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Nucleic Acids Research, 1980, Vol. 8, No. 11 2547-2560
© 1980

Articles

Binding of non-substrate nucleotides to a restriction endonuclease: a model for the interaction of Bam HI with its recognition sequence

Bernhard Hinsch, Hubert Mayer and Maria-Regina Kula

Gesellschaft für Biotechnologische Forschung mbH. Mascheroder Weg I, D-3300 Braunschweig-Stöckheim, GFR

Received March 3, 1980. The kinetic constants of the site-specific endonuclease Bam HI for various substrates were determined and binding of non-substrate nucleotides to the enzyme was studied. Agarose gel assays in combination with an integrated Michaelis-Menten equation were used for the evaluation of data. The turnover number was 2.2 min at 37°C with pJC80 ONA as the substrate. It depends on the conformation and base composition of the substrate. Michaelis constants also depend on substrate conformation. Non-substrate polynucleotides were found to inhibit Bam competitively with KI ranging from 10–6 to 10–6 M depending on base composition, base pairing, and helix conformation. Dinucleotides showed sequence-specific, competitive inhibition with KIs ranging from 10–5to >10–3 M. Mononucleotides and -nucleosides acted non-competitively. Binding was influenced by the extent of phosphorylation, but not by the nature of the base. KIs varied between 10–3 and 10–2 M. The results are discussed with respect to the recognition requirements of Bam HI.


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