Sequence-specific DNA binding by theMspl DNA methyltransferase

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Abstract
The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the first C-residue. We have investigated the sequence-specific DNA-binding properties of M.MspI under equilibrium conditions, using gel-mobility shift assays and DNasel footprinting. M.MspI binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methionine as well as the analogues, sinefungin and S-adenosyl-L-homocysteine. In the presence of S-adenosyl-L-homocysteine, M.Mspl shows the highest binding affinity to DNA containing a hemimethylated recognition sequence (Kd = 3.6 x 10(-7)M), but binds less well to unmethylated DNA (Kd = 8.3 x 10(-7)M). Surprisingly it shows specific, although poor, binding to fully methylated DNA (Kd = 4.2 x 10(-6)M). M.Mspl binds approximately 5-fold 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-fold. DNasel footprinting studies indicate that 16 base pairs of DNA are covered by M.MspI, with the recognition sequence CCGG located asymmetrically within the footprint.