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• W2056075515 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 yeastCandida 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 (Tomás̆ka, 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. 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 yeastCandida 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 (Tomás̆ka, 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. Terminal structures (telomeres) of eukaryotic chromosomes and telomerases (specialized nucleoprotein enzymes that maintain the telomere length) are involved in several important cellular processes as senescence, immortalization, and carcinogenesis. Telomerase activation appears to be critical for cell immortalization and represents a promising target for cancer therapy. However, several studies have demonstrated that cells lacking functional telomerase utilize an alternative mechanism to elongate the chromosome ends, suggesting that some tumor cells may survive following treatment with telomerase inhibitors (1Autexier C. Greider C.W. Trends Biochem. Sci. 1996; 21: 387-391Abstract Full Text PDF PubMed Scopus (150) Google Scholar). Therefore a more detailed understanding of both telomerase-dependent and -independent replication mechanisms is crucial for cancer therapy and the design of therapeutic agents capable of specifically blocking telomere replication. Furthermore, the study of various linear genophores might shed some light on alternative solutions to the end-replication problem. In contrast to the generally held belief that mitochondrial genomes are circular molecules, a large number of organisms contain linear mitochondrial DNA (mtDNA) molecules possessing a homogeneous terminal structure. Sequence analysis of mitochondrial telomeres from various organisms revealed that they do not conform to a single consensus sequence or structural motif (2Nosek J. Tomás̆ka L'. Fukuhara H. Suyama Y. Kovác̆ L. Trends Genet. 1998; 14: 184-188Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). According to their terminal structures, two types of linear mtDNA have been identified in yeasts. Linear mtDNA of the yeast species in closely related generaWilliopsis and Pichia terminates at both ends with an inverted terminal repeat possessing a covalently closed single-stranded hairpin loop resembling the structure of the vaccinia virus genome (3Fukuhara H. Sor F. Drissi R. Dinouël N. Miyakawa I. Rousset S. Viola A.M. Mol. Cell. Biol. 1993; 13: 2309-2314Crossref PubMed Scopus (56) Google Scholar, 4Dinouël N. Drissi R. Miyakawa I. Sor F. Rousset S. Fukuhara H. Mol. Cell. Biol. 1993; 13: 2315-2323Crossref PubMed Scopus (49) Google Scholar). The terminal structures of type 2 linear mtDNA of a pathogenic yeast Candida parapsilosis are represented by inverted terminal repetitions consisting of tandem arrays of a 738-bp 1The abbreviations used are:bp, base pair(s); mtTBP, mitochondrial telomere-binding protein; SSB protein, single-stranded DNA-binding protein; PCR, polymerase chain reaction; GFP, green fluorescence protein; ssDNA, single-stranded DNA; PBS, phosphate-buffered saline.-long repetitive unit. This structure remotely resembles the organization of telomeres of nuclear chromosomes, although their repetitive unit is considerably shorter (5–8 bp). The variable number of tandem units generates a population of mtDNA molecules of heterogeneous size where the shortest molecules containing only incomplete repetitive unit predominate. A more detailed analysis of C. parapsilosis mitochondrial telomeres revealed that mtDNA molecules terminate at a defined position within a repetitive unit, thus generating a 5′ single-stranded extension of about 110 nucleotides (5Nosek J. Dinouël N. Kovác̆ L. Fukuhara H. Mol. Gen. Genet. 