Abstract
The mitochondrial genome in a number of organisms is represented by linear DNA molecules with defined terminal structures. The telomeres of linear mitochondrial DNA (mtDNA) of yeast Candida parapsilosis consist of tandem arrays of large repetitive units possessing single-stranded 5' extension of about 110 nucleotides. Recently we identified the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of C. parapsilosis linear mtDNA and protects it from attack by various DNA-modifying enzymes (Tomáska, L'., Nosek, J., and Fukuhara, H. (1997) J. Biol. Chem. 272, 3049-3059). Here we report the isolation of MTP1, the gene encoding mtTBP of C. parapsilosis. Sequence analysis revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded DNA-binding proteins that nonspecifically bind to single-stranded DNA with high affinity. Recombinant mtTBP displays a preference for the telomeric 5' overhang of C. parapsilosis mtDNA. The heterologous expression of a mtTBP-GFP fusion protein resulted in its localization to the mitochondria but was unable to functionally substitute for the loss of the S. cerevisiae homologue Rimlp. Analysis of the MTP1 gene and its translation product mtTBP may provide an insight into the evolutionary origin of linear mitochondrial genomes and the role it plays in their replication and maintenance.
Highlights
Terminal structures of eukaryotic chromosomes and telomerases are involved in several important cellular processes as senescence, immortalization, and carcinogenesis
Mitochondrial Telomere-binding Protein, Encoded by a Nuclear Gene MTP1, Is a Member of the single-stranded DNA binding (SSB) Protein Family— Previously we reported the purification of a 15-kDa protein designated mitochondrial telomerebinding protein (mtTBP) that selectively bound to an affinity matrix containing the terminal 51 nucleotides of the 5Ј overhang of mitochondrial telomere from C. parapsilosis [7]
The gene coding for mtTBP was assigned MTP1
Summary
Construction of Plasmid Clones—The plasmid pGFP-C-FUS-mtTBP containing the whole MTP1 open reading frame (lacking stop codon) fused with green fluorescent protein was prepared by ligation of PCR product amplified using 5Ј-ATGTTGCGAGCATTCACTAGATCA-3Ј and 5Ј-TTCTGTAGCTTCGGCTCTATCCTCA-3Ј primers into SmaI-digested pGFP-C-FUS vector Digested mtDNA (100 l) was mixed with 5 g of recombinant mtTBP in a final volume of 250 l of the DNA binding buffer (10 mM Tris-HCl pH 7.4, 50 mM NaCl) and incubated for 30 min at room temperature. 10 ng of purified recombinant mtTBP was incubated with 1 ng of labeled oligonucleotide in 10 l of DNA binding buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl) containing 1 mg/ml poly(dI:dC) for 10 min at room temperature. Intact cells of S. cerevisiae were transformed by standard lithium acetate/ssDNA/polyethylene glycol protocol [19]
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