Recombinant Human DNA (Cytosine-5) Methyltransferase

Abstract

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 ∼1 mg of intact recombinant enzyme >95% pure per 1.5 × 109 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 (kcat) 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 (KmCG) andS-adenosyl-l-methionine (AdoMet) (KmAdoMet) ranged from 0.33–1.32 and 2.6–7.2 μm, respectively, whereas the ratio ofkcat/KmCGranged from 3.9 to 44 (237–336 for poly(dI-dC)·poly(dI-dC)) × 106 m−1 h−1. The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7–21-fold. The values of kcat 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.