ROLES OF TYR103 AND TYR264 IN THE REGULATION OF RECA-DNA INTERACTIONSBY NUCLEOTIDE COFACTORS

The DNA-binding mode of the RecA protein, in particular its dependence on nucleotide cofactor, has been investigated by monitoring the fluor escence 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 f luorescence changes upon binding of cofactor and DNA [Morimatsu, K., H orii, T, & Takahashi, M. (1995) Eur. J. Biochem. 228, 779-785]. The su bstitution of these residues with tryptophan does not affect the struc ture or biological function of the complex and can therefore be exploi ted to gain structural information in terms of the orientation and env ironment 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 p resence 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 measuremen ts indicate that the cofactor does not markedly alter the organization of RecA filament. Linear dichroism shows that neither the aromatic mo iety of residue 103 nor that of residue 264 is intercalated between th e DNA bases. The textural changes reported for the helical pitch and c ontour length of RecA fiber upon interaction with cofactor and DNA may derive from a subtle change in orientation of the RecA subunits in th e filament.