The run-on oligomer filament enzyme mechanism of SgrAI: Part 2. Kinetic modeling of the full DNA cleavage pathway

摘要

Filament or run-on oligomer formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms run-on oligomeric filaments with activated DNA cleavage activity and altered DNA sequence specificity. In this two-part work, we measure individual steps in the run-on oligomer filament mechanism to address specific questions of cooperativity, trapping, filament growth mechanisms, and sequestration of activity using fluorophore-labeled DNA, kinetic FRET measurements, and reaction modeling with global data fitting. The final models and rate constants show that the assembly step involving association of SgrAI–DNA complexes into the run-on oligomer filament is relatively slow (3–4 orders of magnitude slower than diffusion limited) and rate-limiting at low to moderate concentrations of SgrAI–DNA. The disassembly step involving dissociation of complexes of SgrAI–DNA from each other in the run-on oligomer filament is the next slowest step but is fast enough to limit the residence time of any one copy of SgrAI or DNA within the dynamic filament. Further, the rate constant for DNA cleavage is found to be 4 orders of magnitude faster in the run-on oligomer filament than in isolated SgrAI–DNA complexes and faster than dissociation of SgrAI–DNA complexes from the run-on oligomer filament, making the reaction efficient in that each association into the filament likely leads to DNA cleavage before filament dissociation.