Colloquium: “Freezing with force: Direct observation of type IA topoisomerase gate dynamics” — Wednesday, December 5, 2018, 4:00 PM

Dr. K. Maria Mills,
Laboratory of Molecular Biophysics, NIH
George P. Williams, Jr. Lecture Hall, (Olin 101)
Wednesday, December 5, 2018, at 4:00 PM

There will be a reception with refreshments at 3:30 PM in the lounge. All interested persons are cordially invited to attend.


Type IA topoisomerases cleave single-stranded DNA and relieve topological strain by passing an intact DNA strand through the broken strand. Although it is assumed that these enzymes accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule magnetic tweezers assay to directly measure gate opening of the E. coli type IA topoisomerases, topo I and topo III. Following cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. The force-dependent kinetics reveal that the gate dynamics of these enzymes are remarkably fast. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes associated with gate-opening obtained from molecular dynamics simulations. These results allow us to develop a complete mechanistic model of type IA topoisomerase-ssDNA interactions.