Hybrid mouse-prokaryotic DNA (cytosine-5) methyltransferases retain the specificity of the parental C-terminal domain

Abstract

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. Hha I (GCGC), M. Hpa II (CCGG) or M. Sss I (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– Hha I 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.