1995; 247: 61-72Crossref PubMed Scopus (65) Google Scholar). This unique telomeric structure has raised several important questions: (i) how the mitochondrial telomere is stabilized, (ii) how the 5′ single-stranded extension is generated, (iii) why DNA polymerase does not fill the protruding 5′ overhang, and (iv) how the shortest mtDNA molecules that do not possess a complete tandem unit restore the missing sequence. Several proteins that specifically bind either double-stranded or single-stranded DNA of telomeres of nuclear chromosomes have been identified. These proteins mediate telomere functions such as capping the ends of chromosomes, preventing nucleolytic degradation and end-to-end fusions, promoting the formation of telomere chromatin structure and nuclear architecture, participating in the replication and regulation of telomere length, etc. (6Fang G. Cech T.R. Blackburn E.H. Greider C.W. Telomeres. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1995: 69-105Google Scholar). To decipher the role of mitochondrial telomeres, we have initiated a search for proteins interacting with terminal sequence of linear mtDNA. Recently, we identified the first protein specifically recognizing the terminal structure of linear mtDNA from C. parapsilosis. Mitochondrial telomere-binding protein (mtTBP) is a heat- and protease-resistant protein that specifically recognizes the synthetic oligonucleotide identical to the terminal 51 nucleotides of the 5′ single-stranded overhang of C. parapsilosis mitochondrial telomere and protects it from various DNA modifying enzymes. Affinity-purified mtTBP exhibits a molecular mass of 15 kDa in its monomeric state under denaturing conditions but forms homo-oligomers under native conditions (7Tomás̆ka L'. Nosek J. Fukuhara H. J. Biol. Chem. 1997; 272: 3049-3056Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The properties of mtTBP suggested that it may play an important role in the stabilization and/or replication of linear mtDNA of C. parapsilosis. In this report we describe the isolation and characterization of a nuclear gene, MTP1, that encodes the mtTBP of the yeastC. parapsilosis. Surprisingly, the sequence analysis of mtTBP revealed a striking homology to a family of bacterial and mitochondrial single-stranded DNA binding (SSB) proteins. Although other members of the SSB protein family bind with high affinity to single-stranded DNA without apparent sequence specificity, mtTBP preferentially binds the terminal 5′ single-stranded overhang of the mitochondrial telomere. It has been proposed that the evolutionary appearance of linear mtDNA led to adaptation of the replication machinery to ensure complete replication of the linear genophore. In mitochondria of C. parapsilosis, such adaptation might have forced the conversion of a sequence-nonspecific mitochondrial SSB protein to a telomere binding factor. C. parapsilosis SR23 (CBS 7157) is a laboratory strain from the collection of the Department of Biochemistry (Comenius University, Bratislava, Slovakia). Escherichia coli DH5α (deoR, endA1, gyrA96, hsdR17 (r k− , m k+ ), recA1, relA1, supE44, thi-1, Δ(lac-argFV169), φ80δlacZΔM15, F−, λ−) and Saccharomyces cerevisiae S150–2B (MATa, leu2–3, 112, his3-Δ, trp1–289, ura3–52) strains were used in cloning experiments. S. cerevisiae strains αEVΔRIM1–8b (MATα, rim1::URA3, ade2–1, leu2–3, 112, his3–11, 15, trp1–1, ura3–1, can1–100, ρ 0) and aDBY754 × αEVRT-7b (MATa/MATα, RIM1/rim1::URA3, leu2/leu2, ura3/ura3, ρ +) were kindly provided by F. Foury (Université Catholique de Louvain, Belgium). S. cerevisiae D22 strain (MATa, ade2, ω +) was used as a source for mitochondrial protein extracts. Mitochondrial protein extracts from C. parapsilosis and S. cerevisiaeD22 were prepared as described previously (7Tomás̆ka L'. Nosek