Hybrid mouse-prokaryotic DNA (cytosine-5) methyltransferases retain the specificity of the parental C-terminal domain.
The mouse (cytosine-5) DNA methyltransferase (Dnmt1) consists of a regulatory N-terminal and a catalytic C-terminal domain, which are fused by a stretch of Gly-Lys dipeptide repeats. The C-terminal region contains all of the conserved motifs found in other cytosine-5 DNA methyltransferases including the relative position of the catalytic Pro-Cys dipeptide. In prokaryotes, the methyltransferases are simpler and lack the regulatory N-terminal domain. We constructed three hybrid methyltransferases, containing the intact N-terminus of the murine Dnmt1 and most of the coding sequences from M.HhaI (GCGC), M.HpaII (CCGG) or M.SssI (CG). These hybrids are biologically active when expressed in a baculovirus system and show the specificity of the parental C-terminal domain. Expression of these recombinant constructs leads to de novo methylation of both host and viral genomes in a sequence-specific manner. Steady-state kinetic analyses were performed on the murine Dnmt1-HhaI hybrid using poly(dG-dC).poly (dG-dC), unmethylated and hemimethylated oligonucleotides as substrates. The enzyme has a slow catalytic turnover number of 4.38 h(-1) for poly(dG-dC). poly(dG-dC), and exhibits 3-fold higher catalytic efficiency for hemimethylated substrates.
Pradhan
S
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New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA.
Roberts
R J
RJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D020134:Catalytic Domain; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005786:Gene Expression Regulation; D016679:Genome, Viral; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D000077183:Occlusion Body Matrix Proteins; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D011387:Prokaryotic Cells / Q000201:enzymology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins
D000077183:Occlusion Body Matrix Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D004247:DNA; C037664:DNA modification methylase HhaI; C029641:DNA modification methylase HpaII; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases