The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease.
The kinetics of the cleavage of superhelical plasmid DNA (pBR322) by the restriction endonuclease, BamHI, have been analyzed in terms a compartmental model consistent with the chemistry first proposed by Rubin and Modrich (Rubin, R. A., and Modrich, P. (1978) Nucleic Acids Res. 5, 2991-2997) for analysis of the kinetics of the restriction endonuclease, EcoRI. The model was defined in terms of two compartments representing DNA substrate (bound and free), two compartments representing nicked intermediate (bound and free), one compartment representing linear product, and one compartment for free enzyme. A simultaneous analysis of concentration changes over time of the three DNA forms (superhelical, nicked, and linear) at six different enzyme concentrations was undertaken employing this compartmental model using SAAM (Simulation Analysis And Modeling) software. Results showed that rate constants characterizing the association of enzyme with superhelical DNA (6.0 x 10(5) M-1 s-1) and nicked DNA (2.8 x 10(5) M-1 s-1) were similar in magnitude and rate constants characterizing cleavage of the first (1.2 x 10(-2) s-1) and second phosphodiester bonds (3.1 x 10(-2) s-1) were also similar. The analysis yields a kinetically determined equilibrium constant of 12.9 nM for the dissociation of nicked intermediate from the enzyme. The rate constant describing the release of the nicked intermediate from the enzyme has a value of 3.7 x 10(-3) s-1. By comparing the value of this release rate constant to the value of the constant describing the second cleavage event, it can be determined that only 10% of the nicked intermediate bound to the enzyme is released as free nicked DNA and that 90% of the nicked intermediate is processed to the linear form without being released. Hence, most of the DNA is cleaved as the result of a single enzyme-DNA recognition event. No steady state assumptions were made in the analysis. The approach was to directly solve the differential equations which described the kinetic processes using an interactive method. This study demonstrates the usefulness of this approach for the analysis of kinetics of protein-DNA interactions for the restriction endonucleases.
Macromolecular Sciences Department, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939.
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P
Hensley
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Nardone
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JG
Chirikjian
M E
ME
Wastney
D004278:DNA, Superhelical / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010957:Plasmids; D013379:Substrate Specificity; D013997:Time Factors
D004278:DNA, Superhelical; D015245:Deoxyribonuclease BamHI