Direct observation of complexes of ssb and recA proteins with a fluorescent single-stranded deoxyribonucleic acid derivative.

Evidence is presented from fluorescence and kinetic experiments that ssb binds to a recA-ssDNA-ATP complex causing a major structural change in which some 40% of the bound recA is released. On addition of ssb to recA-epsilon DNA-ATP (containing the fluorescent analogue of ssDNA epsilon DNA), there is a slow first-order decrease in fluorescence (t1/2 approximately 3 min). This is accompanied by a loss in the ATPase activity of recA protein. The resultant complex does not exchange epsilon DNA for added ssDNA. Measurement of the DNA-stimulated ATPase activity on addition of excess ssDNA reveals that 40% of the previously bound recA has been released. The stoichiometry of recA bound to epsilon DNA thus changes from 1 mol per six nucleotides to 1 per 10 on addition of ssb. Formation of the ssb-recA-epsilon DNA complex is dependent on ATP, and the rate varies with the concentration of ssb.