Single-Molecule Studies Of Restriction Endonuclease Kinetics

Under optimal conditions, restriction endonucleases are capable of mediating remarkably specific DNA cleavage. This quality makes the restriction endonuclease an indispensible tool for genetic modification and manipulation. However, the mechanism by which restriction endonucleases effectively discriminate between their cognate site and other DNA sequences is not fully understood. Under certain conditions, many restriction endonucleases display “star activity” - relaxed specificity resulting in DNA cleavage at sequences that differ from their normal recognition sequence - but the mechanism by which specificity is relaxed is not fully understood. Although at least 600 of the almost 4000 restriction endonucleases that have been identified are commercially available in purified form, DNA cleavage kinetics of only a few of these enzymes have been studied in detail. We have developed a fluorescence-based approach with which we can track the progress of the cleavage reaction in real time, and simultaneously determine the values of the kinetic constants for a particular restriction endonuclease at a specific sequence. Modeling restriction endonuclease-mediated DNA cleavage as a Michaelis-Menten-like process, we expected reaction rates to display a hyperbolic dependence on substrate concentration, but our measurements deviate from this dependence, especially under conditions associated with increased star activity (such as low ionic strength). These observations suggest that substrate inhibition may be a part of the reaction mechanism under normal conditions, and that star activity may be a result of an increase in the population of this pathway. Using high density arrays of femtoliter-sized reaction vessels created by selectively etching bundled optical fibers, we can observe the cleavage activity of hundreds of individual restriction endonuclease molecules in solution. By characterizing the population distribution of single-enzyme turnover rates under a variety of conditions, we hope to gain insight into the reaction mechanisms of both specific cleavage and star activity.