J. Fukuhara H. J. Biol. Chem. 1997; 272: 3049-3056Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). mtTBP was purified using an affinity chromatography step (7Tomás̆ka L'. Nosek J. Fukuhara H. J. Biol. Chem. 1997; 272: 3049-3056Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), except that the protein was concentrated by binding to Fast Q-Sepharose (Amersham Pharmacia Biotech), eluted by 1 m NaCl, dialyzed against 20 mm HEPES-NaOH, pH 7, and stored in 100-μl aliquots at −70 °C. Protein concentration was determined by a method of Bradford (8Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217508) Google Scholar). The sequence of three peptides (HAEIVQWGK, YSLAVNK, and LDKFEDP, respectively) was determined by the Harvard Microchem (Cambridge, MA). The sequences of oligonucleotides (synthesized by Genset, France) used in this study are shown in Table I. Oligonucleotide I was used for priming the synthesis of cDNA on C. parapsilosis total RNA template using the First-strand cDNA synthesis kit (Amersham Pharmacia Biotech) according to the manufacturer instructions. The sequence of peptide HAEIVQWGK was used to designMTP1-specific degenerate oligonucleotide II. The PCR reaction for amplification of the 3′ end of MTP1 cDNA contained 50 mm KCl, 10 mm Tris-HCl, pH 9.0 (at 25 °C), 0.1% Triton X-100, 1.25 mm MgCl2, 0.2 mm dNTP, 2.5 units of Taq DNA polymerase, single-stranded cDNA prepared as described above as the template and 1 μm oligonucleotide I and 5 μmoligonucleotide II as the upstream and downstream primer, respectively. Amplification was performed in DNA Thermal Cycler 480 (Perkin-Elmer) with initial denaturation at 95 °C for 3 min, followed by 30 cycles of 94 °C for 1 min, 53 °C for 1 min, 72 °C for 1 min, and final polymerization at 72 °C for 5 min. The 125-bp PCR product was gel-purified and sequenced. The DNA sequence upstream of the stop codon of putative open reading frame was used to design downstreamMTP1-specific primer (oligonucleotide III). The 5′ end ofMTP1 was amplified using linker-ligation-mediated PCR strategy (9Garrity P.A. Wold B. Mueller P.R. McPherson M.J. Hames B.D. Taylor G.R. PCR 2 : A Practical Approach. IRL Press at Oxford University Press, Oxford1995: 309-322Google Scholar, 10Niedenthal R.K. Riles L. Johnston M. Hegemann J.H. Yeast. 1996; 12: 773-786Crossref PubMed Scopus (366) Google Scholar). Briefly, total genomic DNA of C. parapsilosiswas digested with AvaII endonuclease, denatured, annealed with MTP1 specific downstream primer, then extended with Vent(exo−) DNA polymerase (New England Biolabs), followed by ligation of the primer extension products with a synthetic linker. The MTP1 sequence was then PCR-amplified usingMTP1-specific downstream (oligonucleotide III) and 25-nucleotide-linker primers. The DNA sequence analysis of 600-bp PCR product revealed a putative 399-bp open reading frame containing the sequences corresponding to three known peptides. The integrity ofMTP1 gene was confirmed by sequencing of several independent PCR products obtained using MTP1 oligonucleotides IV and V on C. parapsilosis genomic DNA and cDNA as the template, respectively.Table IThe sequences of synthetic oligonucleotidesOligonucleotideI5′-CAGGAAACAGCTATGACTTTTTTTTTTTTTTTTTV-3′II5′-CAYGCIGARATHGTNCARTGGGGNAA-3′III5′-TTCTGTAGCTTCGGCTCTATCCTCA-3′IV5′-CAGATTTTATGTAACAACCCACG-3′V5′-GACATAGAAATATATTATACCAATAC-3′Linker5′-GCGGTGACCCGGGAGATCTGAATTC-3′3′CTAGACTTAAG-5′TEL315′-TAGGGATTGATTATTTACCTATATATTATCA-3′OLI315′-TAAAAATAGAGAGAAATGTATATATTTTACC-3′ Open table in a new tab 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 5′-ATGTTGCGAGCATTCACTAGATCA-3′ and 5′-TTCTGTAGCTTCGGCTCTATCCTCA-3′ primers into SmaI-digested pGFP-C-FUS vector (10, provided by J. H. Hegemann, Justus-Liebig University, Giessen, Germany). The expression