Description:

Title: The Mechanism of type IA Topoisomerases: From Structural to Single Molecule Studies

Speaker: Alfonso Mondragón, Northwestern University, Molecular Biosciences

Abstract: Topoisomerase I is an important E. coli enzyme that regulates the topology of DNA by removing negative supercoils and catenating/decatenating DNA molecules. Previously we solved the atomic structure of the domain responsible for the DNA strand passage reaction and proposed a mechanism of action for this type of enzymes that explains many of the most salient features of the reaction. To extend and complement our structural work, we used single molecule Magnetic Tweezers studies to reveal that the presence of pauses in between relaxation events is a major feature of the mechanism of topological transformations. In order to determine the origin of these pauses, we recently built a combined Magnetic Tweezers-Total Internal Reflection Fluorescence microscope which can simultaneously report on the topology of DNA and conformation changes of the protein. Using Protein Induced Fluorescent Enhancement we determined that topoisomerase I remains bound during relaxation pauses. Additionally, we observed short bursts of greater fluorescence enhancement correlated to relaxation events, indicating a conformational change in the protein that moves the fluorophore closer to the protein during the relaxation process. We observed more bursts than relaxation events, revealing that not every protein conformational change results in a relaxation event. We infer that the enzyme is constantly changing conformation and attempting to change the topology of DNA, but only succeeds in a fraction of the attempts. The mechanism can be described as a series of attempts to pass one DNA strand through a break in another strand, which generates the pauses, culminating in a successful relaxation event. The experiments demonstrate that conformational changes in a molecular motor can occur with more frequency than successful mechanical steps.

Host: Marko

Speaker Schedule

Keywords: Physics, Astronomy, colloquium