Preferential Methylation of Unmethylated DNA by Mammalian de Novo DNA Methyltransferase Dnmt3a
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m106590200
ISSN1083-351X
AutoresTomoki Yokochi, Keith D. Robertson,
Tópico(s)RNA modifications and cancer
ResumoDNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-l-methionine. DNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-l-methionine. DNA methylation in mammals occurs predominantly at cytosine within the CpG dinucleotide. Numerous studies have revealed that DNA methylation is critical for proper embryonic development (1.Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3235) Google Scholar, 2.Okano M. Bell D.W. Haber D.A. Li W. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4533) Google Scholar), genome stability (3.Chen R.Z. Pettersson U. Beard C. Jackson-Grusby L. Jaenisch R. Nature. 1998; 395: 89-93Crossref PubMed Scopus (794) Google Scholar), X chromosome inactivation (4.Beard C. Li E. Jaenisch R. Genes Dev. 1995; 9: 2325-2334Crossref PubMed Scopus (247) Google Scholar), genomic imprinting (5.Reik W. Walter J. Nat. Rev. Genet. 2001; 2: 21-32Crossref PubMed Scopus (1832) Google Scholar), and the suppression of the detrimental effects of parasitic elements (6.Yoder J.A. Walsh C.P. Bestor T.H. Trends Genet. 1997; 13: 335-340Abstract Full Text PDF PubMed Scopus (1520) Google Scholar). Certain genomic regions, like CpG islands, are usually hypomethylated regardless of whether the associated gene is transcriptionally silent or active (7.Bird A. Nature. 1986; 321: 209-213Crossref PubMed Scopus (3022) Google Scholar). Other genomic regions, such as centromeres and parasitic DNA sequences, are hypermethylated in normal cells (6.Yoder J.A. Walsh C.P. Bestor T.H. Trends Genet. 1997; 13: 335-340Abstract Full Text PDF PubMed Scopus (1520) Google Scholar). During the process of tumor formation, these methylation patterns are frequently disrupted, resulting in improper gene silencing by hypermethylation of promoter regions and genomic instability by global hypomethylation (8.Baylin S.B. Herman J.G. Trends Genet. 2000; 16: 168-174Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar, 9.Jones P.A. Laird P.W. Nat. Genet. 1999; 21: 163-166Crossref PubMed Scopus (2058) Google Scholar). It remains unclear how cellular DNA methylation patterns are established during development and properly maintained in somatic cells. One mechanism may be through the use of several differentially regulated, independently encoded, DNA methyltransferases (DNMTs) 1The abbreviations used are: DNMTsDNA methyltransferasesDNMT1DNA (cytosine-5) methyltransferase 1Dnmt3aDNA methyltransferase 3aAdoMetS-adenosyl-l-methionineAdoHcyS-adenosyl-l-homocysteinedsDNAdouble-stranded DNAssDNAsingle-stranded DNA (10.Robertson K.D. Wolffe A.P. Nat. Rev. Genet. 2000; 1: 11-19Crossref PubMed Scopus (880) Google Scholar). To date, four mammalian DNMTs have been cloned and characterized. Of these four, three have been found to be essential for proper embryonic development in murine knockout models (Dnmt1, -3a, and -3b) (1.Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3235) Google Scholar, 2.Okano M. Bell D.W. Haber D.A. Li W. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4533) Google Scholar). DNMT1 is the most abundant DNA methyltransferase in somatic cells (11.Robertson K.D. Uzvolgyi E. Liang G. Talmadge C. Sumegi J. Gonzales F.A. Jones P.A. Nucleic Acids Res. 1999; 27: 2291-2298Crossref PubMed Scopus (718) Google Scholar) and is targeted to replication foci during S phase (12.Leonhardt H. Page A.W. Weier H. Bestor T.H. Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (829) Google Scholar, 13.Liu Y. Oakeley E.J. Sun L. Jost J.