Studying RecA Homology Search Mechanism using Single-Molecule Methods

Abstract

RecA recombinase plays an important role in repairing the double-strand DNA breaks in E. coli. To repair damaged DNA, RecA first forms a nucleoprotein filament on a single-stranded DNA molecule, this nucleoprotein filament then searches and pairs the homology duplex DNA, and finally carries out the strand exchange reaction to initiate the homologous recombinational repair. To ensure that the repair process is carried out within the limited timescale set by the cell cycle of E. coli, it is important that RecA nucleoprotein filament locates its homologous sequence efficiently. Several search mechanisms have been proposed: a one-dimensional sliding (1D), a three-dimensional hopping (3D), and a facilitated intersegment transfer model. We have used two single-molecule imaging techniques to investigate the homology search mechanism of RecA nucleoprotein filaments. By applying a lateral force of a few pN to a bead-tethered dsDNA, the dsDNA is extended horizontally, and the diffusion trajectories of the labeled RecA nucleoprotein filaments (labeled with a bead or quantum dot) can be visualized at the single-molecule level. From individual searching events, the diffusion coefficient of the RecA nucleoprotein filament is determined to be ∼ 106 nm2/s. The salt dependence of the diffusion coefficients from 0 to 200 mM NaCl allows us to distinguish its major search mechanism.