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Figure 7. One molecule of LigIII binds two molecules of DNA in the bridging complex. (A) The DNA binding activity of LigIII was monitored by fluorescence anisotropy of a labeled DNA under conditions of stoichiometric binding. A 5′ FITC labeled duplex (800 nM) containing a three nucleotide overhang that cannot pair with itself was titrated with increasing concentrations of LigIII and the change in fluorescence anisotropy was measured. The DNA concentration in this assay is ∼5-fold higher than the apparent equilibrium binding constant (Kapp ∼ 154 nM) determined with limiting concentrations of DNA and LigIII in excess. The measured binding stoichiometry of 1:1 for the LigIII:DNA complex was additionally confirmed by repeating the titration experiment at a higher concentration of DNA (1600 nM; not shown). (B) A binding stoichiometry of 1:2 (ratio = 0.5) was measured for LigIII in complex with a 5′ FITC labeled, blunt-ended DNA duplex (800 nM) that can form the DNA bridging complex. The same binding stoichiometry was determined in a binding reaction containing a higher concentration of the DNA (1600 nM; not shown). Image published in: Kukshal V et al. (2015) © The Author(s) 2015. Creative Commons Attribution license Larger Image Printer Friendly View

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