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Fluorescence resonance energy transfer (FRET) based methods to study DNA topology and topoisomerases
Author(s) -
Leng Fenfei
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.775.6
Subject(s) - förster resonance energy transfer , dna supercoil , topoisomerase , dna , chemistry , dna gyrase , dna sequencing , fluorescence , biophysics , topology (electrical circuits) , biology , biochemistry , gene , physics , dna replication , mathematics , escherichia coli , quantum mechanics , combinatorics
Previously, we reported the synthesis of a type of unique fluorescence‐labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET) (Gu et al, Scientific Reports , 2016, 6:36006). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 or FL924 carrying a 42 nt. AT sequence with fluorescein and dabcyl or TAMRA and BHQ2 labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905 or pAB1_FL924. Since the fluorescence intensity of pAB1_FL905 and pAB1_FL924 is dependent on its supercoiling status, these DNA molecules are powerful tools to study DNA topology and topoisomerases by FRET. Recently, we are able to use these unique plasmid DNA molecules to study kinetics of different DNA topoisomerases and find that all DNA topoisomerases follow the classical Michaelis‐Menten kinetics and have unique steady‐state kinetic parameters, K M , V max , and k cat . We also developed high throughput screening (HTS) assays to identify inhibitors/activators targeting different DNA topoisomerases including bacterial DNA gyrase and topoisomerase I. Support or Funding Information 1R21AI125973‐01A1 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .