Restriction endonucleases are enzymes that cleave DNA. Some REases possess a mechanism by which they maneuver from one strand to another in order to cleave both nucleotide strands. Various models have been proposed as explanations for how REases manage to initially bind to a single strand then flip to the to the opposite strand in a single reaction. To test these various models, we developed an assay that utilizes Total Internal Reflection Fluorescence (TIRF) microscopy to visualize hundreds of individual REase cleavage events simultaneously. The resulting dwell-time distributions can be fitted with Gamma probability distributions which in turn provide insights into kinetic intermediate steps between the initial and final states. Through testing BcnI's effectiveness in environments with varying salt concentrations, analysis shows BcnI is significantly inhibited by low screening. Furthermore, analysis of Gamma fit parameters reveals there is a single rate-limiting step that overwhelmingly contributes to the overall reaction time for a single event in low screening environments. In the presence of high screening, BcnI is significantly more effective, and the presence of multiple significant rate-limiting steps was identified. The disparities in enzymatic activity and Gamma fit parameters indicate that BcnI's flipping mechanism is most accurately described by a tumbling model where the enzyme maintains non-covalent contacts throughout the protein flipping process.
"},{"label":"Degree","value":["Bachelor of Arts with Departmental Honors"]},{"label":"Administrative Note","value":"Drupal Node ID: 23786