Figure 3
From: Structural and Biochemical Characterization of Poly-ADP-ribose Polymerase from Trypanosoma brucei
Selectivity, affinity and stoichiometry of DNA binding. (A) DNA templates used in activation and fluorescence polarization assays. (B) Activation of TBP-FL by DNA templates measured with the fluorescence based activity assay. Activated DNA (act. DNA) was used as a control. (C–G) DNA-binding affinities for TBP-FL, TBP-NW, TBP-N, TBP-WRA, and TBP-RA were measured with fluorescence polarization assay and are presented in panels C, D, E, F, and G, respectively. A representative curve for each measurement is shown. (H) Stoichiometry of TBP-FL binding to oligonucleotides 20, 21, 22, and 23 were measured by fluorescence polarization using saturating DNA concentration (250 nM). 1:1 binding is observed with ssDNA, while 2:1 protein:DNA binding occurs with dsDNA and hairpin DNA. (I) Stoichiometry of TBP-FL in low salt (50 mM NaCl) buffer and stoichiometries of TBP-NW and TBP-N constructs using DNA oligonucleotide 20.
