Molecular Enzymology of the Catalytic Domains of the Dnmt3a and Dnmt3b DNA Methyltransferases

The C-terminal domains of the mammalian DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b harbor all the conserved motifs characteristic for cytosine-C5 methyltransferases. Whereas the isolated catalytic domain of Dnmt1 is inactive, we show here that the C-terminal domains of Dnmt3a and Dnmt3b are catalytically active. Neither Dnmt3a nor Dnmt3b shows a significant preference for the satellite 2 sequence, although Dnmt3b is required for methylation of these regions in vivo. However, the catalytic domain of Dnmt3a methylates DNA in a distributive reaction, whereas Dnmt3b is processive, which accelerates methylation of macromolecular DNA in vitro. This property could make Dnmt3b a preferred enzyme for methylation at satellite 2 repeats, since they are highly CG-rich. We have also analyzed the catalytic activities of six different mutations found in ICF (immunodeficiency, centromeric instability, and facial abnormalities) patients in the catalytic domain of Dnmt3b. Five of them display catalytic activities reduced by 10–50-fold; one mutant was inactive in our assay (residual activity <1%). These results confirm that a reduced catalytic activity of Dnm3b causes ICF. However, the mutations in general do not completely abrogate catalytic activity. This finding may explain why ICF patients are viable, whereas nmt3b knock-out mice die during embryogenesis. The C-terminal domains of the mammalian DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b harbor all the conserved motifs characteristic for cytosine-C5 methyltransferases. Whereas the isolated catalytic domain of Dnmt1 is inactive, we show here that the C-terminal domains of Dnmt3a and Dnmt3b are catalytically active. Neither Dnmt3a nor Dnmt3b shows a significant preference for the satellite 2 sequence, although Dnmt3b is required for methylation of these regions in vivo. However, the catalytic domain of Dnmt3a methylates DNA in a distributive reaction, whereas Dnmt3b is processive, which accelerates methylation of macromolecular DNA in vitro. This property could make Dnmt3b a preferred enzyme for methylation at satellite 2 repeats, since they are highly CG-rich. We have also analyzed the catalytic activities of six different mutations found in ICF (immunodeficiency, centromeric instability, and facial abnormalities) patients in the catalytic domain of Dnmt3b. Five of them display catalytic activities reduced by 10–50-fold; one mutant was inactive in our assay (residual activity <1%). These results confirm that a reduced catalytic activity of Dnm3b causes ICF. However, the mutations in general do not completely abrogate catalytic activity. This finding may explain why ICF patients are viable, whereas nmt3b knock-out mice die during embryogenesis. In vertebrate DNA cytosine residues are modified by cytosine-C5 methylation mainly at CG sequences (reviewed in Refs. 1Robertson K.D. Wolffe A.P. Nat. Rev. Genet. 2000; 1: 11-19Crossref PubMed Scopus (880) Google Scholar, 2Jones P.A. Takai D. Science. 2001; 293: 1068-1070Crossref PubMed Scopus (1550) Google Scholar, 3Cheng X. Roberts R.J. Nucleic Acids Res. 2001; 29: 3784-3795Crossref PubMed Scopus (405) Google Scholar). Approximately, 70–80% of all CG sequences are modified in a cell type specific pattern. Methylation is involved in epigenetic processes like gene regulation during the embryonic development and cell differentiation, genomic imprinting, and X-inactivation. By silencing the expression of repetitive sequences, DNA methylation protects the genome against selfish genetic elements and helps to maintain genomic integrity. Hypermethylation and hypomethylation of DNA contributes to cancerogenesis and tumor progression (reviewed in Refs. 4Jones P.A. Laird P.W. Nat. Genet. 1999; 21: 163-167Crossref PubMed Scopus (2058) Google Scholar, 5Warnecke P.M. Bestor T.H. Curr. Opin. Oncol. 2000; 12: 68-73Crossref PubMed Scopus (95) Google Scholar, 6Baylin S.B. Herman J.G. Trends Genet. 2000; 16: 168-174Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar). DNA methylation is introduced by DNA methyltransferases (MTases), 1The abbreviations used are: MTaseDNA methyltransferaseAdoMetS-adenosylmethionineCDcatalytic domainICFimmunodeficiency, centromeric instability, and facial abnormalities which use S-adenosylmethionine (AdoMet) as donor for an activated methyl group (reviewed in Ref. 7Bestor T.H. Hum. Mol. Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1600) Google Scholar). Depending on the developmental state of the cell, the DNA methylation pattern either has to be created de novo, or the existing pattern of methylation has to be maintained. To accomplish this purpose, vertebrates contain a maintenance methyltransferase, Dnmt1, and de novo methyltransferases, Dnmt3a and Dnmt3b, although there is now evidence accumulating that the functions of these proteins overlap (8Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1286) Google Scholar, 9Rhee I. Jair K.W. Yen R.W. Lengauer C. Herman J.G. Kinzler K.W. Vogelstein B. Baylin S.B. Schuebel K.E. Nature. 2000; 404: 1003-1007Crossref PubMed Scopus (372) Google Scholar, 10Liang G. Chan M.F. Tomigahara Y. Tsai Y.C. Gonzales F.A., Li, E. Laird P.W. Jones P.A. Mol. Cell. Biol. 2002; 22: 480-491Crossref PubMed Scopus (448) Google Scholar, 11Guex N. Diemand A. Peitsch M.C. Trends Biochem. Sci. 1999; 24: 364-367Abstract Full Text Full Text PDF PubMed Google Scholar). 2M. Fatemi, A. Hermann, H. Gowher, and A. Jeltsch, submitted for publication. Dnmt1 is responsible for propagation of methylation pattern through cell generations by methylating the hemimethylated sites created after every round of replication. The Dnmt3 MTases have been assigned the role of de novo methylation, since they do not show a preference for hemimethylated DNA (8Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1286) Google Scholar, 12Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (191) Google Scholar). De novo methylation of DNA by Dnmt3a and Dnmt3b was also demonstrated in vivo after expression in human cell lines (13Hsieh C.-L. Mol. Cell. Biol. 1999; 19: 8211-8218Crossref PubMed Scopus (221) Google Scholar) and by expression of Dnmt3a in transgenic Drosophila melanogaster (14Lyko 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). Dnmt3a and Dnmt3b are highly expressed in embryonic tissues, whereas only low expression is observed in differentiated cells (8Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1286) Google Scholar, 15Robertson 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), suggesting that these proteins are involved in the re-methylation of the genome in early embryogenesis that occurs after a massive demethylation immediately after fertilization (reviewed in Ref. 16Reik W. Dean W. Walter J. Science. 2001; 293: 1089-1093Crossref PubMed Scopus (2437) Google Scholar). There are several alternative splice variants of Dnmt3b, two of which are active, one is not (8Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1286) Google Scholar, 17Aoki A. Suetake I. Miyagawa J. Fujio T. Chijiwa T. Sasaki H. Tajima S. Nucleic Acids Res. 2001; 29: 3506-3512Crossref PubMed Scopus (149) Google Scholar). DNA methyltransferase S-adenosylmethionine catalytic domain immunodeficiency, centromeric instability, and facial abnormalities The Dnmt1 and both Dnmt3 proteins consist of an N-terminal part, which has regulatory and targeting functions and a C-terminal catalytic domain, which contains 10 characteristic amino acid motifs that are conserved among all cytosine-C5 MTases (reviewed in Ref. 18Kumar S. Cheng X. Klimasauskas S. Sha M. Posfai J. Roberts R.J. Wilson G.G. Nucleic Acids Res. 1994; 22: 1-10Crossref PubMed Scopus (393) Google Scholar). The Dnmt3a and Dnmt3b proteins from mouse comprise 908 and 859 amino acid residues, respectively, and share 36% amino acid sequence identity with each other (>80% in their C-terminal domains). The N-terminal part of Dnmt1 is an important regulator of enzyme activity (19Pradhan S. Roberts R.J. EMBO J. 2000; 19: 2103-2114Crossref PubMed Scopus (21) Google Scholar, 20Bacolla 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, 21Fatemi M. Hermann A. Pradhan S. Jeltsch A. J. Mol. Biol. 2001; 309: 1189-1199Crossref PubMed Scopus (200) Google Scholar) that has been shown to interact with many other proteins like PCNA, transcription factors, and histone deacetylases (22Chuang 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, 23Rountree M.R. Bachman K.E. Baylin S.B. Nat. Genet. 2000; 25: 269-277Crossref PubMed Scopus (864) Google Scholar, 24Fuks F. Burgers W.A. Brehm A. Hughes-Davies L. Kouzarides T. Nat. Genet. 2000; 24: 88-91Crossref PubMed Scopus (820) Google Scholar, 25Robertson 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, 26Fuks F. Burgers W.A. Godin N. Kasai M. Kouzarides T. EMBO J. 2001; 20: 2536-2544Crossref PubMed Scopus (469) Google Scholar) and to be involved in targeting Dnmt1 to replication foci (27Leonhardt H. Page A.W. Weier H.U. Bestor T.H. Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (829) Google Scholar, 28Liu Y. Oakeley E.J. Sun L. Jost J.-P. Nucleic Acids Res. 1998; 26: 1038-1045Crossref PubMed Scopus (115) Google Scholar). The C-terminal domain of Dnmt1 was cloned several times, always showing that it is catalytically inactive and does not act as an independent methyltransferase despite the presence of all the amino acid motifs characteristic for DNA MTases (21Fatemi M. Hermann A. Pradhan S. Jeltsch A. J. Mol. Biol. 2001; 309: 1189-1199Crossref PubMed Scopus (200) Google Scholar, 29Zimmermann C. Guhl E. Graessmann A. Biol. Chem. 1997; 378: 393-405Crossref PubMed Scopus (34) Google Scholar, 30Margot J.B. Aguirre-Arteta A.M., Di Giacco B.V. Pradhan S. Roberts R.J. Cardoso M.C. H. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). The N-terminal of Dnmt3a and Dnmt3b the to regions in cells K.E. M.R. Baylin S.B. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). contain a that is to the which with the histone F. Burgers W.A. Godin N. Kasai M. Kouzarides T. EMBO J. 2001; 20: 2536-2544Crossref PubMed Scopus (469) Google Scholar) and a domain, which with the DNA C. X. Cheng X. Nat. Biol. 2002; 9: Google Scholar). Dnmt3a also with a found at F. Burgers W.A. Godin N. Kasai M. Kouzarides T. EMBO J. 2001; 20: 2536-2544Crossref PubMed Scopus (469) Google Scholar). The isolated catalytic domains of Dnmt3a and Dnmt3b have not been mice Dnmt3a and Dnmt3b, and in are and die at embryonic or after the the role of these during the development E. Bestor T.H. Jaenisch R. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, M. Li E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). mice show a massive demethylation at satellite in the Dnmt3b gene have been shown to be with the ICF (immunodeficiency, centromeric facial a genetic M. Li E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Bestor T.H. D. C.-L. N. M. X. E. Nature. 1999; PubMed Scopus Google Scholar, C. Sci. S. A. 1999; PubMed Scopus Google Scholar) that is with hypomethylation of 2 and in of and and in regions of the and inactive R. C. M.R. M. R. G. Laird Hum. Mol. Genet. 2000; 9: PubMed Scopus Google Scholar). The hypomethylation of at regions to the of A. F. J. M. Genet. 2000; PubMed Google Scholar). of the DNA causes of gene expression which with development of the M. F. G. Sun B. W. D. B.H. N. R. Hum. Mol. Genet. 2001; PubMed Scopus Google Scholar). The that mutations in the of ICF patients all at or to the catalytic domain of Dnmt3b that reduced catalytic activity of the enzyme is responsible for the M. Li E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Bestor T.H. D. C.-L. N. M. X. E. Nature. 