm, there was a new, strong non-competitive inhibition by AdoHcy that was then reduced by higher levels of AdoMet. This pattern found at greater than 20.0 μm AdoMet is anomalous; the reason for this behavior is uncertain. When these unusual data at >20.0 μm AdoMet were excluded, the pattern of inhibition versus AdoHcy (like substrate and product) was competitive (Fig.8 A).Figure 8Product inhibition by AdoHcy. A, nm [3H]CH3incorporated in supercoiled pRW3602 (6.02 μm CG) at varying AdoMet concentrations and changing-fixed AdoHcy as the inhibitor; AdoHcy concentrations were 2.5 μm(filled circles), 5.0 μm (open circles), 10.0 μm (filled squares), 15.0 μm (open squares), 20.0 μm (filled triangles), and 25.0 μm (open triangles). B, Dixon plot for AdoHcy inhibition; the slopes (Slope1/CG) of 1/v versus 1/CG (μm CG in supercoiled pRW3602: 4.00, 4.81, 6.02, 8.06, 12.19, and 25.0) obtained at fixed AdoMet (2.0, 4.0, 6.67, 10.0, 15.0, and 20.0 μm) and changing-fixed AdoHcy (0, 2.5, 5.0, 10.0, 15.0, 20.0, and 25.0 μm) concentrations were calculated; these results then were replotted as Slope1/CG versus 1/AdoMet for each concentration of AdoHcy (all regressions were linear). The slopes of these last regressions are shown as a function of AdoHcy concentrations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To test whether there was evidence for more than one binding site for AdoMet in DNMT1, a Dixon plot (49Segel I.H. Enzyme Kinetics; Behavior and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems. John Wiley & Sons, Inc., New York1975: 505-845Google Scholar) was constructed. Slopes1/CG were replotted as a function of 1/AdoMet for AdoMet ≤20.0 μm, at each fixed value of AdoHcy. The replots were linear, and their slopes (SlopeCG/AdoMet) were finally graphed as a function of AdoHcy (Fig. 8 B). The result was a linear, rather than a parabolic, curve indicating that only one AdoMet-binding site per DNMT1 molecule was detected. Thex axis intercept gives the K i for AdoHcy, which is ∼14 μm.Product Inhibition with Methylated and Fluorinated DNAThe second part of the inhibition studies consisted of the use of the other product, fully methylated DNA. In conjunction with the data with AdoHcy, fully methylated DNA was expected to give non-c" @default.
• W1982188814 created "2016-06-24" @default.
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• W1982188814 title "Recombinant Human DNA (Cytosine-5) Methyltransferase" @default.
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