PAMS Seminar: "Force-based detection of sub-millisecond topoisomerase IA dynamics" by Dr. Maria Mills

Dr. Maria Mills
Department of Physics & Astronomy
University of Missouri

Abstract:
Single molecule fluorescence and manipulation techniques have enabled measurements of nanometer-scale changes in molecular conformations. These techniques have been particularly useful for the study of biological molecules. However, the timescales of many biomolecular motions remain a challenge for single molecule measurements. Many protein and nucleic acid conformational changes happen on the ns-µs timescale, while single molecule measurements that rely on camera-based detection are limited to ms-s timescales. We have developed a novel magnetic tweezers assay that uses an applied force to probe the fast conformational dynamics of type IA topoisomerases. Type IA topoisomerases bind and cleave single-stranded DNA and pass a second strand through the transient break. While it has long been assumed that these proteins must undergo a conformational change in order to pass this second strand, this gate opening had never been observed. By applying a force against closing, we were able to increase the lifetime of the enzyme’s open state and observe the opening and closing of a protein-mediated DNA gate. We found that following cleavage of single-stranded DNA, the gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. From the force-rate dependence, we calculated an average lifetime of the open state at equilibrium of ~500 µs. These experiments allowed us to delineate the full kinetic pathway of type IA topoisomerase-ssDNA kinetics.

This seminar will be in Kemper 206.

Free