Source:http://linkedlifedata.com/resource/pubmed/id/14556639
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
41
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pubmed:dateCreated |
2003-10-14
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pubmed:abstractText |
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.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Adenosine Triphosphate,
http://linkedlifedata.com/resource/pubmed/chemical/Adenylyl Imidodiphosphate,
http://linkedlifedata.com/resource/pubmed/chemical/DNA-Binding Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Ligands,
http://linkedlifedata.com/resource/pubmed/chemical/Oligonucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Polynucleotide 5'-Hydroxyl-Kinase,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins
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pubmed:status |
MEDLINE
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pubmed:month |
Oct
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pubmed:issn |
0006-2960
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
21
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pubmed:volume |
42
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
12077-84
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pubmed:dateRevised |
2006-11-15
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pubmed:meshHeading |
pubmed-meshheading:14556639-Adenosine Triphosphate,
pubmed-meshheading:14556639-Adenylyl Imidodiphosphate,
pubmed-meshheading:14556639-Binding Sites,
pubmed-meshheading:14556639-Circular Dichroism,
pubmed-meshheading:14556639-DNA-Binding Proteins,
pubmed-meshheading:14556639-Humans,
pubmed-meshheading:14556639-Ligands,
pubmed-meshheading:14556639-Oligonucleotides,
pubmed-meshheading:14556639-Phosphorylation,
pubmed-meshheading:14556639-Polynucleotide 5'-Hydroxyl-Kinase,
pubmed-meshheading:14556639-Protein Conformation,
pubmed-meshheading:14556639-Protein Folding,
pubmed-meshheading:14556639-Protein Structure, Tertiary,
pubmed-meshheading:14556639-Recombinant Proteins,
pubmed-meshheading:14556639-Spectrometry, Fluorescence,
pubmed-meshheading:14556639-Ultracentrifugation
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pubmed:year |
2003
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pubmed:articleTitle |
Spectroscopic studies of DNA and ATP binding to human polynucleotide kinase: evidence for a ternary complex.
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pubmed:affiliation |
Department of Experimental Oncology, Cross Cancer Institute, and Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada. rajam.mani@cancerboard.ab.ca
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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