of fusion protein in this construct is driven by S. cerevisiae MET25 promoter. The plasmid YEplac181-PMET-mtTBP-tCYC1 was constructed as follows. First, the plasmid pGFP-C-FUS was digested withSalI to remove green fluorescent protein (GFP) sequences. The termini were filled with a Klenow enzyme and then ligated with theMTP1 PCR product amplified using 5′-ATGTTGCGAGCATTCACTAGATCA-3′ and oligonucleotide V (Table I) primers. Subsequently, the KpnI-SacI restriction fragment containing a cassette MET25 promoter, MTP1 open reading frame, CYC1 terminator was blunt-ended by T4 DNA polymerase and then ligated into SmaI site of YEplac181 vector (11Gietz R.D. Sugino A. Gene. 1988; 74: 527-534Crossref PubMed Scopus (2528) Google Scholar). pGEX-2T-mTBP designed for production of recombinant mtTBP in E. coli was constructed by insertion of Sau3A fragment of MTP1 gene into the BamHI site of pGEX-2T expression vector (Amersham Pharmacia Biotech). TheMTP1 sequence in the constructs was verified by DNA sequencing. Recombinant mtTBP was purified according to the instructions of the supplier of the expression vector (Amersham Pharmacia Biotech). Briefly, 20 ml of an overnight bacterial culture was inoculated into 1 liter of Superbroth media (3.2% tryptone (Difco), 2% yeast extract (Difco), 0.5% NaCl) and grown at 37 °C to a final A 600 = 0.7. The culture was then induced for 3 h at 30 °C with 1 mmisopropyl-1-thio-β-d-galactopyranoside (Sigma). Cells were washed once with ice-cold double-distilled water, resuspended in 20 ml of buffer G (20 mm HEPES-NaOH, pH 8.0, 500 mm NaCl, 0.1% Triton X-100, 0.1 mm EDTA, protease inhibitor mixture (CompleteTM, Boehringer Mannheim), sonicated, and centrifuged at 25,000 × gfor 30 min at 4 °C to remove insoluble material. The supernatant was mixed with 0.3 ml of glutathione-agarose (Sigma) prewashed with buffer G and incubated for 60 min on ice with occasional mixing. Beads were loaded onto a 5-ml column, washed with 100 ml of buffer G (without protease inhibitor mixture) followed by 50 ml of ice-cold phosphate-buffered saline (PBS, 137 mm NaCl, 2.6 mm KCl, 10 mm Na2HPO4, 1.4 mm KH2PO4). The washed beads were resuspended in 0.45 ml of PBS containing thrombin (0.05 units/μl, Amersham Pharmacia Biotech) and incubated overnight at 4 °C. The cleaved mtTBP was eluted in 3 × 0.5 ml of PBS and stored at −70 °C. Rabbit polyclonal antisera was raised against 100 μg of purified recombinant thrombin-cleaved mtTBP and purified by Protein A-Sepharose (Eurogentec, Belgium). C. parapsilosis SR23 was grown until the late logarithmic phase in YP medium (1% yeast extract, 1% peptone) supplemented with glucose (2% w/v), glycerol (3% w/v), or galactose (2% w/v), and cells (0.1 ml of the culture) were lysed as described by Horváth and Riezman (12Horváth A. Riezman H. Yeast. 1994; 10: 1305-1310Crossref PubMed Scopus (135) Google Scholar). Proteins were separated by 13% SDS-polyacrylamide gel electrophoresis by a method of Laemmli (13Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207523) Google Scholar). Resolved proteins were transferred to nitrocellulose filters in the transfer buffer (25 mm Tris, 192 mmglycine, 20% methanol, pH 8.3) using a semidry electroblotter system (Owl Scientific) for 45 min at 250 mA. Filters were blocked for 2 h at room temperature with blocking solution (20 mmTris-HCl, pH 7.5, 150 mm NaCl, 2% (w/v) skim milk (Difco)) and then incubated overnight at 4 °C in the blocking solution containing anti-mtTBP antibody (1:200). Membranes were washed four times with rinsing buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.05% Tween 20), one time with rinsing buffer without Tween 20, and then incubated with blocking solution containing goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma, 1:3,000) for 2 h at room