Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching

Authors

Yunke Wang, Biomolecular Sciences Institute, Department of Chemistry & Biochemistry, and Enviromental and Occupational Health, Robert Stempel College of Public Health & Social Work, Florida International UniversityFollow
Samantha Rakela, Biomolecular Sciences Institute, Department of Chemistry & Biochemistry, and Enviromental and Occupational Health, Robert Stempel College of Public Health & Social Work, Florida International University
Jeremy W. Chambers, Biomolecular Sciences Institute, Department of Chemistry & Biochemistry, and Enviromental and Occupational Health, Robert Stempel College of Public Health & Social Work, Florida International University
Zi-Chun Hua, School of Life Sciences, Nanjing University
Mark T. Muller, TopoGEN, Inc.
John L. Nitiss, Pharmaceutical Sciences Department, College of Pharmacy at Rockford, University of Illinois at Chicago
Yuk-Ching Tse-Dinh, Biomolecular Sciences Institute, Department of Chemistry & Biochemistry, and Enviromental and Occupational Health, Robert Stempel College of Public Health & Social Work, Florida International University, Miami, Florida 33199, United States.
Fenfei Leng, Biomolecular Sciences Institute, Department of Chemistry & Biochemistry, and Enviromental and Occupational Health, Robert Stempel College of Public Health & Social Work, Florida International University

Date of this Version

11-5-2019

Document Type

Article

Rights

default

Abstract

DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes ( and DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes ( DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, , , and . We found that for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5'-triphosphate binding to DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors.

DOI

10.1021/acsomega.9b02676

Identifier

31720544

Comments

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Recommended Citation

Wang Y, Rakela S, Chambers JW, Hua ZC, Muller MT, Nitiss JL, Tse-Dinh YC, Leng F. Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching. ACS Omega. 2019 Oct 24;4(19):18413-18422. doi: 10.1021/acsomega.9b02676. PMID: 31720544; PMCID: PMC6844113.