-P. Nucleic Acids Res. 1998; 26: 1038-1045Crossref PubMed Scopus (115) Google Scholar). DNMT1 has been shown to have a 7–21-fold preference for hemimethylated DNA and is therefore believed to be the primary enzyme responsible for copying methylation patterns from the parental to the daughter strand following DNA replication (14.Pradhan S. Bacolla A. Wells R.D. Roberts R.J. J. Biol. Chem. 1999; 274: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). The DNMT3 family of enzymes appears to be responsible for the de novo establishment of DNA methylation patterns during embryonic development. These enzymes are more highly expressed during early developmental stages where de novo methylation is believed to occur. Limited substrate preference studies revealed that these enzymes have an equal affinity for unmethylated and hemimethylated DNA. The sequence specificity of the mammalian DNMTs is less restricted than most bacterial methylases, requiring only a CpG dinucleotide (2.Okano M. Bell D.W. Haber D.A. Li W. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4533) Google Scholar, 15.Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1286) Google Scholar). DNA methyltransferases DNA (cytosine-5) methyltransferase 1 DNA methyltransferase 3a S-adenosyl-l-methionine S-adenosyl-l-homocysteine double-stranded DNA single-stranded DNA Structurally, mammalian DNMTs can be divided into an N-terminal regulatory domain and a C-terminal catalytic domain. The N-terminal domains of the DNMT3 family enzymes and DNMT1 are unrelated to each other and are completely lacking in bacterial cytosine methylases (16.Kumar S. Cheng X. Klimasauskas S. Mi S. Posfai J. Roberts R.J. Wilson G.G. Nucleic Acids Res. 1994; 22: 1-10Crossref PubMed Scopus (393) Google Scholar). The N-terminal region of the Dnmt3 family contains a domain with similarity to the plant homeodomain, common to many chromatin-associated proteins, which may mediate protein-protein interactions (15.Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1286) Google Scholar, 17.Aasland R. Gibson T.J. Stewart A.F. Trends Biochem. Sci. 1995; 20: 56-59Abstract Full Text PDF PubMed Scopus (755) Google Scholar). The N-terminal domain of DNMT1 participates in the interaction with histone deacetylases (18.Fuks F. Bergers W.A. Brehm A. Hughes-Davies L. Kouzarides T. Nat. Genet. 2000; 24: 88-91Crossref PubMed Scopus (820) Google Scholar, 19.Robertson K.D. Ait-Si-Ali S. Yokochi T. Wade P.A. Jones P.L. Wolffe A.P. Nat. Genet. 2000; 25: 338-342Crossref PubMed Scopus (807) Google Scholar, 20.Rountree M.R. Bachman K.E. Baylin S.B. Nat. Genet. 2000; 25: 269-277Crossref PubMed Scopus (864) Google Scholar), pRb (19.Robertson K.D. Ait-Si-Ali S. Yokochi T. Wade P.A. Jones P.L. Wolffe A.P. Nat. Genet. 2000; 25: 338-342Crossref PubMed Scopus (807) Google Scholar), co-repressors (20.Rountree M.R. Bachman K.E. Baylin S.B. Nat. Genet. 2000; 25: 269-277Crossref PubMed Scopus (864) Google Scholar), and proliferating cell nuclear antigen (21.Chuang L.S.-H. Ian H.-I. Koh T.-W. Ng H.-H. Xu G. Li B.F.L. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (787) Google Scholar). An allosteric DNA-binding site in this domain was shown to influence enzymatic activity of the C-terminal catalytic domain (22.Bacolla A. Pradhan S. Larson J.E. Roberts R.J. Wells R.D. J. Biol. Chem. 2001; 276: 18605-18613Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) that is highly conserved in all cytosine methyltransferases from bacteria to mammals. Structural studies with bacterial cytosine methylases have shown that the cytosine base is extruded from the DNA double helix into the active site of the enzyme where it can be reacted upon without steric hindrance (23.Klimasauskas S. Kumar S. Roberts R.J. Cheng X. Cell. 1994; 76: 357-369Abstract Full Text PDF PubMed Scopus (923) Google Scholar). The active site cysteine attacks the 6-position of the pyrimidine ring and then activates the 5-carbon for nucleophilic attack of the methyl group donor S-adenosyl-l-methionine (AdoMet) (24.Smith S.S. Kaplan B.E. Sowers L.C. Newman E.