title: Interaction of Tyr103 and Tyr264 of the RecA protein with DNA and nucleotide cofactors. Fluorescence study of engineered proteins.
abstract: To obtain structural insight on the interaction of the RecA protein with nucleotide cofactors (ATP and ADP) and DNA, we have made two engineered RecA proteins, in which either Tyr103 or Tyr264 was replaced with tryptophan. The fluorescence of tryptophan residues (two/subunit) of wild-type RecA is not significantly altered upon the binding of cofactor or DNA. Therefore, any detectable fluorescence change of the engineered proteins could be directly related to interaction with the particular inserted tryptophan residue. The fluorescence of Trp103 is almost completely quenched upon ADP binding, supporting a stacking interaction of adenine base of ADP with Tyr103. By contrast, with ATP the quenching of fluorescence of Trp103 is not complete (75%), possibly indicating that there is no stacking interaction with ATP. Such a difference could explain the antagonistic effects of ATP and ADP. Both nucleotides partially quench the fluorescence of Trp264 (about 70%), confirming that this residue is in the vicinity of the cofactor-binding site. The binding of ssDNA also decreases the fluorescence of both Trp103 and Trp264, the degree of quenching depending upon base composition and decreasing in the following order: poly(dT) > poly(dI) > M13 ssDNA > poly(dA). This order coincides with that of the binding affinities of these polynucleotides to RecA reported by Cazenave et al. [Cazenave, C., Chabbert, M., Toulmé, J. J. & Hélène, C. (1984) Biochim. Biophys. Acta 781, 7-13]. This correlation supports the finding that a region very close to Tyr103 interacts with DNA.
authors.affiliation: Department of Molecular Protozoology, Osaka University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Morimatsu
authors.forename: T
authors.initials: T
authors.lastname: Horii
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding; D015202:Protein Engineering; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D009838:Oligodeoxyribonucleotides; D014443:Tyrosine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases