TIRF-Based Single-Molecule Detection of the RecA Presynaptic Filament Dynamics

RecA is a key protein in homologous DNA repair process. On a single-stranded (ss) DNA, which appears as an intermediate structure at a double-strand break site, RecA forms a kilobase-long presynaptic filament that mediates homology search and strand exchange reaction. RecA requires adenosine triphosphate as a cofactor that confers dynamic features to the filament such as nucleation, end-dependent growth and disassembly, scaffold shift along the ssDNA, and conformational change. Due to the complexity of the dynamics, detailed molecular mechanisms of functioning presynaptic filament have been characterized only recently after the advent of single-molecule techniques that allowed real-time observation of each kinetic process. In this chapter, single-molecule fluorescence resonance energy transfer assays, which revealed detailed molecular pictures of the presynaptic filament dynamics, will be discussed.