Kinetics of presynaptic filament assembly in the presence of SSB and mediator proteins

Abstract

Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and DSB repair. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on ssDNA, a process that is regulated by ssDNA‐binding (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate‐limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. Here we present a detailed kinetic analysis of presynaptic filament assembly using the well‐defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator). We demonstrate that the ATP‐dependent assembly of UvsX presynaptic filaments on Gp32‐covered ssDNA is limited by a salt‐sensitive nucleation step in the absence of mediator. Mediator protein UvsY selectively enhances filament nucleation, allowing UvsX assembly and Gp32 displacement to occur at higher salt concentrations. UvsY promotes filament nucleation by selectively stabilizing the pre‐nucleation complex that forms prior to a slow isomerization step. These and other results provide a rigorous kinetic model for presynaptic filament assembly involving all three major protein components—recombinase, SSB, and mediator, on native ssDNA.