A study on the mechanism of RecA in homologous recognition by using single molecule fluorescence tracking

Abstract

RecA plays an important role in homologous recognition in prokaryotes, and it has become a hot point in homologous recognition related research since its discovery. We establish an assay by combining total internal reflection fluorescence and flow stretching to visualize in real time the motion of single RecA-ssDNA filaments which are tagged with fluorescent beads. This enables us to study the interaction of RecA-ssDNA filaments with their templates in the homologous recognition process. It is found that the searching and binding is a short-time (τ=0.2 s) and short-distance (l=1.05 μm) process. Two distinguished motion modes for the RecA-ssDNA filament are observed, a Brownian motion and a directed motion. The observations suggest a model that a RecA-ssDNA filament just interacts weakly with the template DNA before it binds firmly to the template DNA. If no homologous site is found in a searching process, the filament drops off the template and repeats the searching process again until it finally finds its target.