Roles of Tyr103 and Tyr264 in the regulation of RecA-DNA interactions by nucleotide cofactors.

The DNA-binding mode of the RecA protein, in particular its dependence on nucleotide cofactor, has been investigated by monitoring the fluorescence and linear-dichroism signals of a tryptophan residue inserted in the RecA to replace tyrosine at position 103 or 264. These residues are important for cofactor and DNA binding, as evidenced from their fluorescence changes upon binding of cofactor and DNA [Morimatsu, K., Horii, T. & Takahashi, M. (1995) Eur. J. Biochem. 228, 779-785]. The substitution of these residues with tryptophan does not affect the structure or biological function of the complex and can therefore be exploited to gain structural information in terms of the orientation and environment of the inserted reporter chromophore. The fluorescence change upon formation of the ternary cofactor.RecA. DNA complex was much smaller than the sum of the changes induced by cofactor or DNA alone. This difference indicates that the cofactor and DNA interact with RecA via common components. The fluorescence change caused by DNA in the presence of cofactor was almost independent of the base composition of DNA, in contrast to the interaction in the absence of cofactor. Hence, the contact mode between the selected residues and DNA in the complex may depend significantly on the cofactor. Linear-dichroism measurements indicate that the cofactor does not markedly alter the organization of RecA filament. Linear dichroism shows that neither the aromatic moiety of residue 103 nor that of residue 264 is intercalated between the DNA bases. The textural changes reported for the helical pitch and contour length of RecA fiber upon interaction with cofactor and DNA may derive from a subtle change in orientation of the RecA subunits in the filament.