Single strand DNA specificity analysis of human nucleoside diphosphate kinase B.
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.
Agou
F
F
Unité de Régulation Enzymatique des Activités Cellulaires, Institut Pasteur, CNRS-URA 1773, 25 rue du Docteur Roux 75724, Paris cedex 15, France.
Raveh
S
S
Mesnildrey
S
S
Véron
M
M
D001665:Binding Sites; D001667:Binding, Competitive; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000235:genetics; D016259:Genes, myc / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009690:Nucleic Acid Conformation; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009994:Osmolar Concentration; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D015533:Transcriptional Activation; D014461:Ultracentrifugation
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D009701:Nucleoside-Diphosphate Kinase