Single-Molecule Tethered Particle Motion Studies on the DNA Recombinase Filament Assembly and Disassembly
Journal
Methods in Molecular Biology
Journal Volume
2281
Pages
135-149
Date Issued
2021
Author(s)
Abstract
Bacterial RecA and eukaryotic Rad51 are recombinases indispensable for DNA homologous recombination and repair of double-stranded DNA breaks. Understanding the functions and biophysical properties of the DNA recombinases benefits the research in human medicine such as cancer biology. Single-molecule techniques provide the mechanistic details of complex biological reactions. Tethered particle motion (TPM) experiment is a simple and multiplex single-molecule tool to monitor DNA-protein interactions. We have developed a single-molecule TPM assay to study DNA recombinase filament assembly and disassembly on individual DNA molecules in real time. Characterization of the temporal change of the Brownian motion of DNA tethers during recombinase assembly and disassembly in real time allows the determination of multiple kinetic parameters of nucleation rate, extension rate, dissociation rate, and length of the recombinase-DNA filament. ? 2021, Springer Science+Business Media, LLC, part of Springer Nature.
Subjects
DNA homologous recombination; Rad51; RecA; Single-molecule microscopy; SSB; Tethered particle motion
SDGs
Other Subjects
DNA; nucleoprotein; polystyrene; Rad51 protein; RecA protein; recombinase; single stranded DNA; controlled study; enzyme phosphorylation; homologous recombination; human; kinetic parameters; microscopy; protein assembly; protein DNA interaction; double st
Type
book part