Atypical DNA-binding properties of class-IIS restriction endonucleases : evidence for recognition of the cognate sequence by a FokI monomer

• Published in: Gene Vol. 125; no. 1; pp. 1 - 10
• Main Authors: SKOWRON, P, KACZOROWSKI, T, TUCHOLSKI, J, POHDAJSKA, A. J
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier 15.03.1993; Amsterdam; New York, NY
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA - metabolism; DNA-Binding Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Flavobacterium - enzymology; Flavobacterium okeanokoites; Fundamental and applied biological sciences. Psychology; Hydrolases; Kinetics; Magnesium - physiology; Methylation; Sequence specificity; Enzyme; DNA binding protein; Restriction endonuclease; Molecular interaction; Molecular complex; Binding site; Cooperative phenomenon
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(93)90738-O

The DNA-binding properties of the FokI restriction endonuclease were studied using the gel-mobility-shift assay. Specific recognition of the cognate sequence and cleavage of DNA are distinguishable functions and can be separated. FokI binds to its recognition site predominantly as a monomer. At high concentrations, FokI exhibits a cooperative recognition sequence-dependent aggregation. In 20 mM KCl/10 mM Tris.HCl buffer, the binding constant of FokI to its cognate site is equal 6.0-7.9 x 10(8)/mol and is lower than the values for most gene-regulatory proteins. FokI binding is 600-1500 times weaker to non-cognate double-stranded DNA than to the GGATG site, and 30,000 times weaker to single-stranded DNA or tRNA. The method of Bading [Nucleic Acids Res. 16 (1988) 5241-5248], used for determining the stoichiometry of protein bound to DNA by gel-mobility-shift assay, is extended.