Investigation of binding between recA protein and single-stranded polynucleotides with the aid of a fluorescent deoxyribonucleic acid derivative

• Published in: Biochemistry (Easton) 1983-06, Vol.22 (12), p.2860-2866
• Main Authors: Silver, Marc S, Fersht, Alan R
• Format: Article
• Language: English
• Subjects: Adenosine - analogs & derivatives; Adenosine - metabolism; Adenosine Triphosphatases - metabolism; Animals; Bacterial Proteins - metabolism; Cattle; Cytidine - analogs & derivatives; Cytidine - metabolism; DNA; DNA, Single-Stranded - metabolism; Escherichia coli; fluorescence; Kinetics; Poly T - metabolism; poly(dT); Polydeoxyribonucleotides - metabolism; Protein Binding; Rec A Recombinases; recA gene protein; Spectrometry, Fluorescence; Thymus Gland
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00281a014

The availability of epsilon DNA, a fluorescent ssDNA derivative, has made it possible to examine quantitatively the interactions between recA protein and single-stranded polynucleotides. Fluorescence titrations of epsilon DNA with recA protein and vice versa establish that each recA protein monomer covers 5.5 epsilon DNA nucleotides and that the dissociation constant of the recA-epsilon DNA complex is 10 nM. Fluorescence titrations of recA protein-epsilon DNA mixtures with poly(dT) establish that each recA protein monomer covers 5.1 poly(dT) nucleotides and that the dissociation constant of the recA-poly(dT) complex is 0.03 nM. Observations on how the addition of ssDNA affects the fluorescence of recA protein-epsilon DNA mixtures establish that the dissociation constant of the recA-ssDNA complex exceeds 20 microM. Stopped-flow kinetics in which excess recA protein binds to epsilon DNA indicate that k2 = 6 X 10(6) M-1 s-1 for the process. A more approximate kinetic technique indicates that recA protein binds to epsilon DNA at least one-tenth as fast as to poly(dT); the rate constant for dissociation of recA-epsilon DNA exceeds that for recA-poly(dT) by at least 30-fold. epsilon DNA is proven to be a versatile reagent for studying single-stranded polynucleotide-protein interactions. Not only can its own complexes with protein be investigated but also, under suitable circumstances, it can be used as a fluorescent probe to explore complexes incorporating nonfluorescent polynucleotides.