Interaction of EcoP15I DNA methyltransferase with oligonucleotides containing the asymmetric sequence 5'-CAGCAG-3'.
EcoP15I DNA methyltransferase (Mtase) recognizes the asymmeteric sequence CAGCAG and catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the second adenine residue. We have investigated the DNA binding properties of EcoP15I DNA Mtase using gel mobility shift assays. EcoP15I DNA Mtase binds approximately threefold more tightly to DNA containing its recognition sequence, CAGCAG, than to non-specific sequences in the absence or presence of cofactors. Interestingly, in the presence of ATP the discrimination between specific and non-specific sequences increases significantly. These results suggest for the first time a role for ATP in DNA recognition by type III restriction-modification enzymes. In addition, we have shown that bromodeoxyuridine-containing oligonucleotides form complexes with EcoP15I DNA Mtase that are crosslinked upon irradiation. More importantly, we have shown that the crosslink site is at the site of DNA binding, since it can be suppressed by an excess of unmodified oligonucleotide. EcoP15I DNA Mtase exhibited Michaelis-Menten kinetics with both unmodified and bromodeoxyuridine-substituted DNA, with a higher specificity constant for the latter. Furthermore, gel mobility shift assays showed that proteolyzed EcoP15I DNA Mtase formed a specific complex with DNA, which had similar mobility as the native protein-DNA complex. Taken together these results form the basis for a detailed structure-function analysis of EcoP15I DNA Mtase.
Ahmad
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I
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Rao
D N
DN
D001483:Base Sequence; D002413:Cations, Divalent; D004247:DNA / Q000378:metabolism; D015263:Deoxyribonucleases, Type III Site-Specific / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010777:Photochemistry; D011485:Protein Binding; D012436:S-Adenosylmethionine / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*
D002413:Cations, Divalent; D009838:Oligodeoxyribonucleotides; D012436:S-Adenosylmethionine; D004247:DNA; C089712:DNA modification methylase EcoP15I; D008780:Methyltransferases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C030432:endodeoxyribonuclease EcoP1; D015263:Deoxyribonucleases, Type III Site-Specific