Inhibition of the restriction endonuclease BanII using modified DNA substrates. Determination of phosphate residues critical for the formation of an active enzyme-DNA complex

• Published in: The Journal of biological chemistry Vol. 265; no. 24; pp. 14389 - 14394
• Main Authors: OLSEN, D. B, KOTZOREK, G, SAYERS, J. R, ECKSTEIN, F
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-08-1990
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Coliphages - genetics; Deoxyribonucleases, Type II Site-Specific - antagonists & inhibitors; DNA, Viral - genetics; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Hydrolysis; Kinetics; Molecular Sequence Data; Mutation; Oligonucleotide Probes - chemical synthesis; Substrate Specificity; Substrate; Enzymatic activity; Enzyme; DNA; Site directed mutagenesis; Restriction endonuclease; Inhibition
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)77314-6

The restriction endonuclease BanII catalyzes the cleavage of double-stranded DNA and recognizes the degenerate sequence 5'-GPuGCPyC-3'.
The poly-linker of M13mp18 contains one such sequence, 5'-GAGCTC-3'. The three other possible sites recognized by the enzyme
were prepared by site-directed muta-genesis. The substitution of phosphate groups by phosphorothioate residues at some positions
within the various recognition sites had relatively little effect on the rate of cleavage of the DNA. However, when the DNA
contained a phosphorothioate group at the site of cleavage the rate of linearization of the DNA was decreased by a factor
of 9. Interestingly, DNA which contained an additional phosphorothioate internucleotidic linkage immediately 3'-outside the
recognition site could not be linearized by the enzyme. The results indicate that an important contact between enzyme and
substrate is perturbed by the presence of the sulfur atom at this position.