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 ϳ1 mg of intact recombinant enzyme >95% pure per 1.5 ؋ 10 9 insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two Cterminal 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(dIdC)⅐poly(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)⅐poly(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 M, respectively, whereas the ratio of k cat /K m CG ranged from 3.9 to 44 (237-336 for poly(dI-dC)⅐poly(dI-dC)) ؋ 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.Methylated cytosine is found in the genome of organisms ranging from prokaryotes to mammals (1). Methylation of DNA in eukaryotes is implicated in various biological and developmental processes, such as gene regulation (2), DNA replication (3), genomic imprinting (4), embryonic development (5), carcinogenesis (6), and genetic diseases (7). The bulk of the methylation takes place during DNA replication in the S-phase of the cell cycle (8). The maintenance methylation ensures the propagation of tissue-specific methylation patterns established during mammalian development. The methyl transfer reaction proceeds via nonspecific binding of the enzyme to DNA, recognition of the specific DNA target site, and recruitment of the methyl group donor S-adenosyl-L-methionine (AdoMet) 1 to the active site of the enzyme. DNA (cytosine-5) methyltransferases (m 5 C MTase) introduce a methyl group onto carbon 5 of the target cytosine through a covalent intermediate between the protein and the target cytosine (9). During this process, the cytosine is flipped 180°out of the DNA backbone into an active site pocket of the enzyme (10). After completion of the methyl transfer reaction, the products, methylated DNA and S-adenosyl-L-homocysteine (AdoHcy), are released. Previous studies on the mechanism of methylation were mainly limited to prokaryotic m 5 C MTases although some limited kinetic studies have also been reported with mouse and human DNMT1 (11,12...

Global delay in nascent strand DNA methylation

“…More conceptually, accurate reestablishment of the human methylome requires catalytic activity at ~45 million heterogeneously distributed CpGs (roughly 80% of CpG sites within the diploid genome) that must be completed within a single cell cycle 13 . Given this scale, it may not be surprising that some earlier studies have observed a lag in nascent strand methylation in somatic and transformed cells 14–18 , which presumably reflects the kinetic discrepancy between rapid polymer extension from the 3′-OH of the previously incorporated base versus the multistep transfer of a methyl-group to hemi-methylated CpG dyads 19,20 . However, the global scale, kinetics and possible implications of this disconnect between copying genetic versus epigenetic information remain to be determined.…”

DNA methylation, chromatin inheritance, and cancer

“…Indeed, this enzyme has a 10 ± 40-fold preference for hemimethylated CpGs over unmethylated CpGs (Pradhan et al, 1997(Pradhan et al, , 1999Yoder et al, 1997b). This portion of DNMT1 also contains a cysteine-rich (CXXC) Zn-binding motif, that resembles motifs found in MBD1 and the human Trithorax proteins (Cross et al, 1997).…”

DNA methylation, methyltransferases, and cancer

“…DNMT1 has been shown to have a 10 ± 40-fold preference for hemimethylated DNA (Pradhan et al, 1999;1997) and is the most abundant methyltransferase in somatic cells (Robertson et al, 1999). While the enzyme was cloned 12 years ago (Bestor et al, 1988) its sequence remained incomplete for many years (Yoder et al, 1996) which complicated studies of the DNMT1 promoter (Rouleau et al, 1992) as well as interpretation of the e ect of its overexpression on cell growth (Tucker et al, 1996;Vertino et al, 1996).…”