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Sequence-specific DNA binding by the Msp l DNA methyltransferase

1992; Oxford University Press; Volume: 20; Issue: 12 Linguagem: Inglês

10.1093/nar/20.12.3167

1362-4962

Ashok K. Dubey, Richard J. Roberts,

Bacterial Genetics and Biotechnology

The Msp l methyltransferase (M. Msp l) 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. Msp l under equilibrium conditions, using gel-mobility shift assays and DNasei footprlnting. M. Msp l 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. Msp l shows the highest binding affinity to DNA containing a hemimethyiated recognition sequence (Kd=3.6×10 −7 M), but binds less well to unmethylated DNA (Kd=8.3×10 −7 M). Surprisingly it shows specific, although poor, binding to fully methyiated DNA (Kd=4.2×8M). M. Msp l 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. Msp l, with the recognition sequence CCGG located asymmetrically within the footprint.