Spectroscopic studies of DNA and ATP binding to human polynucleotide kinase: evidence for a ternary complex.
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 K(d) value of 1.3 microM and with 1:1 stoichiometry. A 5'-phosphorylated oligonucleotide with the same sequence exhibited an almost 6-fold lower affinity (K(d) value, 7.2 microM). ATP and AMP-PNP bound with high affinity (K(d) values, respectively, of 1.4 and 1.6 microM), 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 K(d) value of 2.5 microM, 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.
Mani
Rajam S
RS
Department of Experimental Oncology, Cross Cancer Institute, and Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada. rajam.mani@cancerboard.ab.ca
Karimi-Busheri
Feridoun
F
Fanta
Mesfin
M
Cass
Carol E
CE
Weinfeld
Michael
M
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry*; D001665:Binding Sites; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry*; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D004268:DNA-Binding Proteins; D008024:Ligands; D009841:Oligonucleotides; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase