Characterization of a thermostable archaeal polynucleotide kinase homologous to human Clp1

Effect of monovalent salts. (A) Reaction mixtures (10 μL) containing 50 mM Tris-acetate (pH 6.5), 10 mM MgCl₂, 500 μM [γ³²P]ATP, 5 μM 5′-OH d(A)₃₀ or r(A)₃₀ as specified, 0.9 pmol PhoClp1, and either no added salt or 250 mM NaCl as indicated were incubated for 15 min at 55°C. The products were analyzed by denaturing PAGE and visualized by autoradiography. (B) Reaction mixtures (10 μL) containing 50 mM Tris-acetate (pH 6.5), 10 mM MgCl₂, 500 μM [γ³²P]ATP, 5 μM 5′-OH r(A)₃₀, 0.4 pmol PhoClp1, and the indicated concentrations of NH₄Cl, KCl, or NaCl were incubated at 55°C for 15 min. The extents of product formation are plotted as a function of salt concentration. (C) Reaction mixtures (10 μL) containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 500 μM [γ³²P]ATP, 5 μM 5′-OH d(A)₃₀, 0.45 pmol PhoClp1, and and the indicated concentrations of NH₄Cl, KCl, or NaCl were incubated at 55°C for 15 min. The extents of product formation are plotted as a function of salt concentration.