Protein interactions in genetic recombination in Escherichia coli. Interactions involving RecO and RecR overcome the inhibition of RecA by single-stranded DNA-binding protein.
RecA promotes homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA). This reaction occurs inefficiently if the ssDNA substrate is preincubated with Escherichia coli ssDNA-binding protein (SSB). However, RecO and RecR can act together as accessory factors for RecA to overcome this inhibition by SSB (Umezu, K., Chi, N.-W., and Kolodner, R. D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3875-3879). To elucidate the mechanism that underlies this process, we examined protein-protein interactions between RecA, RecF, RecO, RecR, and SSB, and characterized the structure and activity of the ssDNA complexes formed with different combinations of these proteins. We obtained the following results. (i) RecO physically interacts with both RecR and SSB. The interaction between RecO and SSB is stronger than the RecO-RecR interaction. (ii) RecO and RecR do not remove SSB from SSB.ssDNA complexes, but instead bind to these complexes. The resulting RecO.RecR.SSB.ssDNA complexes were more active in RecA-mediated joint molecule formation than were SSB.ssDNA complexes. (iii) RecA can nucleate on the RecO.RecR.SSB.ssDNA complexes more efficiently than on SSB.ssDNA complexes. (iv) When RecA presynaptic filaments were formed in the presence of SSB, RecO, and RecR, the protein-DNA complexes obtained contained 70% of the amount of RecA required to saturate ssDNA. These complexes, however, can mediate joint molecule formation and strand exchange as efficiently as presynaptic filaments which are fully saturated with RecA. Based on these results, we propose dual roles for RecO and RecR in joint molecule formation. First, RecO and RecR bind to SSB.ssDNA complexes and modify their structure to allow RecA to nucleate on them efficiently. Second, RecO and RecR are retained in RecA presynaptic filaments and play a role in the subsequent homologous pairing process promoted by RecA.
Umezu
K
K
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Kolodner
R D
RD
D001426:Bacterial Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D011938:Rec A Recombinases / Q000037:antagonists & inhibitors*; D011995:Recombination, Genetic; D013395:Sucrose; D013569:Synapses / Q000378:metabolism
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C081006:RecO protein, E coli; C489318:RecR protein, E coli; C065831:RecR protein, Bacteria; D013395:Sucrose; D011938:Rec A Recombinases