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4744-4748Crossref PubMed Scopus (125) Google Scholar). Unlike the maintenance methyltransferase DNMT1, mammalian Dnmt3a has been poorly characterized biochemically, because the enzymatic activity of Dnmt3a is considerably lower than that of DNMT1 (15.Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1286) Google Scholar, 25.Lyko F. Ramsahoye B.H. Kashevsky H. Tudor M. Mastrangelo M.A. Orr-Weaver T.L. Jaenisch R. Nat. Genet. 1999; 23: 363-366Crossref PubMed Scopus (163) Google Scholar, 26.Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (191) Google Scholar). To understand how cellular DNA methylation patterns are regulated enzymatically, we have cloned and purified both enzymes using a baculovirus expression system. Also, we have established a novel and highly reproducible methyltransferase assay system utilizing magnetic beads to determine the precise enzymatic activity of Dnmt3a. Here we report the substrate specificity and molecular mechanism for the methylation reaction of Dnmt3a compared with DNMT1. Most importantly, our results show that Dnmt3a prefers unmethylated DNA more than 3-fold over hemimethylated DNA. Initial velocity analysis, product inhibition, and substrate inhibition studies indicated that the methylation reaction mediated by Dnmt3a followed an Ordered Bi Bi reaction mechanism, with DNA as the first substrate to bind and methylated DNA as the last product to be released. Radioactive materials ([3H]AdoMet, [32P]dATP, and [32P]dCTP) were purchased from Amersham Bioscience. Streptoavidin-coated magnetic beads were purchased from Dynal. General chemicals were purchased from Sigma, Invitrogen, and Roche Molecular Biochemicals. DNA oligonucleotides were synthesized by MWG Biotech, Sigma Genosys, and New England Biolabs. Sequences of oligonucleotides are listed in Table I. Equal amounts of complementary single-stranded oligonucleotides were mixed, heated at 95 °C for 5 min, and annealed by slowly cooling to room temperature to make double-stranded oligonucleotides. Annealing was confirmed by electrophoresis of samples on a 20% nondenaturing PAGE gel. The DNMT1 anti-peptide antibody has been previously described (19.Robertson K.D. Ait-Si-Ali S. Yokochi T. Wade P.A. Jones P.L. Wolffe A.P. Nat. Genet. 2000; 25: 338-342Crossref PubMed Scopus (807) Google Scholar). The DNMT3a antibody was raised against the synthetic peptide DLLPNGDLEKRSEPQ(C) injected into rabbits and affinity purified against the same peptide (Affinity BioReagents).Table ISynthetic DNA oligonucleotides used in this studyName appearing in text and figuresSequenceaM: 5-methyl-2′-deoxycytosine; I: deoxyinosine.Unmethylated dsDNA5′-Biotin-GATCCGACGACGACGCGCGCGCGACGACGAGATCCTAGGCTGCTGCTGCGCGCGCGCTGCTGCTCTAGHemimethylated-1 dsDNA5′-Biotin-GATCCGACGACGACGCGCGCGCGACGACGAGATCCTAGGMTGMTGMTGMGMGMGMGMTGMTGMTCTAGHemimethylated-2 dsDNA5′-Biotin-GATCMGAMGAMGAMGMGMGMGMGAMGAMGAGATCCTAGGCTGCTGCTGCGCGCGCGCTGCTGCTCTAGFully methylated dsDNA5′-Biotin-GATCMGAMGAMGAMGMGMGMGMGAMGAMGAGATCCTAGGMTGMTGMTGMGMGMGMGMTGMTGMTCTAGUnmethylated ssDNA5′-Biotin-GATCCGACGACGACGCGCGCGCGACGACGAGATCMethylated ssDNA5′-Biotin-GATCMGAMGAMGAMGMGMGMGMGAMGAMGAGATC(CGG/CCG)75′-Biotin-GATCCGGGCGGACGGTCGGTCGGACGGGCGGGATCCTAGGCCCGCCTGCCAGCCAGCCTGCCCGCCCTAGGGGG(CAG/CTG)75′-Biotin-GATCCAGGCAGACAGTCAGTCAGACAGGCAGGATCCTAGGTCCGTCTGTCAGTCAGTCTGTCCGTCCTAGGGGGAT/TA5′-Biotin-AAATATATATATATAATTAATATATATATATAAATTTATATATATATATTAATTATATATATATATTTTTTTSynthetic d(I-C)5′-Biotin-GATCICICICICICICICICICICGATCCTAGCICICICICICICICICICICTAG-biotina M: 5-methyl-2′-deoxycytosine; I: deoxyinosine. Open table in a new tab Recombinant His-tagged DNMT1 and Dnmt3a were produced in Sf9 cells using the Bac-to-Bac Baculovirus expression system (Invitrogen). The full-length human DNMT1 cDNA was cloned into pFastBacHTa as an EcoRI-SalI fragment from plasmid pKR30–1. The Dnmt3a cDNA was created by PCR using primers (sense) 5′-GATCTACTAGTATGCCCTCCAGCGGCCCCGG and (antisense) 5′-TCTAGACTAGTTTACACACAAGCAAAATATTCC, full-length murine Dnmt3a cDNA as template, and Pfu polymerase (Stratagene). The PCR product was cloned into the SpeI site of pFastBacHTc and clones were screened for the proper insert orientation. The cloned genes were sequenced entirely to ensure the absence of mutations. Recombinant baculovirus was made according to the manufacturer's instructions and clones were amplified twice to achieve a high titer stock (∼109 pfu/ml). Sf9 cells grown in Sf900II medium containing 5% fetal calf serum (Invitrogen) were infected with baculovirus stocks. The multiplicity of infection was varied between 1 and 10 and times of infection were varied between 24 and 96 h. For both DNMT1 and Dnmt3a, a multiplicity of infection of 5 and an infection time of 72 h were determined to be optimal. Infected Sf9 cells were pelleted and washed once with 1 × phosphate-buffered saline. The cell pellet was sonicated in RIPA buffer (1 × phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 0.5 mmphenylmethylsulfonyl fluoride, 5 μg/ml pepstatin A, 5 μg/ml antipanin, 5 μg/ml chymostatin, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 μg/ml E-64) and centrifuged at 3000 rpm for 20 min. The supernatants were saved as whole cell extracts. Nickel-agarose resin (Novagen) was charged with 50 mm NiSO4 for 3 h and washed with distilled water and then binding buffer (20 mm phosphate, pH 7.6, 10% glycerol, 500 mmNaCl, 5 mm imidazole). Whole cell extract (250 μl) was mixed with Ni-agarose resin (200 μl in bed volume) and rotated gently for 30 min at 4 °C. Nickel-agarose resin was washed three times with 400 μl of washing buffer-1 (20 mm phosphate, pH 7.6, 10% glycerol, 500 mm NaCl, 40 mm imidazole) and then two times with 400 μl of washing buffer-2 (20 mmphosphate, pH 7.6, 10% glycerol, 10 mm NaCl). His-tagged protein was eluted with 280 μl of elution buffer (20 mmphosphate, pH 7.6, 10% glycerol, 10 mm NaCl, 1m imidazole) and snap-frozen in liquid nitrogen. Protein solutions were stored at −70 °C. Enzyme concentration was determined with the Protein Assay Kit (Bio-Rad). A 96-well microtiter plate was pretreated with a 10 mg/ml bovine serum albumin solution for 30 min just prior to use. Magnetic beads (10 mg/ml, Dynal) were washed with TENT2M (20 mm Tris, pH 8.0, 2 mm EDTA, 0.01% Triton X-100, 2 m NaCl) three times. To prepare magnetic beads suspension, the final concentration of magnetic beads was adjusted to 2.5 mg/ml with TENT2M and 1/10 volume of 100 mg/ml nonradioactive AdoMet (Sigma) was added. A typical methylation reaction (40 μl) contained enzyme (30 nm for DNMT1 or 300 nm for Dnmt3a), 125 nm DNA oligonucleotides, and 900 nm tritium-labeled AdoMet (Amersham Bioscience, 1 mCi/ml) in reaction buffer (50 mm Tris, pH 8.0, 5 mm EDTA, 10% glycerol, 10 mm2-mercaptoethanol, 0.5 mm phenylmethylsulfonyl fluoride). After incubation at 37 °C for 30 min, reactions were quenched with an equal volume of the magnetic beads suspension and gently shaken on a rocking shaker for 15 min at room temperature. Magnetic beads were separated on a magnet and washed with TENT1M (10 mm Tris, pH 8.0, 1 mm EDTA, 0.005% Triton X-100, 1 mNaCl) as follows: 300, 200, 150, and 100 μl. Magnetic beads were suspended in 25 μl of TENT1M and tritium incorporation was measured by scintillation counting. A detailed protocol will be published elsewhere. All experiments were performed in duplicate or triplicate independently. For a substrate (DNA) inhibition experiment (Fig. 5C, left panel), the concentration of magnetic beads was increased 4-fold (10 mg/ml) to ensure a steady recovery of large amounts of DNA substrate. The reaction mixture (20 μl) contains 50 mm Hepes (pH 7.9), 5 mm EDTA, 10% glycerol, 100 nm DNMTs, and dsDNA (2 × 105counts/min). 10 mm AdoMet, 10 mm AdoHcy, or 1.2 μg of poly d(I-C) (Roche Molecular Biochemicals) was added to selected reactions. After incubation at room temperature for 30 min, an equal volume of a loading buffer (0.5 × TBE, 10% glycerol, 0.02% bromphenol blue) was added and protein-DNA complexes were resolved on a 6% nondenaturing PAGE gel run in 0.5 × TBE. The gel was dried and visualized by autoradiography. The murine de novo methyltransferase Dnmt3a and the human maintenance methyltransferase DNMT1 were cloned, expressed, and purified to directly compare their substrate specificities and kinetic behaviors in vitro. Since we observed that the expression of DNMT1 in bacteria is highly inefficient, both enzymes were expressed in Sf9 insect cells using a baculovirus expression system to ensure a similar condition for protein production. Recombinant enzymes were purified via nickel-agarose resin by virtue of a His6 affinity tag at their N termini. This method yielded recombinant proteins that were nearly homogenous as determined by SDS-PAGE followed by Coomassie Blue staining (Fig. 1, left panel). Further confirmation of the identity of the purified recombinant proteins was carried out by Western blotting with antibodies specific for each DNMT (right panel). The low level of methyltransferase activity for Dnmt3a reported by others (15.Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1286) Google Scholar, 25.Lyko F. Ramsahoye B.H. Kashevsky H. Tudor M. Mastrangelo M.A. Orr-Weaver T.L. Jaenisch R. Nat. Genet. 1999; 23: 363-366Crossref PubMed Scopus (163) Google Scholar, 26.Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (191) Google Scholar), coupled with our data, necessitated the development of a reproducible assay with a higher signal-to-noise ratio than currently available methods. A flow chart of the DNMT magnetic beads assay is shown in Fig. 2A, top panel. Briefly, 5′-terminal biotinylated DNA oligonucleotides are incubated with DNA methyltransferase in the presence of AdoMet that has a tritium-labeled methyl group. After the reaction, biotinylated DNA is immobilized onto streptavidin-coated magnetic beads. DNA containing tritium can be easily separated from the unreacted radioactive AdoMet using a magnet and washed with the buffer containing 1 mNaCl and a detergent. Tritium incorporation is then counted using a liquid scintillation system. Synthetic ssDNA and dsDNA oligonucleotides utilized in this report are listed in Table I. Mammalian maintenance DNA methyltransferase DNMT1 has been extensively studied biochemically and enzymatically (14.Pradhan S. Bacolla A. Wells R.D. Roberts R.J. J. Biol. Chem. 1999; 274: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 27.Flynn J. Glickman J.F. Reich N.O. Biochemistry. 1996; 35: 7308-7315Crossref PubMed Scopus (72) Google Scholar, 28.Glickman J.F. Flynn J. Reich N.O. Biochem. Biophys. Res. Commun. 1997; 230: 280-284Crossref PubMed Scopus (25) Google Scholar, 29.Pradhan 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 (88) Google Scholar, 30.Flynn J. Reich N. Biochemistry. 1998; 37: 15162-15169Crossref PubMed Scopus (53) Google Scholar, 31.Bacolla A. Pradhan S. Roberts R.J. Wells R.D. J. Biol. Chem. 1999; 274: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). DNMT1 prefers hemimethylated dsDNA and its kinetic constants have been reported (14.Pradhan S. Bacolla A. Wells R.D. Roberts R.J. J. Biol. Chem. 1999; 274: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar,28.Glickman J.F. Flynn J. Reich N.O. Biochem. Biophys. Res. Commun. 1997; 230: 280-284Crossref PubMed Scopus (25) Google Scholar, 29.Pradhan 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 (88) Google Scholar). Therefore, we employed recombinant human DNMT1 as a reference enzyme to evaluate our new DNMT assay system. To demonstrate the efficiency and reproducibility, the substrate specificity of DNMT1 (Fig. 2A, bottom panel) and kinetics analysis (Fig. 2, B–E) were examined. DNMT1 showed a 20-fold preference for hemimethylated dsDNA substrates as compared with unmethylated dsDNA and ssDNA, whereas no methylation was detected in fully methylated DNA controls (Fig. 2A, bottom panel, columns 1–6). These results are consistent with previous work (14.Pradhan S. Bacolla A. Wells R.D. Roberts R.J. J. Biol. Chem. 1999; 274: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 28.Glickman J.F. Flynn J. Reich N.O. Biochem. Biophys. Res. Commun. 1997; 230: 280-284Crossref PubMed Scopus (25) Google Scholar, 29.Pradhan 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 (88) Google Scholar). Since poly d(I-C) has been used as the standard DNA substrate for traditional DNMT assays (14.Pradhan S. Bacolla A. Wells R.D. Roberts R.J. J. Biol. Chem. 1999; 274: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar), a synthetic d(I-C) oligonucleotide was also tested. It was, however, shown that a synthetic d(I-C) substrate was less efficiently methylated (Fig. 2A, bottom panel, column 7). Kinetic analysis of DNMT1 was pursued to examine the accuracy of this assay system. The linearity of time course and the dose dependence of the methylation reaction by DNMT1 were investigated first. The reaction was found to be linear up to 140 nm DNMT1 and for the first 60 min (Fig. 2, B and C). Initial enzymatic velocities were then measured by varying one substrate concentration while holding the other substrate concentration constant. The data are displayed in double-reciprocal plots (Fig. 2, D and E). The kinetic constants obtained from secondary plots (not shown) are summarized in Table II. These results showed that the DNMT magnetic beads assay is very reproducible and confers a higher signal-to-noise ratio. Therefore, it is applicable to investigate the enzymatic properties of any DNA methyltransferase that may have relatively weak activity.Table IIComparison of steady-state kinetic parameters for DNMT1 and Dnmt3aDNA methyltransferasedsDNA substratekcatKmAdoMetKmCGkcat/KmCGh−1μm106× m−1 h−1DNMT1Unmethylated0.150.630.360.42±0.01±0.14±0.03±0.03Hemimethylated1.770.430.08919.9±0.23±0.07±0.010±3.4 Dnmt3aUnmethylated0.0260.520.250.10±0.001±0.10±0.03±0.01Hemimethylated0.00720.600.170.042±0.0002±0.02±0.01±0.001 Open table in a new tab Using the DNMT magnetic beads assay, the substrate specificity of Dnmt3a was examined in vitro. As expected, Dnmt3a efficiently incorporated tritium into unmethylated dsDNA (Fig. 3A, column 1). Interestingly, the enzymatic activity of Dnmt3a with hemimethylated DNA was over 3-fold lower than unmethylated dsDNA under the same conditions (columns 2 and 3). The methylation efficiency with a hemimethylated dsDNA substrate was even lower than ssDNA substrates (column 5). These results strongly suggest that Dnmt3a can transfer a methyl group preferentially to unmethylated dsDNA in the presence of AdoMet. In the absence of DNA substrates, less than 100 cpm were detected as the background, which is comparable with the signal detected using fully methylated DNA substrate controls (columns 4, 6, and 8). Synthetic d(I-C) was also tested as a substitute for poly d(I-C), however, no significant stimulation was observed (column 7). In this experiment, saturating conditions for DNA substrates were essential to minimize the difference in CpG concentrations between different substrates. Since the number of CpG dinucleotides in unmethylated dsDNA is twice that of hemimethylated dsDNA or unmethylated ssDNA substrates, it was possible that the difference in the activity between unmethylated and hemimethylated dsDNA by Dnmt3a was a reflection of these different CpG concentrations. To exclude this possibility, we carried out reactions containing twice the concentration of hemimethylated dsDNA and unmethylated ssDNA substrates (columns 9–11, compare with columns 2, 3, and 5). We found little difference in the methylation efficiency when 125 or 250 nm DNA (corresponding to 1.25 or 2.5 μm CpG dinucleotides) was used, indicating that the DNA concentrations were close to saturating. Since the methylation activity was shown to be independent of DNA dosage in these reaction conditions, preferential methylation of unmethylated dsDNA was indeed caused by the substrate specificity of Dnmt3a, rather than resulting from a dose dependence artifact. To further confirm this property, we examined internal substrate competition assays under nonsaturating conditions (Fig. 3B). The methylation reactions were carried out in the presence of both unmethylated and hemimethylated dsDNA substrates, in which one DNA substrate concentration is varied while the other is held constant. Since only the variable substrate is conjugated to biotin, this assay measures the competitive methylation efficiency of the biotinylated dsDNA in the presence of a fixed amount of another substrate. Increasing amounts of unmethylated dsDNA (with biotin) versus a fixed amount of hemimethylated dsDNA (without biotin) gave higher methylation activities than increasing amounts of hemimethylated dsDNA (with biotin) versus a fixed amount of unmethylated dsDNA (without biotin), suggesting that Dnmt3a has a small but significant preference for unmethylated dsDNA even in the presence of hemimethylated dsDNA. The methylation reaction by Dnmt3a involves two substrates, AdoMet and DNA. To evaluate the enzymatic properties of Dnmt3a quantitatively, bi-substrate steady-state kinetic analysis was performed using the DNMT magnetic beads assay. The rate of methylation catalyzed by Dnmt3a was linear at protein concentrations between 30 nm and 1.5 μm and for the first 90 min (data not shown). Initial velocity experiments were carried out at varying or fixed concentrations of AdoMet and either of two DNA substrates, unmethylated (Fig. 4A) or hemimethylated dsDNA (Fig. 4B). The kinetic parameters such as kcat, Km for AdoMet, and Km for CpG were estimated from these double-reciprocal plots of initial velocity versus substrate concentration, followed by secondary plots with their slopes and intercepts (not shown). Values for all kinetic constants for the two different DNA substrates and AdoMet are listed in Table II. The purpose of kinetic analysis are not only to represent the catalytic activity of an enzyme as numbers such as kcat and Km, but also to elucidate the molecular mechanism of the enzymatic reaction (32.Segel I.H. Enzyme Kinetics. John Wiley & Sons, Inc., New York1993Google Scholar). Kinetic patterns of bi-substrate analysis provide a good clue to distinguish between a ternary complex mechanism and a ping-pong mechanism. The double-reciprocal plots of the initial velocities of Dnmt3a obtained at various AdoMet and fixed CpG concentrations yielded an intersecting pattern (Fig. 4, A and B, top panels). The same pattern was obtained when various concentrations of CpG dinucleotide and fixed concentrations of AdoMet were used (Fig. 4, A and B, bottom panels). Together, these results suggest that Dnmt3a proceeds by a random or compulsory ordered Bi Bi type rather than a ping-pong type reaction mechanism. Unlike bacterial cytosine DNA methylases such as M. HhaI (33.Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar) and M. MspI (34.Bhattacharya S.K. Dubey A.K. J. Biol. Chem. 1999; 274: 14743-14749Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), the binding of substrates to mammalian Dnmt3a cannot be described by a rapid equilibrium-ordered mechanism during turnover because the intersection points do not lie on the 1/v axis. Although the data clearly indicate the requirement for a ternary complex, the order of substrate addition and product release cannot be elucidated from these
Referência(s)