Single-Molecule Magnetic Tweezer Analysis of Topoisomerases.
Methods Mol Biol
; 1703: 139-152, 2018.
Article
en En
| MEDLINE
| ID: mdl-29177739
ABSTRACT
Magnetic tweezers (MT) provide a powerful single-molecule approach to study the mechanism of topoisomerases, giving the experimenter the ability to change and read out DNA topology in real time. By using diverse DNA substrates, one can study different aspects of topoisomerase function and arrive at a better mechanistic understanding of these fascinating enzymes. Here we describe methods for the creation of three different DNA substrates used in MT experiments with topoisomerases double-stranded DNA (dsDNA) tethers, "braided" (intertwined or catenated) DNA tether pairs, and dsDNA tethers with single-stranded DNA (ssDNA) regions. Additionally, we discuss how to build flow cells for bright-field MT microscopy, as well as how to noncovalently attach anti-digoxigenin to the coverslip surface for tethering digoxigenin-labeled DNAs. Finally, we describe procedures for the identification of a suitable DNA substrate for MT study and data collection.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN-Topoisomerasas de Tipo I
/
Imagen Individual de Molécula
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2018
Tipo del documento:
Article
País de afiliación:
Estados Unidos