Partial Unwrapping of SSB from SsDNA Facilitates RecA Filament Formation

Abstract

DNA breaks can be repaired by homologous recombination, a process that maintains genetic stability in an organism. A protein that is essential for this mechanism in E. coli is RecA. During repair, RecA must bind and form nucleoprotein filaments on single-stranded DNA (ssDNA) in direct competition with single-stranded DNA binding protein (SSB). Despite extensive studies, the mechanism behind this competitive process remains unclear.Here, we use high-resolution optical tweezers with simultaneous fluorescence microscopy to observe directly the mechanical properties of ssDNA-SSB, ssDNA-RecA, and ssDNA-SSB-RecA complexes under a range of tensions. This single-molecule assay allows us to probe and visualize simultaneously the interactions of RecA and SSB with ssDNA in real time and with nanometer resolution. Using a 70-nucleotide, poly-dT ssDNA construct designed to accommodate a single SSB, we investigated different scenarios of protein-DNA complex formation under a range of tensions between 0-10 pN.Individual SSBs on their own bind and condense ssDNA in discrete steps, the size of which depend on tension. Under low tensions (1-3 pN), an SSB wraps 50-70 nt of ssDNA in a single step. At higher tensions (>4 pN), SSB exhibit transient, partially wrapped states on ssDNA. In the absence of SSB, RecA filaments nucleate rapidly on ssDNA regardless of tension. In contrast, when RecA is added to ssDNA coated with an SSB, filament formation is inhibited at low tensions. At higher tensions, where the SSB can partially unwrap from ssDNA, we observe RecA filaments form after an extended lag time. Our results suggest a mechanism in which SSBs inhibit RecA by sequestering ssDNA. Tension-induced unwrapping of the SSB makes ssDNA available for RecA to bind and nucleate a filament.