Figure 2. NDP acceptor specificity of the reverse kinase reaction.

(A) Reverse kinase reactions were performed as described in Experimental Procedures. The reaction mixtures included 0.1 μM 5′-³²P labeled 15-mer DNA oligonucleotide (pCCGACCAACGAAGGT), 100 μM ADP, UDP, GDP, CDP or dADP as specified, and 0.25 μM kinase (Pnk +). The radiolabeled products were resolved by TLC and visualized by autoradiography, which showed label transfer from the ³²P-DNA oligonucleotide to the added NDP acceptor to form the respective ³²P-labeled NTPs. The extents of label transfer to the NDPs are indicated below the lanes. (B) NDP dependence. Reaction mixtures (10 μl) containing 50 mM Tris-HCl (pH 7.0), 10 mM MgCl₂, 5 mM DTT, 0.1 μM 5′ ³²P-labeled 15-mer oligonucleotide, 0.125 μM kinase and NDP as indicated were incubated at 45°C for 30 min. The products were analyzed by TLC. The extent of ³²P-NTP formation is plotted as a function of NDP concentration. Each datum is the average of three independent titration experiments (±SEM). Nonlinear regression fitting of the data to the Michaelis-Menten equation was performed in Prism. (C) Kinetics. Reaction mixtures containing 50 mM Tris-HCl (pH 7.0), 10 mM MgCl₂, 5 mM DTT, 100 μM ADP, 0.1 μM of a 15-mer 5′-³²P labeled 15-mer oligonucleotide, and 0.25, 0.5, 1.0 or 2.0 μM kinase as indicated were incubated at 45°C. Aliquots (10 μl) were withdrawn at times specified and quenched immediately with formic acid. The time 0 sample was withdrawn prior to adding kinase. The products were analyzed by TLC. The extents of label transfer to form ³²P-ATP are plotted versus reaction time. Each datum is the average of three independent time course experiments (±SEM). The data were fit by non-linear regression to a one-phase association function in Prism.