Characterization of a thermostable archaeal polynucleotide kinase homologous to human Clp1

Effect of oligo(dA) chain length on PhoClp1 kinase activity. Reaction mixtures (10 μL) containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 500 μM [γ³²P]ATP, 5 μM each of 5′-OH oligodeoxyadenylates d(A)₄₀, d(A)₃₀, and d(A)₂₀, 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 r(A)_n substrate. The products were analyzed by denaturing PAGE and visualized by autoradiography (A). The extents of DNA and RNA 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.