Real-time observation of RecA filament nucleation and dynamics

Abstract

The RecA protein helps maintain genomic integrity through recombination. Using new single-mol. fluorescence assays and hidden Markov modeling, we first show the most direct evidence that a RecA filament grows and shrinks primarily one monomer at a time and only at the extremities. Both ends grow and shrink, contrary to expectation, but a higher binding rate at one end is responsible for directional filament growth. Next, we est. that about five monomers are sufficient for filament nucleation. We further find that the nucleation is likely to occur through the binding of a preassembled oligomer rather than the simultaneous binding of several monomers, using a vesicle encapsulation assay. Finally, although ordinarily SSB prevents filament nucleation, single RecA monomers can easily be added to an existing filament and displace SSB from DNA at the rate of filament extension. This supports the proposal for a passive role of RecA-loading machineries in SSB removal.