The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylatedDNA

The mammalian DNA methyltransferase Dnmt1 is responsible for the maintenanc e of the pattern of DNA methylation in vivo. It is a large multidomain enzy me comprising 1620 amino acid residues. We have purified and characterized individual domains of Dnmt1 (NLS-containing domain, NlsD, amino acid residu es: 1-343; replication foci-directing domain, 350-609; Zn-binding domain (Z nD), 613-748; polybromo domain, 746-1110; and the catalytic domain (CatD), 1124-1620). CatD, ZnD and NlsD bind to DNA, demonstrating the existence of three independent DNA-binding sites in Dnmt1. CatD shows a preference for b inding to hemimethylated CpG-sites; ZnD prefers methylated CpGs; and NlsD s pecifically binds to CpG-sites, but does not discriminate between unmethyla ted and methylated DNA. These results are not compatible with the suggestio n that the target recognition domain of Dnmt1 resides in the N terminus of the enzyme. We show by protein-protein interaction assays that ZnD and CatD interact with each other. The isolated catalytic domain does not methylate DNA, neither alone nor in combination with other domains. Full-length Dnmt 1 was purified from baculovirus-infected insect cells. Under the experiment al conditions, Dnmt1 has a strong (50-fold) preference for hemimethylated D NA. Dnmt1 is stimulated to methylate unmodified CpG sites by the addition o f fully methylated DNA. This effect is dependent on Zn, suggesting that bin ding of methylated DNA to ZnD triggers the allosteric activation of the cat alytic center of Dnmt1. The allosteric activation model can explain kinetic data obtained by others. It suggests that Dnmt1 might be responsible for s preading of methylation, a process that is observed during aging and carcen ogenesis but may be important for de novo methylation of DNA. (C) 2001 Acad emic Press.