Physical properties of human polynucleotide kinase: Hydrodynamic and spectroscopic studies

Human polynucleotide kinase (hPNK) is a putative DNA repair enzyme in the b ase excision repair pathway required for processing and rejoining strand-br eak termini. This study represents the first systematic examination of the physical properties of this enzyme. The protein was produced in Escherichia coli as a His-tagged protein, and the purified recombinant protein exhibit ed both the kinase and the phosphatase activities. The predicted relative m olecular mass (M-r) of the 521 amino acid polypeptide encoded by the sequen ced cDNA for PNK and the additional 21 amino acids of the His tag is 59 538 . The M-r determined by low-speed sedimentation equilibrium under nondenatu ring conditions was 59 600 +/- 1000, indicating that the protein exists as a monomer, in contrast to T4 phage PNK, which exists as a homotetramer. The size and shape of hPNK in solution were determined by analytical ultracent rifugation studies. The protein was found to have an intrinsic sedimentatio n coefficient, s(0)20,w, of 3.54 S and a Stokes radius, R-S, of 37.5 Angstr om. These hydrodynamic data, together with the M-r of 59 600, suggest that hPNK is a moderately asymmetric protein with an axial ratio of 5.51. Analys is of the secondary structure of hPNK on the basis of circular dichroism sp ectra, which revealed the presence of two negative dichroic bands located a t 218 and 209 nm, with ellipticity values of -7200 +/- 300 and -7800 300 de g.cm(2).dmol(-1), respectively, indicated the presence of approximately 50% beta -structure and 25% alpha -helix. Binding of ATP to the protein induce d an increase in beta -structure and perturbed tryptophan, tyrosine, and ph enylalanine signals observed by aromatic CD and UV difference spectroscopy.