A RECOMBINATIONAL DEFECT IN THE C-TERMINAL DOMAIN OF ESCHERICHIA-COLIRECA2278-5 PROTEIN IS COMPENSATED BY PROTEIN-BINDING TO ATP

RecA2278-5 is a mutant RecA protein (RecAmut) bearing two amino acid s ubstitutions, Gly-278 to Thr and Val-275 to Phe, in the cy-helix H of the C-terminal subdomain of the protein. recA2278-5 mutant cells are u nusual in that they are thermosensitive for recombination but almost n ormal for DNA repair of UV damage and the SOS response. Biochemical an alysis of purified RecAmut protein revealed that its temperature sensi tivity is suppressed by prior binding of this protein to its ligand. I n fact, the preheating of RecAmut protein for several minutes at a res trictive temperature (42 degrees C) in the absence of ATP resulted in inhibition at 42 degrees C of many activities related to homologous re combination including ss- and dsDNA binding, high-affinity binding for ATP, ss- or dsDNA-dependent ATPase, RecA-RecA interaction, and strand transfer capability. The binary complex RecAmut::ATP under the same c onditions showed a decrease in only two activities, i.e. dsDNA binding and high-affinity binding for ATP. Besides ATP, sodium acetate (1.5 M ) was shown to be another factor that can stabilize the RecAmut protei n at 42 degrees C, judging by restoration of its DNA-free ATPase activ ity. The similarity of influence of high salt (with its non-specific b inding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C-termin al domain is one of the conditions for correct interaction between Rec A protein and ATP in the RecA::ATP::ssDNA presynaptic complex formatio n. The decrease in affinity for ATP was suggested to be the factor tha t determined a particular recombinational (but not repair) thermosensi tivity of the RecAmut protein. Finally, we show that the stability of C-terminal domain appeared to be necessary for the dsDNA-binding activ ity of the protein.