Measurement of DNA-protein equilibria using gel chromatography: application to the HinfI restriction endonuclease.
A method is described for measuring equilibrium constants of DNA-protein interactions using gel chromatography. This technique has been used to study the sequence-specific interaction of the HinfI restriction endonuclease with DNA. HinfI has a monomeric molecular weight of 31000 and exists as a dimer in its active form. The protein binds to supercoiled DNA molecules containing its recognition site with an apparent free energy of -13.9 kcal/mol of sites. This interaction is highly salt sensitive and causes a release of 3.4 ion pairs. The affinity of the nuclease for its recognition site is largely independent of both pH (6.5-8.5) and temperature (7-35 degrees C) and was not affected by variations in the degenerate middle position of the site. Linear DNA fragments containing the HinfI recognition site were bound as tightly as supercoiled molecules. Binding to nonspecific DNA sites or to methylated DNA sites was approximately 6 orders of magnitude weaker. In general, enzyme activity and binding affinity paralleled each other.
Frankel
A D
AD
Ackers
G K
GK
Smith
H O
HO
D002850:Chromatography, Gel / Q000379:methods; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010957:Plasmids; D011485:Protein Binding
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D046911:Macromolecular Substances; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases