Structure of DNA-RecA complexes studied by residue differential linear dichroism and fluorescence spectroscopy for a genetically engineered RecA protein.

The structure of complexes of RecA with double-stranded and single-stranded DNA was studied by linear dichroism spectroscopy, fluorescence quenching and fluorescence anisotropy measurements. One of the two tryptophan residues (Trp291) of RecA was replaced by genetic engineering for an ultraviolet light-transparent threonine. This modified RecA protein shows, within experimental errors, the same DNA-binding kinetics and stoichiometry as the wild-type protein and no significant variation with respect to in vivo repair function was observed between cells with the two protein forms. By comparing the dichroic and fluorescence properties of the wild-type versus the modified protein, when bound to DNA, information about orientation and environment of the Trp291 chromophore in the complex could be obtained. The indole chromophore of Trp291Z was found to be oriented with its pseudo-long axis tilted 61 degrees and the aromatic plane is tilted 27 degrees relative to the fibre axis. Trp291 shows low mobility within the protein and therefore the deduced orientation may be used as a "handle" on the protein at the construction of three-dimensional models of RecA-DNA complexes. Comparison with the orientation for this residue in the crystal structure of the RecA homopolymer fibre indicates no measurable reorientation of the C-terminal subdomain of RecA upon DNA binding. Whereas the accuracy of the orientation determination of tryptophan, in absolute terms, is rather poor, changes of its orientation can be detected with high precision. Thus, similar Trp291 orientations are obtained in the complexes with single-stranded and double-stranded DNA, indicating similar structures of the protein fibres. The fluorescence quenching results indicate that the protein region of Trp291 is not involved in the binding of DNA.