Binding of ATP and DNA to polynucleotide kinase

Full text: Human polynucleotide kinase (hPNK) is a DNA repair enzyme required for processing and rejoining of single and double strand-break termini. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the purified recombinant protein exhibited both the kinase and the phosphatase activities. The protein undergoes a conformational change upon binding ATP (Mani et al., Biochemistry, 2001). In this study, effect of ligand binding on protein intrinsic fluorescence was measured to determine binding affinity (Kd) and the stoichiometry. Titration of hPNK with ATP indicated tight binding (Kd 1.4 μM) and the observed binding stoichiometry was 1:1. AMP-PNP, a nonhydrolyzable form of ATP, which is an inhibitor of kinases exhibited unimodal binding with a Kd value of 1.6 μM. We also studied the binding of an oligonucleotide (20 residues in length) to mimic DNA binding. This oligonucleotide bound with high affinity (Kd = 1.3 μM) in a stoichiometric manner. The oligonucleotide was also able to bind to hPNK in the presence of AMP-PNP with a Kd value of 2.5 μM, indicating the formation of a ternary complex between hPNK, AMP-PNP and the oligonucleotide. Sedimentation equilibrium measurements indicated the protein exists as a monomer in solution and the protein did not undergo any aggregation in the presence of the oligonucleotide. Both far and near-UV CD measurements also revealed a conformational change in the protein upon binding the oligonucleotide. In this study for the first time, we have demonstrated hPNK to bind the oligonucleotide with high affinity, thus fulfilling its functional role