Interaction of Ada protein with DNA examined by fluorescence anisotropy of the protein.
We made use of enhancement of fluorescence anisotropy of protein upon DNA binding to analyze interactions between Ada protein and DNA. Ada protein is a DNA repair enzyme that also acts as a transcription regulator. The isotropic fluorescence was not significantly affected upon interaction with DNA and could not be used as a signal for detection of the binding. The anisotropy did became larger because the binding to DNA reduces diffusion of the protein. The change was reproducible and independent of protein concentration and also independent of the degree of saturation of DNA with the protein when DNA was large; these values can readily be converted to the proportion of the complexed protein. The binding parameters were then determined by direct comparison between experimental and theoretical variations of anisotropy, with increasing concentrations of DNA. The theoretical variations were computed by considering the overlap of potential binding sites on the DNA lattice [McGhee & von Hippel (1974) J. Mol. Biol. 86, 469-489]. Binding does not seem to occur in a cooperative manner. The number of base pairs covered by a protein monomer was 7 +/- 1; this number is independent of the salt concentration. The equilibrium association constant decreased from 4 X 10(7) to 3 X 10(5) M-1 for an increase of NaCl concentration from 0.1 to 0.2 M, thereby indicating the possible involvement of ionic interactions between phosphate groups of DNA and the protein.
Takahashi
M
M
Groupe de Cancérogénèse et de Mutagénèse Moléculaire et Structurale, CNRS, Strasbourg, France.
Sakumi
K
K
Sekiguchi
M
M
D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D005454:Fluorescence Polarization; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase