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Nat Struct Mol Biol. 2018 Dec;25(12):1111-1118. doi: 10.1038/s41594-018-0158-x. Epub 2018 Nov 26.

Direct observation of topoisomerase IA gate dynamics.

Author information

1
Laboratory of Single Molecule Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
2
Biomolecular Sciences Institute, Florida International University, Miami, FL, USA.
3
Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.
4
Laboratory of Single Molecule Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA. neumankc@mail.nih.gov.

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.

PMID:
30478267
PMCID:
PMC6379066
DOI:
10.1038/s41594-018-0158-x
[Indexed for MEDLINE]
Free PMC Article

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