1. Direct Observation of Topoisomerase IA Gate Dynamics
- Author
-
Yuk-Ching Tse-Dinh, Maria Mills, and Keir C. Neuman
- Subjects
0301 basic medicine ,DNA, Bacterial ,Models, Molecular ,Magnetic tweezers ,DNA, Single-Stranded ,Cleavage (embryo) ,Article ,03 medical and health sciences ,Molecular dynamics ,chemistry.chemical_compound ,Structural Biology ,Cleave ,Escherichia coli ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Topoisomerase ,030302 biochemistry & molecular biology ,Kinetics ,030104 developmental biology ,DNA Topoisomerases, Type I ,Biophysics ,biology.protein ,DNA supercoil ,Ligation ,AND gate ,DNA - Abstract
Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after 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. 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 obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA. Single-molecule magnetic tweezers analyses and supporting MD simulations provide evidence for protein-gate opening of type IA topoisomerases.
- Published
- 2018