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Mechanism of Stimulation of Catalytic Activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-methyltransferases by Dnmt3L

2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês

10.1074/jbc.m413412200

ISSN

1083-351X

Autores

Humaira Gowher, Kirsten Liebert, Andrea Hermann, Guoliang Xu, Albert Jeltsch,

Tópico(s)

RNA modifications and cancer

Resumo

Dnmt3L has been identified as a stimulator of the catalytic activity of de novo DNA methyltransferases. It is essential in the development of germ cells in mammals. We show here that Dnmt3L stimulates the catalytic activity of the Dnmt3A and Dnmt3B enzymes by directly binding to their respective catalytic domains via its own C-terminal domain. The catalytic activity of Dnmt3A and -3B was stimulated ∼15-fold, and Dnmt3L directly binds to DNA but not to S-adenosyl-l-methionine (AdoMet). Complex formation between Dnmt3A and Dnmt3L accelerates DNA binding by Dnmt3A 20-fold and lowers its Km for DNA. Interaction of Dnmt3L with Dnmt3A increases the binding of the coenzyme AdoMet to Dnmt3A, and it lowers the Km of Dnmt3A for AdoMet. On the basis of our data we propose a model in which the interaction of Dnmt3A with Dnmt3L induces a conformational change of Dnmt3A that opens the active site of the enzyme and promotes binding of DNA and the AdoMet. We demonstrate that the interaction of Dnmt3A and Dnmt3L is transient, and after DNA binding to Dnmt3A, Dnmt3L dissociates from the complex. Following dissociation of Dnmt3L, Dnmt3A adopts a closed conformation leading to slow rates of DNA release. Therefore, Dnmt3L acts as a substrate exchange factor that accelerates DNA and AdoMet binding to de novo DNA methyltransferases. Dnmt3L has been identified as a stimulator of the catalytic activity of de novo DNA methyltransferases. It is essential in the development of germ cells in mammals. We show here that Dnmt3L stimulates the catalytic activity of the Dnmt3A and Dnmt3B enzymes by directly binding to their respective catalytic domains via its own C-terminal domain. The catalytic activity of Dnmt3A and -3B was stimulated ∼15-fold, and Dnmt3L directly binds to DNA but not to S-adenosyl-l-methionine (AdoMet). Complex formation between Dnmt3A and Dnmt3L accelerates DNA binding by Dnmt3A 20-fold and lowers its Km for DNA. Interaction of Dnmt3L with Dnmt3A increases the binding of the coenzyme AdoMet to Dnmt3A, and it lowers the Km of Dnmt3A for AdoMet. On the basis of our data we propose a model in which the interaction of Dnmt3A with Dnmt3L induces a conformational change of Dnmt3A that opens the active site of the enzyme and promotes binding of DNA and the AdoMet. We demonstrate that the interaction of Dnmt3A and Dnmt3L is transient, and after DNA binding to Dnmt3A, Dnmt3L dissociates from the complex. Following dissociation of Dnmt3L, Dnmt3A adopts a closed conformation leading to slow rates of DNA release. Therefore, Dnmt3L acts as a substrate exchange factor that accelerates DNA and AdoMet binding to de novo DNA methyltransferases. In vertebrates DNA is methylated at the 5-position of cytosine residues within CG dinucleotides, which are methylated to 70–80% in a tissue- and cell-specific pattern (1.Hermann A. Gowher H. Jeltsch A. Cell Mol. Life Sci. 2004; 61: 2571-2587Crossref PubMed Scopus (424) Google Scholar, 2.Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5332) Google Scholar, 3.Jones P.A. Takai D. Science. 2001; 293: 1068-1070Crossref PubMed Scopus (1514) Google Scholar). Methylation is involved in epigenetic regulation of gene expression, X-chromosome inactivation, genomic imprinting, and development (4.Li E. Nat. Rev. Genet. 2002; 3: 662-673Crossref PubMed Scopus (1549) Google Scholar, 5.Jaenisch R. Bird A. Nat. Genet. 2003; 33: 245-254Crossref PubMed Scopus (4584) Google Scholar); aberrant methylation contributes to aging and cancer (6.Jones P.A. Oncogene. 2002; 21: 5358-5360Crossref PubMed Scopus (247) Google Scholar, 7.Feinberg A.P. Tycko B. Nat. Rev. Cancer. 2004; 4: 143-153Crossref PubMed Scopus (1775) Google Scholar). The methyl group of the coenzyme S-adenosyl-l-methionine (AdoMet) 1The abbreviations used are: AdoMet, S-adenosyl-l-methionine; MTase, methyltransferase; SPR, surface plasmon resonance. 1The abbreviations used are: AdoMet, S-adenosyl-l-methionine; MTase, methyltransferase; SPR, surface plasmon resonance. is transferred to cytosine residues of the DNA by DNA methyltransferases (MTases) three families of which have been identified to date in mammals: Dnmt1, Dnmt2, and Dnmt3 (8.Jeltsch A. Chembiochem. 2002; 3: 274-293Crossref PubMed Google Scholar). All these enzymes contain a domain of ∼400–500 amino acid residues, which is characterized by the presence of 10 conserved amino acid motifs, shared between prokaryotic and eukaryotic enzymes of DNA-(cytosine-C5)-methyltransferases (8.Jeltsch A. Chembiochem. 2002; 3: 274-293Crossref PubMed Google Scholar, 9.Cheng X. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 293-318Crossref PubMed Scopus (283) Google Scholar). In addition, the Dnmt1 and the Dnmt3 enzymes harbor large N-terminal regulatory parts (8.Jeltsch A. Chembiochem. 2002; 3: 274-293Crossref PubMed Google Scholar, 10.Chen T. Li E. Curr. Top. Dev. Biol. 2004; 60: 55-89Crossref PubMed Scopus (254) Google Scholar). Dnmt1 has a strong preference for hemimethylated CG sites implicating a function in maintenance of the methylation pattern of the DNA after replication (11.Gruenbaum Y. Cedar H. Razin A. Nature. 1982; 295: 620-622Crossref PubMed Scopus (304) Google Scholar, 12.Bestor T.H. EMBO J. 1992; 11: 2611-2617Crossref PubMed Scopus (386) Google Scholar, 13.Pradhan 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, 14.Fatemi M. Hermann A. Pradhan S. Jeltsch A. J. Mol. Biol. 2001; 309: 1189-1199Crossref PubMed Scopus (197) Google Scholar, 15.Hermann A. Goyal R. Jeltsch A. J. Biol. Chem. 2004; 279: 48350-48359Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). Dnmt2 is the smallest enzyme among the eukaryotic methyltransferases comprising only the catalytic domain. It has been shown to be an active methyltransferase, with a very slow turnover (16.Hermann A. Schmitt S. Jeltsch A. J. Biol. Chem. 2003; 278: 31717-31721Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 17.Tang L.Y. Reddy M.N. Rasheva V. Lee T.L. Lin M.J. Hung M.S. Shen C.K. J. Biol. Chem. 2003; 278: 33613-33616Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 18.Kunert N. Marhold J. Stanke J. Stach D. Lyko F. Development. 2003; 130: 5083-5090Crossref PubMed Scopus (180) Google Scholar, 19.Liu K. Wang Y.F. Cantemir C. Muller M.T. Mol. Cell. Biol. 2003; 23: 2709-2719Crossref PubMed Scopus (126) Google Scholar).The Dnmt3 family consists of three different proteins, Dnmt3A, Dnmt3B, and Dnmt3L (see Fig. 1) (20.Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1260) Google Scholar, 21.Aapola U. Kawasaki K. Scott H.S. Ollila J. Vihinen M. Heino M. Shintani A. Minoshima S. Krohn K. Antonarakis S.E. Shimizu N. Kudoh J. Peterson P. Genomics. 2000; 65: 293-298Crossref PubMed Scopus (208) Google Scholar). Dnmt3A and -3B have a regulatory N-terminal domain that in contrast to Dnmt1 is not essential for catalysis (22.Gowher H. Jeltsch A. J. Biol. Chem. 2002; 277: 20409-20414Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 23.Reither S. Li F. Gowher H. Jeltsch A. J. Mol. Biol. 2003; 329: 675-684Crossref PubMed Scopus (60) Google Scholar) but is involved in enzyme targeting (24.Bachman K.E. Rountree M.R. Baylin S.B. J. Biol. Chem. 2001; 276: 32282-32287Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 25.Fuks F. Hurd P.J. Deplus R. Kouzarides T. Nucleic Acids Res. 2003; 31: 2305-2312Crossref PubMed Scopus (587) Google Scholar, 26.Ge Y.Z. Pu M.T. Gowher H. Wu H.P. Ding J.P. Jeltsch A. Xu G.L. J. Biol. Chem. 2004; 279: 31277-31286Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 27.Chen T. Tsujimoto N. Li E. Mol. Cell. Biol. 2004; 24: 9048-9058Crossref PubMed Scopus (197) Google Scholar). The N-terminal parts of Dnmt3A and -3B contain an ATRX-like Cys-rich domain (also called PHD domain) and a PWWP domain, which are involved in interactions of the enzymes with other proteins and in targeting to heterochromatin (24.Bachman K.E. Rountree M.R. Baylin S.B. J. Biol. Chem. 2001; 276: 32282-32287Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 25.Fuks F. Hurd P.J. Deplus R. Kouzarides T. Nucleic Acids Res. 2003; 31: 2305-2312Crossref PubMed Scopus (587) Google Scholar, 26.Ge Y.Z. Pu M.T. Gowher H. Wu H.P. Ding J.P. Jeltsch A. Xu G.L. J. Biol. Chem. 2004; 279: 31277-31286Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 27.Chen T. Tsujimoto N. Li E. Mol. Cell. Biol. 2004; 24: 9048-9058Crossref PubMed Scopus (197) Google Scholar, 28.Aapola U. Liiv I. Peterson P. Nucleic Acids Res. 2002; 30: 3602-3608Crossref PubMed Scopus (74) Google Scholar). Dnmt3A and -3B also methylate cytosines in a non-CG context, however the biological function of this activity is not known (29.Aoki A. Suetake I. Miyagawa J. Fujio T. Chijiwa T. Sasaki H. Tajima S. Nucleic Acids Res. 2001; 29: 3506-3512Crossref PubMed Scopus (144) Google Scholar, 30.Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (184) Google Scholar, 31.Hsieh C.L. Mol. Cell. Biol. 1999; 19: 8211-8218Crossref PubMed Scopus (217) Google Scholar, 32.Ramsahoye B.H. Biniszkiewicz D. Lyko F. Clark V. Bird A.P. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5237-5242Crossref PubMed Scopus (729) Google Scholar). Both enzymes do not distinguish between unmethylated and hemimethylated substrates and are involved in de novo methylation in vivo (20.Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1260) Google Scholar, 30.Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (184) Google Scholar, 33.Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4413) Google Scholar). Despite these biochemical similarities the Dnmt3A and -3B enzymes have distinct biological roles. Dnmt3B is responsible for methylation of pericentromeric satellite regions (33.Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4413) Google Scholar, 34.Hansen R.S. Wijmenga C. Luo P. Stanek A.M. Canfield T.K. Weemaes C.M. Gartler S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14412-14417Crossref PubMed Scopus (593) Google Scholar, 35.Xu G.L. Bestor T.H. Bourc'his D. Hsieh C.L. Tommerup N. Bugge M. Hulten M. Qu X. Russo J.J. Viegas-Pequignot E. Nature. 1999; 402: 187-191Crossref PubMed Scopus (527) Google Scholar). Dnmt3B–/– knock-out mice die during late embryonic stage and the embryos lack methylation in pericentromeric repeat region (33.Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4413) Google Scholar). Loss of Dnmt3B activity in human leads to the ICF syndrome, a genetic disorder that is accompanied by low methylation in pericentromeric satellite region of chromosomes 1, 9, and 16 (36.Ehrlich M. Clin. Immunol. 2003; 109: 17-28Crossref PubMed Scopus (159) Google Scholar). Dnmt3A knock-out mice show developmental abnormalities and die few weeks after birth (33.Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4413) Google Scholar). This enzyme has been associated with the methylation of single copy genes and retrotransposons (37.Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar, 38.Bourc'his D. Bestor T.H. Nature. 2004; 431: 96-99Crossref PubMed Scopus (876) Google Scholar, 39.Bourc'his D. Xu G.L. Lin C.S. Bollman B. Bestor T.H. Science. 2001; 294: 2536-2539Crossref PubMed Scopus (1052) Google Scholar), and it is required for the establishment of the genomic imprint during germ cell development (40.Kaneda M. Okano M. Hata K. Sado T. Tsujimoto N. Li E. Sasaki H. Nature. 2004; 429: 900-903Crossref PubMed Scopus (1006) Google Scholar).The third member of the Dnmt3 family, Dnmt3L, shows clear homology to the Dnmt3A and -3B enzymes (21.Aapola U. Kawasaki K. Scott H.S. Ollila J. Vihinen M. Heino M. Shintani A. Minoshima S. Krohn K. Antonarakis S.E. Shimizu N. Kudoh J. Peterson P. Genomics. 2000; 65: 293-298Crossref PubMed Scopus (208) Google Scholar). Its N-terminal part contains the PHD domain, but not the PWWP domain, and the C-terminal part extends up to conserved motif VIII (Fig. 1). Strikingly, Dnmt3L carries mutations within all conserved motifs that contain the catalytic residues of DNA-(cytosine-C5)-methyltransferases, suggesting that Dnmt3L adopts the typical MTase fold, but it does not have catalytic activity. In cotransfection experiments Dnmt3L has been shown to stimulate DNA methylation by Dnmt3A in human cell lines (41.Chedin F. Lieber M.R. Hsieh C.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16916-16921Crossref PubMed Scopus (375) Google Scholar). Stimulation of Dnmt3B was not detectable in these experiments. However, biochemical studies revealed an interaction of Dnmt3L with Dnmt3A and -3B (37.Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar). In addition, Dnmt3L has been shown to interact with histone deacetylases via its PHD-like Zn domain and to contribute to gene silencing that is not mediated by DNA methylation (28.Aapola U. Liiv I. Peterson P. Nucleic Acids Res. 2002; 30: 3602-3608Crossref PubMed Scopus (74) Google Scholar, 42.Deplus R. Brenner C. Burgers W.A. Putmans P. Kouzarides T. de Launoit Y. Fuks F. Nucleic Acids Res. 2002; 30: 3831-3838Crossref PubMed Scopus (144) Google Scholar). Dnmt3L is expressed during gametogenesis and embryonic stages (37.Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar, 38.Bourc'his D. Bestor T.H. Nature. 2004; 431: 96-99Crossref PubMed Scopus (876) Google Scholar, 39.Bourc'his D. Xu G.L. Lin C.S. Bollman B. Bestor T.H. Science. 2001; 294: 2536-2539Crossref PubMed Scopus (1052) Google Scholar) showing a similar expression pattern as the Dnmt3A and -3B enzymes. Dnmt3L knock-out mice display a normal phenotype (37.Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar, 38.Bourc'his D. Bestor T.H. Nature. 2004; 431: 96-99Crossref PubMed Scopus (876) Google Scholar, 39.Bourc'his D. Xu G.L. Lin C.S. Bollman B. Bestor T.H. Science. 2001; 294: 2536-2539Crossref PubMed Scopus (1052) Google Scholar). Homozygous female mice are fertile, but when crossed with wild-type males their pups die at embryonic day 10.5. Analysis of the DNA methylation pattern showed that the female imprint was not properly established in oocytes of Dnmt3L knockout females (37.Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar, 39.Bourc'his D. Xu G.L. Lin C.S. Bollman B. Bestor T.H. Science. 