| Abstract: | RecA protein requires ATP and its hydrolysis to ADP to complete the DNA strand-exchange reaction. We investigated how the nucleotides activate RecA by examining their effect on urea-induced unfolding, which could reflect domain-domain contact of protein. RecA is folded into three continuous domains: the N-terminal, central and C-terminal domains. The fluorescence of tyrosine residues, which lie mainly in the central domain, was modified in 1-3 M urea, while the red shift of fluorescence peak of the tryptophan residues located in the C-terminal domain occurred only in 3-6 M urea. Thus, the C-terminal domain of RecA is unfolded after the central part unfolds. The change in intensity of tryptophan fluorescence without a large shift in the peak at low concentrations of urea suggests that there are weak interactions between the central and C-terminal domains. This is supported by our observation that RecA protein lacking the C-terminal tail unfolded at lower concentrations of urea than the entire RecA, and with clear transitions, unlike the entire RecA. ATP and its unhydrolyzable analog (ATPgammaS), which enhance the binding of RecA to DNA, facilitated the urea-induced change in RecA tryptophan fluorescence, while ADP, an antagonist of ATP, prevented the change. ATP probably weakens the domain-domain contact and facilitates the DNA binding, while ADP stabilizes the contact and inhibits it. Supporting this conclusion, the binding of RecA lacking the C-terminal tail to DNA was not inhibited by ADP, while that of the intact RecA was. |
