The effects of base analogue substitutions on the methylation by the EcoRI modification methylase of octadeoxyribonucleotides containing modified EcoRI recognition sequences.
We have examined the DNA-protein interactions involved in the recognition of a specific hexameric sequence, GAATTC, by the EcoRI modification methylase by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues 2-aminopurine, 5-bromocytosine, 5-bromouracil, 2,6-diaminopurine, hypoxanthine, 5-methylcytosine, N6-methyladenine, and uracil were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the ability of the enzyme to methylate the modified substrates were monitored by determining the steady state kinetic values of the reaction in the presence of the cosubstrate, S-adenosylmethionine. The substitutions resulted in effects ranging from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those analogues that were active, whereas the octanucleotides containing hypoxanthine at the guanine site, N6-methyladenine at the first or 2-aminopurine at the second adenine site, or uracil at the second thymine site were completely inactive. The results are discussed in terms of the possible interactions between the methylase and its recognition sequence. In addition, the interactions are compared to those of the EcoRI restriction endonuclease, which has been similarly tested with the same analogue oligonucleotides. The results of that study are reported in an accompanying paper. Although both enzymes recognize the same sequence, they do so in different ways.
D000225:Adenine; D001483:Base Sequence; D001665:Binding Sites; D003596:Cytosine; D004247:DNA / Q000032:analysis; D004262:DNA Restriction Enzymes / Q000494:pharmacology*; D015246:Deoxyribonuclease EcoRI; D006147:Guanine; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D013329:Structure-Activity Relationship; D013941:Thymine
D009838:Oligodeoxyribonucleotides; D006147:Guanine; D003596:Cytosine; D004247:DNA; C020098:DNA modification methylase EcoRI; D008780:Methyltransferases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D000225:Adenine; D013941:Thymine