Spectroscopic Studies of DNA and ATP Binding to Human Polynucleotide Kinase: Evidence for a Ternary Complex

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Abstract
Human polynucleotide kinase (hPNK), which possesses both 5‘-DNA kinase and 3‘-DNA phosphatase activities, is a DNA repair enzyme required for processing and rejoining of single- and double-strand-break termini. Full-length hPNK was subjected to sedimentation and spectroscopic analyses in association with its ligands, a 20-mer oligonucleotide, ATP, and AMP-PNP (a nonhydrolyzable analogue of ATP). Sedimentation equilibrium measurements indicated that hPNK was a monomer in the presence and absence of the ligands. Circular dichroism measurements revealed that the ligands induced different conformational changes in hPNK, although AMP-PNP induced the same conformational changes as ATP. CD also indicated that the oligonucleotide could bind to the protein-AMP-PNP complex. Protein−ligand binding affinities and stoichiometries were determined by measuring changes in protein intrinsic fluorescence. Titrating hPNK with the oligonucleotide indicated tight binding with a Kd value of 1.3 μM and with 1:1 stoichiometry. A 5‘-phosphorylated oligonucleotide with the same sequence exhibited an almost 6-fold lower affinity (Kd value, 7.2 μM). ATP and AMP-PNP bound with high affinity (Kd values, respectively, of 1.4 and 1.6 μM), and the observed binding stoichiometries were 1:1. Furthermore, the nonphosphorylated oligonucleotide was able to bind to hPNK in the presence of AMP-PNP with a Kd value of 2.5 μM, confirming the formation of a ternary complex. This study provides the first direct physical evidence for such a ternary complex involving a polynucleotide kinase, AMP-PNP, and an oligonucleotide, and supports a reaction mechanism in which ATP and DNA bind simultaneously to the enzyme.

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