EcoRV restriction endonuclease binds all DNA sequences with equal affinity.
In the presence of MgCl2, the EcoRV restriction endonuclease cleaves its recognition sequence on DNA at least a million times more readily than any other sequence. In this study, the binding of the EcoRV restriction enzyme to DNA was examined in the absence of Mg2+. With each DNA fragment tested, several DNA-protein complexes were detected by electrophoresis through polyacrylamide. No differences were observed between isogenic DNA molecules that either contained or lacked the EcoRV recognition site. The number of complexes with each fragment varied with the length of the DNA. Three complexes were formed with a DNA molecule of 55 base pairs, corresponding to the DNA bound to 1, 2, or 3 molecules of the protein, while greater than 15 complexes were formed with a DNA of 381 base pairs. A new method was developed to analyze the binding of a protein to multiple sites on DNA. The method showed that the EcoRV enzyme binds to all DNA sequences, including the EcoRV recognition site, with the same equilibrium constant, though two molecules of the protein bind preferentially to adjacent sites on the DNA in a cooperative fashion. All of the complexes with a substrate that contained the EcoRV site dissociated upon addition of competitor DNA, but when the competitor was mixed with MgCl2, a fraction of the substrate was cleaved at the EcoRV site. The fraction cleaved was due mainly to the translocation of the enzyme from nonspecific sites on the DNA to the specific site.
Taylor
J D
JD
Department of Biochemistry, University of Bristol, U.K.
Badcoe
I G
IG
Clarke
A R
AR
Halford
S E
SE
D001667:Binding, Competitive; D002021:Buffers; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D004492:Edetic Acid; D004587:Electrophoresis, Agar Gel; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity / Q000187:drug effects
D002021:Buffers; D004268:DNA-Binding Proteins; D015636:Magnesium Chloride; D004247:DNA; D004492:Edetic Acid; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D008274:Magnesium