Abstract In DNA, cytosine methylation is one of the major epigenetic gene silencing marks in the human genome facilitated by DNA methyltransferases. DNA (cytosine -5) methyltransferase 1 (DNMT1) performs maintenance methylation in somatic and even in cancer cells. It is composed of a large (~1120amino acids) amino-terminal region and a smaller (~500aa) carboxy-terminal region containing conserved, catalytic domains. In this study, Site-Directed Mutagenesis (SDM) approach was used to mutant the DNMT1 site using the phosphorylated primer and established an expression and purification system to produce active recombinant DNMT1 protein. Bioinformatics tool was used for the stimulation of DNMT1 site, to examine whether the mutation alters the DNMT1 structure, activity and what mechanism underlies the alternation. Our bioinformatics predictions show there was no difference between wild and mutant DNMT1 structure. Truncated mouse DNMT1, fused with hexahistidine tagged was expressed by infecting Spodoptera frugiperda (Sf21) cells for 72hours with a recombinant baculovirus carrying the DNMT1 DNA. The DNMT1 was (wild type and mutant (G500C/A526C) expressed through bacterial expression system too. Our data shows, DNMT1 was overexpress through baculovirus expression system in compared to bacterial expression system (E.coli) with correctly predicted size of protein. Western blot was used to analyse the data. Amicon purified recombinant proteins obtained via the baculovirus expression system was analysed in kinase assay. The characterization of hexahistidine-tagged DNA (cytosine-C5)-methyltransfese-1 through DNA methylation, screening assays indicate that the wild DNMT1 protein had higher enzymatic efficiency in compared to mutant DNMT1. Our assay clearly shows the mutant had loss its enzymatic activity.