Single-Molecule FRET Studies on Nucleoprotein Filament Dynamics of RecA E38K Mutant Protein

RecA plays a key role in homologous recombination pathway to repair double-stranded DNA break damage. In homologous recombination, the assembly of RecA onto the ssDNA to form nucleoprotein filaments includes a slow nucleation and a rapid extension step. The assembly of RecA-ssDNA nucleoprotein filaments is the key regulatory step for homologous recombination, and the assembled nucleoprotein filament is responsible for mediating DNA homology search and downstream strand exchange. The previous study showed that a recA strain surviving high doses of UV radiation includes a dominative E38K point mutation. Single-molecule biochemical studies showed that E38K mutants have faster kinetics in forming nucleoprotein filaments compared to wild-type RecA. In this study, we used a single-molecule fluorescence resonance energy transfer (smFRET) experiment and hidden Markov analysis to study the assembly of wild-type RecA and RecA E38K at different ssDNA length (for wild-type RecA, (dT)n, n = 19, 21, 23, 27; for RecA E38K, (dT)n, n = 9, 16-23). We found that RecA E38K has smaller nucleation unit and higher nucleation frequency at the same ssDNA length ((dT)n, n = 19, 21, 23) compared to wild-type RecA. We also showed that RecA E38K filament likely assemble and disassemble with one monomer at the 5′ end of nucleoprotein filament.