Cloning and characterisation of mtDBP, a DNA-binding protein which binds two distinct regions of sea urchin mitochondrial DNA.
The cDNA for the sea urchin mitochondrial D-loop-binding protein (mtDBP), a 40 kDa protein which binds two homologous regions of mitochondrial DNA (the D-loop region and the boundary between the oppositely transcribed ND5 and ND6 genes), has been cloned. Four different 3'-untranslated regions have been detected that are related to each other in pairs and do not contain the canonical polyadenylation signal. The in vitro synthesised mature protein (348 amino acids), deprived of the putative signal sequence, binds specifically to its DNA target sequence and produces a DNase I footprint identical to that given by the natural protein. mtDBP contains two leucine zippers, one of which is bipartite, and two small N- and C-terminal basic domains. A deletion mutation analysis of the recombinant protein has shown that the N-terminal region and the two leucine zippers are necessary for the binding. Furthermore, evidence was provided that mtDBP binds DNA as a monomer. This rules out a dimerization role for the leucine zippers and rather suggests that intramolecular interactions between leucine zippers take place. A database search has revealed as the most significative homology a match with the human mitochondrial transcription termination factor (mTERF), a protein that also binds DNA as a monomer and contains three leucine zippers forming intramolecular interactions. These similarities, and the observation that mtDBP-binding sites contain the 3'-ends of mtRNAs coded by opposite strands and the 3'-end of the D-loop structure, point to a dual function of the protein in modulating sea urchin mitochondrial DNA transcription and replication.
Loguercio Polosa
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Dipartimento di Biochimica e Biologia Molecolare, Università di Bari and the Centro Studi sui Mitocondri e Metabolismo Energetico, CNR, Via Orabona 4, 70125 Bari, Italy.
Roberti
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Musicco
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Gadaleta
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MN
Quagliariello
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Cantatore
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016350:Leucine Zippers; D008928:Mitochondria; D008969:Molecular Sequence Data; D012617:Sea Urchins / Q000235:genetics; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; C119291:mitochondrial D-loop-binding protein, Paracentrotus lividus