Sequence-specific DNA binding by the MspI DNA methyltransferase.
The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the fist 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.MspI 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.MspI 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.
Dubey
A K
AK
Cold Spring Harbor Laboratory, NY 11724.
Roberts
R J
RJ
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases