Effects of co-factor and deoxycytidine substituted oligonucleotides upon sequence-specific interactions between MspI DNA methyltransferase and DNA.
MspI methyltransferase (M.MspI) catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C-5 position of the outer deoxycytidine base in the DNA sequence 5'-CCGG-3'. Recombinant M.MspI when expressed and purified as a translational fusion with glutathione-S-transferase, shows all of the properties of the wild-type enzyme. We report the kinetic analysis of M.MspI binding to DNA, which suggests a two-stage methylation process, whose initial DNA binding rate is governed by the presence of a positively charged sulphonium centre on the cofactor. Results are also presented that indicate that M.MspI binds preferentially to hemi-methylated DNA and that full methylation of either deoxycytidine on both strands significantly impairs sequence-specific protein-DNA interactions. Furthermore, the importance of the 4-amino group of the inner deoxycytidine for sequence-specific protein-DNA interactions is demonstrated by substituting deoxycytidine with 2-pyrimidinone-1-beta-D-2-deoxyriboside. In addition, we detail the intrinsic structural elements of a cofactor, required to enhance the binding of M.MspI to its recognition sequence, by using S-adenosyl-L-methionine and a range of derivatives.
Ford
K
K
Krebs Institute, Department of Molecular Biology, University of Sheffield, U.K.
Taylor
C
C
Connolly
B
B
Hornby
D P
DP
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D003841:Deoxycytidine / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding
D009838:Oligodeoxyribonucleotides; D003841:Deoxycytidine; D004247:DNA; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases