Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.
A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.
Pradhan
S
S
New England Biolabs, Beverly, Massachusetts 01915, USA.
Bacolla
A
A
Wells
R D
RD
Roberts
R J
RJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry / Q000235:genetics*; D019175:DNA Methylation; D051860:Fragile X Messenger Ribonucleoprotein 1; D006801:Humans; D007700:Kinetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D019154:Protein Splicing; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics; D012436:S-Adenosylmethionine / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics
D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D011089:Polydeoxyribonucleotides; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017411:Ribonucleoproteins, Small Nuclear; D012436:S-Adenosylmethionine; C497683:FMR1 protein, human; C497684:Fmr1 protein, mouse; C031156:poly d(I-C); C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse