Effect of DNA length. Reaction mixtures (10 μL) containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 500 μM [γ32P]ATP, 5 μM each of 24-mer, 18-mer, and 12-mer 5′-OH DNA oligonucleotides with identical 5′-sequences (depicted at the bottom of A), and increasing amounts of PhoClp1 (as specified in B) were incubated for 15 min at 55°C. Control reactions contained 10 U of T4 Pnk plus the indicated DNA substrate. The products were analyzed by denaturing PAGE and visualized by autoradiography (A). The extents of DNA phosphorylation were quantified by scanning the gel and are plotted as a function of input PhoClp1 in B. Each datum is the average of three separate titration experiments. Error bars denote the standard deviation.
