The study of the interaction of (cytosine-5)-DNA methyltransferase SsoII with DNA by acoustic method

Abstract

The interaction of (cytosine-5)-DNA methyltransferase SsoII (M.SsoII) with double-stranded DNA was studied by means of thickness shear mode acoustic method (TSM) and gel electrophoresis. M.SsoII recognizes in double-stranded DNA the methylation site 5′-CCNGG-3′ (N = A, C, G, T) and methylates the inner cytosine residue. M.SsoII also acts as a transcription factor via binding to the regulatory site 5′-AGGACAAATTGTCCT-3′ in the promoter region of SsoII restriction–modification system. We designed three 60-mer biotinylated DNA duplexes: with the methylation site (60met), with the regulatory site (60reg), and without a specific binding site (60oct). A strong binding of M.SsoII with each one of the studied DNA immobilized on the TSM transducer has been shown. The equilibrium dissociation constants, K_D, of the M.SsoII–DNA complexes decreased in the order 60oct > 60reg > 60met, suggesting a higher stability of M.SsoII–60met complex in comparison with the others. The association rate constant, k_a, was also higher for 60met, while similar values were obtained for 60reg and 60oct. The difference in the kinetic parameters for 60met and 60reg suggested a possible way of coordination between the two M.SsoII functions in a cell.