1999; PubMed Scopus Google Scholar, C. Sci. S. A. 1999; PubMed Scopus Google Scholar, C. G. E.J. D. B.H. E. C. P.L. Hum. 2000; 16: PubMed Scopus Google Scholar). the results with mice and ICF patients that Dnmt3b is responsible for methylation of satellite sequences like satellite 2 in vivo and that Dnmt3a Dnmt3b in this In this we the isolated C-terminal domains of Dnmt3a and Dnmt3b are catalytically DNA We the and show that the of Dnmt3a and Dnmt3b to the of Dnmt3b to DNA at satellite 2 in vivo and explain why Dnmt3a Dnmt3b in this we the catalytic activities of several Dnmt3b which amino acid observed in ICF from by of contains one CG To for methylation of satellite sequences, we used which has the sequence of human satellite 2 The a that also has two CG sites was used as a for CG methylation in a sequence Dnmt3a and Dnmt3b and by Li domains of Dnmt3a and Dnmt3b cloned as N-terminal variants the mutations in ICF patients and in mouse a M. S. T. Jeltsch A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. Mol. Biol. 2002; Google Scholar). expression was in cells by of at The proteins as for the Dnmt3a H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (191) Google Scholar). In was from for each an to the of the DNA methylation was by the of methyl from as M. Jeltsch A. Biol. Chem. 2000; PubMed Scopus Google Scholar). The methylation at of and of DNA and respectively, in methylation at enzyme of The of 2 of DNA was Methylation of was analyzed by the assay of DNA at an enzyme of in 10 of methylation The on which with by a with to The and in for the of The was an and a of the Dnmt3b gene as a for These contain X. Roberts R.J. Nucleic Acids Res. 2001; 29: 3784-3795Crossref PubMed Scopus (405) Google Scholar) sites and a of of each with was with enzyme in methylation at times, and the was by of of the DNA was in 10 and the with 10 of for at on a in The was analyzed an and of the Dnmt3a and Dnmt3b catalytic domains cloned as N-terminal proteins The proteins and to as from The of the proteins by The catalytic activity was by of methyl from the DNA an with a the proteins active. However, both Dnmt3a their the Dnmt3b This is in to the of the A. Suetake I. Miyagawa J. Fujio T. Chijiwa T. Sasaki H. Tajima S. Nucleic Acids Res. 2001; 29: 3506-3512Crossref PubMed Scopus (149) Google Scholar). the of and the of these proteins used for The catalytic activity of the was at different and different and the activity was found at in and at low not We to use in our methylation to to and catalytic activity of the and of the catalytic domains of Dnmt3a and The shows a of the and of the catalytic domains of Dnmt3a and Dnmt3b The shows the methylation of a by the The that the catalytic domains of Dnmt3a and Dnmt3b are DNA MTases that these domains cytosine-C5 which in general do not have a N-terminal part (reviewed in Refs. 3Cheng X. Roberts R.J. Nucleic Acids Res. 2001; 29: 3784-3795Crossref PubMed Scopus (405) Google Scholar, X. Rev. 24: PubMed Scopus Google Scholar, and Cheng X. and Google Scholar). is in to the that the C-terminal part of Dnmt1 was found not to be an methyltransferase, although it all the catalytic amino acid motifs characteristic for cytosine-C5 MTases (21Fatemi M. Hermann A. Pradhan S. Jeltsch A. J. Mol. Biol. 2001; 309: 1189-1199Crossref PubMed Scopus (200) Google Scholar, 29Zimmermann C. Guhl E. Graessmann A. Biol. Chem. 1997; 378: 393-405Crossref PubMed Scopus (34) Google Scholar, 30Margot J.B. Aguirre-Arteta A.M., Di Giacco B.V. Pradhan S. Roberts R.J. Cardoso M.C. H. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). the catalytic domains of Dnmt3a and Dnmt3b DNA which in general DNA at sequences, the preference of Dnmt3b for methylation at satellite 2 sequences could be to an preference of catalytic domain for the sequence of the satellite To this a was to have a satellite 2 This two CG sites and one at the the was that also two sites in a sequence shown in the catalytic domain of Dnmt3b shows preference for methylation at the satellite 2 of the satellite sequence explain the in vivo role of Dnmt3b. The of is an important for like DNA which act on several for the and distributive of DNA methylation be on the that they in a completely different of and modified during the methylation the modified the to whereas a distributive methylation to an of DNA as We the of DNA methylation by and two in one and one We the of a these DNA against by CG The DNA contains of the in to 10 different one of which be observed in shown in methylates the in a distributive There is a in the of the DNA during the of the reaction, and DNA is only observed after of the This shows that each and is distributive as shown by for the Dnmt3a H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (191) Google Scholar). In not many after methylation of the by and of DNA and are at the in the be results with an that contains and CG sites not This in the of and was observed with different enzyme and also with a is not to the that we used the of the catalytic domain of Dnmt3b and the of Dnmt3a for these with the of Dnmt3a also a distributive not and also the Dnmt3a enzyme methylates DNA in a distributive H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (191) Google Scholar). To the of DNA methylation of both in we the of all in methylation with and and the of all the different are in the of all and all are to a the is The to a which six different to the distributive methylation at methylation at and methylation at two methylation at sites and methylation at sites 2 and and one one methylation at all be that this does not make on the of of the methylation could be and the for This shows that methylates DNA in an completely distributive reaction, of the activity is assigned to and and only to a methylation of two sites This the of the that in an distributive a completely different was of the activity is assigned to which to a methylation of all results in activity of and of was the contains CG and the sites are a of all the CG our results that methylates all CG sites on the of DNA methylation also was observed with the we that is to at CG sites from the These also show that the activity of is that of with the macromolecular although with that contain only one is This that a accelerates methylation of macromolecular which contain one mutations have been in the gene of ICF which are in or to the catalytic domain of the Dnmt3b M. Li E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Bestor T.H. D. C.-L. N. M. X. E. Nature. 1999; PubMed Scopus Google Scholar, C. Sci. S. A. 1999; PubMed Scopus Google Scholar). We to the ICF mutant of for catalytic activity and to six of The proteins different of all of them could be from E. cells and the methylation activity the We analyzed the of after with the in the presence of To for methylation, was from and the used for methylation The observed in the However, of the six variants in of in the that these are To the of DNA methylation by the ICF methylation and with with of the are shown in and the results of at independent are in The of the observed in the always was of with the type that catalytic activities of be in this shown in of the show a catalytic activity that is and of the type activity with the with a activity of of We could not activity that was the with the However, the of the this does not that this is catalytically We our that all ICF have a reduced catalytic suggesting that Dnmt3b activity causes ICF. However, ICF variants be as catalytically inactive in In vertebrates the pattern of DNA methylation is used to epigenetic that has important The methylation pattern of the DNA is during embryogenesis by de novo methylation (reviewed in Ref. 16Reik W. Dean W. Walter J. Science. 2001; 293: 1089-1093Crossref PubMed Scopus (2437) Google Scholar). The Dnmt3a and Dnmt3b in this regulation and is of de novo methylation is the methylation of the satellite 2 These are in cells in L. J. N. J. Nature. PubMed Scopus Google Scholar, J. L. A. N. Nucleic Acids Res. 12: PubMed Scopus Google Scholar). 2 sequences are in patients with ICF that have a gene M. Li E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Bestor T.H. D. C.-L. N. M. X. E. Nature. 1999; PubMed Scopus Google Scholar, C. Sci. S. A. 1999; PubMed Scopus Google Scholar, C. G. E.J. D. B.H. E. C. P.L. Hum. 2000; 16: PubMed Scopus Google Scholar) and in Dnmt3b knock-out mice M. Li E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Dnmt3b is responsible for methylation at these sites during and Dnmt3a Dnmt3b in this We have shown that the C-terminal catalytic domains of the Dnmt3a and Dnmt3b DNA MTases are independent of their N-terminal This that Dnmt3a and Dnmt3b, Dnmt1, do not part of N-terminal domain for catalytic activity. the catalytic domains of Dnmt3a and Dnmt3b DNA which in general DNA at sequences (reviewed in Refs. 3Cheng X. Roberts R.J. Nucleic Acids Res. 2001; 29: 3784-3795Crossref PubMed Scopus (405) Google Scholar, X. Rev. 24: PubMed Scopus Google Scholar, and Cheng X. and Google Scholar). We have the catalytic activities of six ICF and show that of them have of the type and one mutant has activity. In a the not show catalytic activity in an in vivo assay Bestor T.H. D. C.-L. N. M. X. E. Nature. 1999; PubMed Scopus Google Scholar), although this is only reduced to our This be to different expression of the type and mutant in vivo the of the in vivo These results confirm that a reduced catalytic activity of Dnmt3b to hypomethylation at satellite 2 sequences and causes ICF. the activity of the ICF variants may explain why a knock-out of Dnmt3b in mice is during whereas ICF patients are This is in with the finding that in ICF patients methylation of the satellite is not completely only reduced with T. G. Hum. Genet. 2001; PubMed Scopus Google Scholar). results show that the catalytic domains of Dnmt3a and Dnmt3b DNA a different whereas the catalytic domain of Dnmt3a, like Dnmt3a H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (191) Google Scholar), methylates DNA in a distributive reaction, the catalytic domain of Dnmt3b is the that the Dnmt3a and Dnmt3b catalytic domains are in amino acid sequence and in share a of it is to a these two However, among the amino acid residues that are not human and Dnmt3a and Dnmt3b in the C-terminal acid residues of the The observed among these residues are highly that Dnmt3b six Dnmt3b has a DNA Dnmt3a which could explain why Dnmt3b methylates DNA in a reaction, whereas Dnmt3a is The in the of the catalytic domains of Dnmt3a and Dnmt3b could be to the functions the in the cell, satellite 2 are among the sequences in the human satellite 2 DNA with a of and T. only CG sequences are in the sequence to a of observed to of the for CG sequences is in DNA F. A. Sci. S. A. PubMed Scopus Google Scholar, B. C. M.C. J. M. W. R. D. A. J. L. J. R. Nature. 2001; PubMed Scopus Google Scholar), the in CG sequences has a for and to which to a of CG sequences in the genome (reviewed in Ref. M.F. Jones P.A. DNA Methylation and Scholar). 2 DNA is not CG it is in the are as a of of M. M. J. Mol. Biol. PubMed Scopus Google Scholar, N. Peitsch M.C. 1997; PubMed Scopus Google Scholar), and only regions in the human genome of all have of for the CG B. C. M.C. J. M. W. R. D. A. J. L. J. R. Nature. 2001; PubMed Scopus Google Scholar). the satellite are in CG sites with the of the The of Dnmt3b it to these after targeting to the DNA it several cytosine residues in a In Dnmt3a methylates DNA in a distributive and from the DNA after each which could explain why Dnmt3a Dnmt3b at satellite This is on our results and does not that Dnmt3b could be to satellite sequences by with other In both of these after targeting Dnmt3b to satellite the methylation methylation of the satellite the on the of our results with isolated domains of Dnmt3a and Dnmt3b has to be The different domains in proteins the two different that domains be completely independent of each other or that the activity of one domain is of We show here that the Dnmt3a and Dnmt3b proteins do not the since the isolated catalytic domains are catalytically active. This the of these proteins by their catalytic domains as This to the of the catalytic part of the MTases in the of the of the N-terminal domains and of other the of the be which the for of by with the N-terminal part and other the results of our an for of the N-terminal part or other proteins on the of the Dnmt3a and Dnmt3b only be by with the of the our results to that are also to the the catalytic domain of Dnmt3b is it is that the enzyme this is a highly property that is to be by of a In Dnmt3a is distributive both as a and This does not that Dnmt3a could by with other Dnmt3b ICF variants are catalytically as isolated domains it is that the variants also display catalytic activity. We E. Li for of Dnmt3a and Dnmt3b. with