Single turnovers of the EcoRI restriction endonuclease.
Single turnovers of the EcoRI restriction endonuclease, cleaving its recognition site on the covalently closed form of plasmid pMB9, were examined. Two methods were used to monitor the progress of the reactions: one involved quenching the reaction at various times followed by the electrophoretic separation of the products cleaved in one and in both strands of the duplex; the other employed a stopped-flow fluorimeter to measure the amount of ethidium bromide bound to the DNA as it changes when the DNA, cleaved in at least one strand, dissociates from the enzyme. Two procedures were used to initiate the reactions. For some, one solution containing the enzyme was mixed with a second containing both DNA and MgCl2: in these reactions, the fluorescence changed at the same rate as the cleavage of the first strand of the duplex. Other reactions were started by the addition of MgCl2 to a pre-equilibrium of enzyme and DNA: here, both strands of the DNA were cleaved faster than before, with the fluorescence signal now occurring at the same time as the cleavage of the second strand. The different kinetics from the two assays and the two mixing procedures are consistent with the rates of these reactions being controlled by protein conformational changes. These may affect either one subunit alone within the dimeric EcoRI enzyme, allowing the enzyme to cleave only one strand of the DNA in each turnover. Alternatively, both subunits of the dimer may change, so that the enzyme then cleaves both strands during the life-time of one enzyme-DNA complex.
D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI; D003851:Deoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D015636:Magnesium Chloride; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence
D046911:Macromolecular Substances; D015636:Magnesium Chloride; D004247:DNA; D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D008274:Magnesium