Sequence-specific DNA binding by the Mspl DNA methyltransferase

Abstract

The Mspl methyltransferase (M.Mspl) recognizes the sequence CCGG and catalyzes the formation of 5-methyicytosine at the first C-residue. We have investigated the sequence-specific DNA-binding properties of M.Mspl under equilibrium conditions, using gel-mobility shift assays and DNasei footprlnting. M.Mspl binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methlonine as well as the anaiogues, sinefungin and S-adenosyl-L.-homocystelne. in the presence of S-adenosyl-L-homocysteine, M.Mspl shows the highest binding affinity to DNA containing a hemimethyiated recognition sequence (Kd=3.6×10-7M), but binds less well to unmethylated DNA (Kd=8.3×10-7M). Surprisingly it shows specific, although poor, binding to fully methyiated DNA (Kd=4.2×8M). M.Mspl binds approximately 5-told more tightly to DNA containing its recognition sequence, CCGG, than to nonspecific sequences in the absence of cofactors. in the presence of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine or sinefungin the discrimination between specific and non-specific sequences increases up to 100-toid. DNasel footprlnting studies indicate that 16 base pairs of DNA are covered by M.Mspl, with the recognition sequence CCGG located asymmetrically within the footprint. © 1992 IRL Press at Oxford University Press.