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

Abstract

Evidence is presented from fluorescence and kinetic experiments that ssb binds to a recA-ss[single-stranded]DNA-ATP complex [from Escherichia coli], 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 .apprx. 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 was released. The stoichiometry of recA bound to .epsilon.DNA changes from 1 mol/6 nucleotides to 1/10 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.