Abstract
Nucleoside diphosphate kinases (NDP kinases) form a family of oligomeric enzymes present in all organisms. Eukaryotic NDP kinases are hexamers composed of identical subunits (approximately 17 kDa). A distinctive property of human NDPK-B encoded by the gene nm23-H2 is its ability to stimulate the gene transcription. This property is independent of its catalytic activity and is possibly related to the role of this protein in cellular events including differentiation and tumor metastasis. In this paper, we report the first characterization of human NDPK-B.DNA complex formation using a filter-binding assay and fluorescence spectroscopy. We analyzed the binding of several oligonucleotides mimicking the promoter region of the c-myc oncogene including variants in sequence, structure, and length of both strands. We show that NDPK-B binds to single-stranded oligonucleotides in a nonsequence specific manner, but that it exhibits a poor binding activity to double-stranded oligonucleotides. This indicates that the specificity of recognition to DNA is a function of the structural conformation of DNA rather than of its specific sequence. Moreover, competition experiments performed with all nucleotides provide evidence for the contribution of the six active sites in the DNA.protein complex formation. We propose a mechanism through which human NDPK-B could stimulate transcription of c-myc or possibly other genes involved in cellular differentiation.
Highlights
Nucleoside diphosphate kinases are essential oligomeric enzymes that play a key role in the maintenance of the intracellular pool of all (d)NTPs and NTPs
We found that NDPK-B binds to oligonucleotides with considerably higher affinity as compared to its enzymatic substrates
The oligonucleotide concentration used was near the dissociation constant, conditions in which the stoichiometry could be in error [47]
Summary
(Received for publication, December 30, 1998, and in revised form, March 22, 1999). From the Unitede Regulation Enzymatique des Activites Cellulaires, Institut Pasteur, CNRS-URA 1773, 25 rue du Docteur Roux 75724, Paris cedex 15, France. A distinctive property of human NDPK-B encoded by the gene nm23-H2 is its ability to stimulate the gene transcription This property is independent of its catalytic activity and is possibly related to the role of this protein in cellular events including differentiation and tumor metastasis. The crystal structures of NDP1 kinases from several organisms including prokaryotes [2] and eukaryotes [3,4,5] have been determined They all share a common subunit fold, built around a ␣␣ motif, present in the “palm domain” of DNA polymerases [5]. NDPK-B was identified as a transcription factor based on its ability to stimulate the transcription in vitro of the c-myc gene [22] This stimulatory activity is independent from the enzymatic activity, because it was retained in a catalytically inactive mutant [23]. We propose a mechanism through which human NDPK-B could stimulate transcription of c-myc and possibly of other genes involved in cellular differentiation
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