2001; 294: 2536-2539Crossref PubMed Scopus (1052) Google Scholar). These data suggest that Dnmt3A and -3L together are required for the establishment of an imprint during gametogenesis. Homozygous male knock-out animals are sterile because of defects in spermatogenesis. Methylation analysis showed major loss of methylation in spermatogonial stem cells leading to a demethylation of the DNA and male infertility (37.Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar, 38.Bourc'his D. Bestor T.H. Nature. 2004; 431: 96-99Crossref PubMed Scopus (876) Google Scholar).Although several studies have been carried out that elucidate the role of Dnmt3L in vivo, the mechanism by which Dnmt3L stimulates de novo methylation has not been thoroughly investigated. Recently, Suetake et al. (43.Suetake I. Shinozaki F. Miyagawa J. Takeshima H. Tajima S. J. Biol. Chem. 2004; 279: 27816-27823Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) demonstrated that Dnmt3L stimulates Dnmt3A and -3B in vitro 1.5- to 3-fold independent of the sequence of the substrate DNA. In this work we extend these findings by demonstrating that Dnmt3L stimulates the catalytic activity of the Dnmt3A and -3B enzymes ∼15-fold in vitro. Furthermore, we investigate the mechanism of activation and find that binding to Dnmt3L induces a conformational change of Dnmt3A that facilitates DNA and AdoMet binding. The interaction of Dnmt3A and Dnmt3L is transient and Dnmt3L dissociates from Dnmt3A-DNA complexes. Therefore, Dnmt3L functions as a substrate exchange factor for Dnmt3A and -3B.EXPERIMENTAL PROCEDURESProteins—Dnmt1, Dnmt3A, Dnmt3B, and their catalytic domains were expressed and purified as described (14.Fatemi M. Hermann A. Pradhan S. Jeltsch A. J. Mol. Biol. 2001; 309: 1189-1199Crossref PubMed Scopus (197) Google Scholar, 23.Reither S. Li F. Gowher H. Jeltsch A. J. Mol. Biol. 2003; 329: 675-684Crossref PubMed Scopus (60) Google Scholar, 30.Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (184) Google Scholar). Murine Dnmt3L (421 amino acid residues) was cloned into pET28a+ by reverse transcription-PCR using RNA isolated from mouse testis, and its sequence was confirmed. A truncated version of Dnmt3L (CD-Dnmt3L comprising amino acids 208–421) was prepared by PCR mutagenesis as described previously (44.Jeltsch A. Lanio T. Methods Mol. Biol. 2002; 182: 85-94PubMed Google Scholar). Dnmt3L and CD-Dnmt3L were purified as described for Dnmt3A. All proteins were >90% pure as determined from Coomassie-stained SDS-gels.Oligonucleotide Substrates—The following oligodeoxynucleotide substrates were used in this study (written in 5′ → 3′ direction): CG30: Bt-GAA GCT GGG ACT TCCGGG AGG AGA GTG CAA/TTG CAC TCT CCT CCC GGA AGT CCC AGC TTC; CA30: Bt-GAA GCT GGG ACT TCC AGG AGG AGA GTG CAA/TTG CAC TCT CCT CCT GGA AGT CCC AGC TTC; CT30: Bt-GAA GCT GGG ACT TCC TGG AGG AGA GTG CAA/TTG CAC TCT CCT CCA GGA AGT CCC AGC TTC; CG16: Bt-GG ACT TCC GGG AGG AG/CT CCT CCC GGA AGT CC; CG 12: Bt-ACT TCCGGG AGG/CCT CCC GGA AGT; CG45: Bt-ATC GCT GAG AAG CTG GGA CTT CCG GGA GGA GAG TGC AAT AGA TTC/GAA TCT ATT GCA CTC TCC TCC CGG AAG TCC CAG CTT CTC AGC GAT.Purified oligonucleotides were purchased from MWG (Ebersberg, Germany). The quality of the oligonucleotide synthesis was confirmed by denaturing polyacrylamide gel electrophoresis, demonstrating all oligonucleotides had the expected length and were pure to >95%. Duplex oligonucleotides were prepared by adding equimolar amounts of complementary strands, heating to 95 °C, and slowly cooling to room temperature. Extinction coefficients were used as provided by the supplier.Biochemical Assays—DNA methylation reactions were carried out in methylation buffer containing 20 mm HEPES (pH 7.5) and 1 mm EDTA basically as described previously (45.Roth M. Jeltsch A. Biol. Chem. 2000; 381: 269-272Crossref PubMed Scopus (80) Google Scholar). If not indicated differently, DNA and labeled [methyl-3H]AdoMet (3048 GBq/mmol, PerkinElmer Life Sciences) were used at concentrations of 0.5 μm and 0.76 μm, respectively. DNA binding was analyzed by nitrocellulose filter binding assay in 50 mm HEPES, pH 7.5, 1 mm EDTA as described (46.Jeltsch A. Friedrich T. Roth M. J. Mol. Biol. 1998; 275: 747-758Crossref PubMed Scopus (44) Google Scholar). UV-cross-linking experiments to analyze AdoMet binding to MTases were performed by mixing 1.3 μm CD-Dnmt3A with [methyl-3H]Adomet (1.4 μm) in the absence or presence of 12 μm CD-Dnmt3L in methylation buffer supplemented with 10% glycerol and 1% β-mercaptoethanol. The mixture was incubated on ice for 5 min and then exposed to UV in a UV cross-linker (Hoefer UVC 500) for 5 min at 7 × 104 mJ/cm2. The samples were then mixed with SDS loading dye, heated at 80°C for 5 min, and separated on a denaturing 15% polyacrylamide gel. After the run, the gel was treated with Amplify solution (NAMP 100, Amersham Biosciences) according to the manufacturer's protocol and dried. This was followed by exposure to a hyperfilm (Amersham Biosciences) at –80 °C for 3–4 days.Surface Plasmon Resonance Experiments—Biomolecular interaction analysis was performed by surface plasmon resonance (SPR) experiments using a Biacore X instrument in buffer HBS-EP (10 mm HEPES, pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.005% surfactant P20) as recommended by the supplier. Experiments were performed at a flow rate of 10 μl/min at ambient temperature. DNA binding was analyzed using streptavidin-coated chips (Sensor Chip SA, Biacore), which were loaded with 400–800 resonance units of biotinylated oligonucleotide as recommended by the supplier (typically 1–3 μlof1 μm oligonucleotide solution was applied to the chip). Protein-protein interaction was analyzed after covalent immobilization of one protein on the surface of Sensor Chip CM5 (Biacore) using amine coupling kit (Biacore). SPR data were analyzed by standard procedures (47.Pingoud A. Urbanke C. Hogget J. Jeltsch A. Biochemical Methods. Wiley-VCH, Weinheim, Germany2002: 327-344Google Scholar). Briefly, a non-linear least-squares minimization of the deviation of the experimental data from theoretical data was performed using the Microsoft Excel solver module. Theoretical data were generated by numerical integration of the differential equations given by the different binding models using time integration intervals of 1 s. Three to five data sets obtained at different concentrations of ligands were fitted simultaneously in global analyses. In these fits, kinetic constants were treated as global variables. The accuracy of the numerical integration was confirmed by simulations of simplified systems that could be analytically solved. Error analysis was performed by extensive simulations in which the value of one individual rate constant was fixed and all others varied.RESULTSActivation of Dnmt3A and -3B by Dnmt3L—It has been shown previously that Dnmt3L stimulates DNA methylation by Dnmt3A and -3B ∼1.5- to 3-fold in vitro (43.Suetake I. Shinozaki F. Miyagawa J. Takeshima H. Tajima S. J. Biol. Chem. 2004; 279: 27816-27823Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). As an initial step in investigating the mechanism of this process we purified Dnmt3A, -3B, and -3L and studied the stimulation of DNA methylation by Dnmt3L using synthetic oligonucleotide substrates. As shown in Fig. 2A, in the presence of 4 μm Dnmt3L the initial rate of the enzymatic reaction of Dnmt3A was stimulated about 5-fold. We confirmed the absence of DNA methylation activity of Dnmt3L (data not shown), which is in agreement with the observation that all the essential DNA-(cytosine-C5)-MTase amino acid sequence motifs are disrupted in Dnmt3L. Therefore, the increased rates of DNA methylation by Dnmt3A observed in the presence of Dnmt3L must be due to a stimulation of the activity of Dnmt3A. Similar experiments using purified Dnmt1 did not show any influence of Dnmt3L on its methylation activity (Fig. 2F). To examine if the stimulation of Dnmt3A by Dnmt3L requires the interaction of the N-terminal part of Dnmt3A, we have investigated the stimulation of the isolated catalytic domain of Dnmt3A (CD-Dnmt3A), which is active even in the absence of the N-terminal domain (22.Gowher H. Jeltsch A. J. Biol. Chem. 2002; 277: 20409-20414Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 23.Reither S. Li F. Gowher H. Jeltsch A. J. Mol. Biol. 2003; 329: 675-684Crossref PubMed Scopus (60) Google Scholar). The results demonstrate a similar level of stimulation of CD-Dnmt3A by addition of Dnmt3L as observed for the full-length Dnmt3A protein (Fig. 2B). To test for an involvement of the PHD domain of Dnmt3L in the stimulation process, we deleted the N-terminal part of Dnmt3L and studied stimulation of Dnmt3A by the resulting C-terminal fragment of Dnmt3L. Again a strong increase in methylation activity was observed (Fig. 2D), indicating that the stimulation is caused by an interaction of the catalytic domain of Dnmt3A with the C-terminal domain of Dnmt3L. Titration experiments using the same amounts of Dnmt3L and Cd-Dnmt3L demonstrated a comparable degree of stimulation. Similar experiments were also performed with Dnmt3B and its isolated catalytic domain, which was activated to a similar degree as Dnmt3A (Fig. 2, C and E). In summary, addition of Dnmt3L or CD-Dnmt3L caused a 5- to 10-fold stimulation of the DNA methylation activity of Dnmt3A, Dnmt3B, CD-Dnmt3A, and CD-Dnmt3B but not of Dnmt1.Fig. 2Stimulation of DNA methylation of DNA MTases by Dnmt3L. In A–C stimulation of Dnmt3A (100 nm), the catalytic domain of Dnmt3A (150 nm), and the catalytic domain of Dnmt3B (500 nm) by Dnmt3L (4 μm) is shown. D and E, stimulation of CD-Dnmt3A (150 nm) and Dnmt3B (100 nm) by addition of CD-Dnmt3L (10 μm). F, Dnmt1 (100 nm) enzyme is not stimulated by addition of Dnmt3L (4 μm). Unmethylated CG30 was used as substrate (0.5 μm) for all the experiments, except F, where hemimethylated CG30 was used. In all figures the data points without Dnmt3L are labeled by squares (▪), the data points with Dnmt3L are labeled by diamonds (♦).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Determination of the Maximum Level of Stimulation—We were interested to determine the maximum level of stimulation of Dnmt3A and -3B by Dnmt3L. Because this process involves a functional interaction of Dnmt3A and Dnmt3L, the degree of stimulation should be dependent on the concentration of Dnmt3L and the ratio of Dnmt3L and active methyltransferase (Dnmt3A or -3B). Higher amounts of Dnmt3L will increase the level of stimulation until saturation is reached. Therefore, we performed methylation experiments with CD-Dnmt3A and CD-Dnmt3B in the presence of increasing amounts of Dnmt3L (Fig. 3A). The stimulation effects were analyzed as binary binding experiments to estimate the apparent binding affinity of both proteins and the intrinsic level of stimulation of Dnmt3L on CD-Dnmt3A and CD-Dnmt3B. With CD-Dnmt3A and CD-Dnmt3B apparent binding constants of Dnmt3L were in the lower micromolar range indicating a low affinity interaction: Ka(CD-Dnmt3A) = 8 × 104 m–1 and Ka(CD-Dnmt3B) = 9 × 104 m–1. A binary binding model was chosen for analysis, because it is the simplest model that allowed fitting the activity curves. Given that a more complicated reaction mechanism is likely (see below), these numbers can only be interpreted as phenomenological values that do not reflect the real binding affinities of the two proteins. These titration experiments allow extrapolation to the stimulation level that would be observed at saturating concentrations of Dnmt3L. This stimulation level reflects the absolute difference between the catalytic activities of free Dnmt3A or -3B and the Dnmt3A- or -3B-Dnmt3L complexes. On the basis of our data the molecular activation level of CD-Dnmt3A and -3B by Dnmt3L is estimated 15-fold in the case of CD-Dnmt3A and 13-fold in the case of CD-Dnmt3B. We conclude that saturating amounts of Dnmt3L induce a considerable (∼15-fold) stimulation of the catalytic activity of the catalytic domains of Dnmt3A and -3B.Fig. 3A, titration of the catalytic activity of CD-Dnmt3A (150 nm) and CD-Dnmt3B (500 nm) with Dnmt3L. Rates were determined by linear regression from kinetics similar as shown in Fig. 2. The data points represent averages of three independent experiments, and variations of the individual stimulation levels were below 10%. The stimulation is defined as the ratio of the reaction rate CD-Dnmt3A in the presence Dnmt3L versus CD-Dnmt3A alone. B, stimulation of CD-Dnmt3A (150 nm) by Dnmt3L (4 μm) in the presence of various concentrations of oligonucleotide substrate (CG30) and AdoMet. The lines represent global fits of the data to the Michaelis-Menten model. Data points without Dnmt3L are labeled by diamonds (♦), the data points with Dnmt3L are labeled by squares (▪). C, stimulation of AdoMet binding to CD-Dnmt3A (1.3 μm) by CD-Dnmt3L (12 μm) as observed by UV-cross-linking experiments carried out with 3H-labeled AdoMet (1.4 μm). The figure compares the efficiency of cross-linking in the presence and absence of Dnmt3L. The lanes labeled C and A show the Coomassie stain (C) and autoradiogram (A) of the same gel lanes. The apparent molecular masses of Dnmt3A and 3L are 36 and 28 kDa, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Biochemical Analysis of the Stimulation of CD-Dnmt3A— Next we attempted to understand the mechanistic basis of the activation of the de novo DNA MTases by Dnmt3L. For technical reasons, we performed these experiments with the catalytic domain of Dnmt3A. First, we have determined the rates of DNA methylation at different concentrations of DNA and AdoMet (Fig. 3B). A global fit of the data to the Michaelis-Menten model yielded Km(AdoMet) = 2.55 μm, Km(DNA) = 0.76 μm, and kcat = 0.65 min–1 in the absence of Dnmt3L. In the presence of Dnmt3L these values were determined as Km(AdoMet) = 0.96 μm, Km(DNA) = 0.25 μm, and kcat = 0.78 min–1. Simulations revealed Km values were valid within ±20%, and the accuracy of kcat values was ±30%. These results demonstrate that the apparent Km values of CD-Dnmt3A for DNA and AdoMet were improved 3.0- and 2.7-fold, respectively, whereas kcat was only marginally changed (1.2-fold).Because the Michaelis-Menten analysis suggested an influence of Dnmt3L on DNA and AdoMet binding of Dnmt3A, we directly tested the effect of Dnmt3L on AdoMet binding using a UV-cross-linking assay. In this assay, 3H-labeled AdoMet was incubated with Dnmt3A, cross-linked to the protein by UV-irradiation, and the binding was analyzed by autoradiography of SDS-polyacrylamide gels. As shown in Fig. 3C, AdoMet binding by Dnmt3A was improved in the presence o

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