The molecular role of the RecF protein in loading RecA protein onto single-stranded DNA (ssDNA)-binding proteincoated ssDNA has been obscured by the facility with which the RecO and RecR proteins alone perform this function. We now show that RecFOR and RecOR define distinct RecA loading functions that operate optimally in different contexts. RecFOR, but not RecOR, is most effective when RecF(R) is bound near an ssDNA/double-stranded (dsDNA) junction. However, RecF(R) has no enhanced binding affinity for such a junction. RecO and RecR proteins are both required under all conditions in which the RecFOR pathway operates. The RecOR pathway is uniquely distinguished by a required interaction between RecO protein and the ssDNA binding protein C terminus. The RecOR pathway is more efficient for RecA loading onto ssDNA when no proximal dsDNA is available. A merger of new and published results leads to a new model for RecFOR function.Recombination reactions catalyzed by the RecA protein form an integral part of DNA metabolism in Escherichia coli. The major role for recombination in bacteria is the repair of stalled replication forks (1-4). E. coli strains lacking intact recombination systems exhibit sensitivity to DNA damaging agents, hypermutability, and impaired growth rates (5).RecA is found in all eubacteria with the exception of a few endosymbionts (Buchnera sp.) (6 -8). It functions as a helical nucleoprotein filament, formation of which requires ATP and Mg 2ϩ ion. When bound to single-stranded DNA (ssDNA), 2RecA hydrolyzes ATP. The active filament can promote the exchange of DNA strands between homologous DNA molecules in vitro. RecA filament formation occurs in at least two distinct phases (9). Filament nucleation occurs first and involves binding of a few (probably 4 -5 (10)) RecA protomers to DNA. Nucleation is rate-limiting and is followed by rapid filament extension in the 5Ј to 3Ј direction along the DNA (11-14). RecA filament disassembly requires ATP hydrolysis and also occurs in the 5Ј to 3Ј direction along ssDNA (14). Recombinase function is highly regulated in all cells, and the regulation occurs at many levels. The LexA repressor protein controls expression of the recA gene (15, 16). The 17 C-terminal amino acids of RecA function in autoregulation of most protein activities (17-19). Finally, E. coli possesses many additional proteins that function as recombination regulators, with RecA as their target (20 -22). Most of these proteins affect the kinetics of filament formation and disassembly. Proteins that facilitate the formation of the filaments of RecA-class recombinases are called recombination mediator proteins (21). Recombination mediator proteins are as ubiquitous as are the recombinases themselves (21,(23)(24)(25)(26)(27)(28). The E. coli RecF, RecO, and RecR proteins are perhaps the prototypical recombination mediator proteins (12, 21, 29 -32), part of a larger network of bacterial proteins that regulate almost every aspect of RecA function (22,30,33).The ssDNA-binding protein (SSB; 18.2 kDa) ex...