temperature. Blots were washed as described above and developed by incubating with 0.3 mg/ml p-nitro blue tetrazolium chloride (Sigma) and 0.15 mg/ml 5-bromo-4-chloro-3-indolylphosphate toluidine salt (Sigma) in alkaline phosphatase buffer (100 mm NaHCO3, 1 mm MgCl2, pH 9.8) for 5–20 min at room temperature. Purified mitochondrial DNA (3 μg) from C. parapsilosis was digested with BglII (30 units) in a final volume of 250 μl, followed by heat-inactivation of the restriction enzyme by incubation of the sample for 10 min at 65 °C. Digested mtDNA (100 μl) was mixed with 5 μg of recombinant mtTBP in a final volume of 250 μl of the DNA binding buffer (10 mm Tris-HCl pH 7.4, 50 mm NaCl) and incubated for 30 min at room temperature. To immunoprecipitate mtTBP-DNA complexes, the binding reaction was combined with 50 μl of anti-mtTBP antibody for 2 h at 4 °C. Immune complexes were immobilized by incubation with Protein A-Sepharose 4B (Sigma) for an additional 2 h at 4 °C and centrifuged for 5 s in a microcentrifuge at maximal speed, and the supernatant was removed and placed on ice. The pellet was washed 5 times with 1 ml of PBS containing 200 mm NaCl. The immunoprecipitated complexes were separated from the beads by boiling for 5 min in 300 μl of PBS containing 0.1% SDS. This step was repeated two more times, and the resulting fractions were saved as eluates 1, 2, and 3, respectively. Oligonucleotides were 5′ end-labeled using [γ32P]ATP by T4 polynucleotide kinase and separated from nonincorporated nucleotides by chromatography on a BioSpin-6 column (Bio-Rad). 10 ng of purified recombinant mtTBP was incubated with 1 ng of labeled oligonucleotide in 10 μl of DNA binding buffer (10 mm Tris-HCl, pH 7.4, 50 mmNaCl) containing 1 mg/ml poly(dI:dC) for 10 min at room temperature. DNA-protein complexes were resolved by electrophoresis on 4% polyacrylamide gel in 0.5× TBE (4.5 mm Tris borate, 1 mm EDTA) at 10 V/cm for 90 min. Gels were dried under vacuum at 80 °C and exposed to Kodak X-Omat film at −70 °C between intensifying screens. 1 μg of recombinant mtTBP was incubated with various concentrations of glutaraldehyde in 10 μl of PBS for 10 min at room temperature. The cross-linking reaction was stopped by heating the samples in 1× Laemmli sample buffer for 5 min at 95 °C. Proteins were separated by 13% SDS-polyacrylamide gel electrophoresis by a method of Laemmli (13Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207523) Google Scholar) and transferred to nitrocellulose filters, and mtTBP was detected by immunoblot analysis as described above. Alternatively, cAMP-dependent protein kinase assays were performed as described previously (14Resnick R.J. Racker E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2474-2478Crossref PubMed Scopus (34) Google Scholar, 15Tomás̆ka L'. Biochem. Biophys. Res. Commun. 1998; 242: 457-460Crossref PubMed Scopus (11) Google Scholar). Briefly, reaction mixtures (10 μl) contained 10 mm HEPES-NaOH, pH 6.5, 4 mm MgCl2, 20 mm dithiothreitol, 1 μg of recombinant mtTBP, 50 ng of the purified catalytic subunit of protein kinase A (kindly provided by E. Fisher, Seattle, WA), and 250 μm [γ32P]ATP (500 cpm/pmol) and were incubated for 240 min at 30 °C. After the phosphorylation of mtTBP, glutaraldehyde and bis(sulfosuccinimidyl)suberate were added to the corresponding samples to final concentrations indicated in legend to Fig. 4. After a 10-min incubation at room temperature, cross-linking reactions were stopped by the addition of an equal volume of 2× SDS-polyacrylamide gel electrophoresis Laemmli sample buffer. Proteins were separated by 13% SDS-polyacrylamide gel electrophoresis by a method of Laemmli (13Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207523) Google Scholar). Gels were heat-dried under vacuum and exposed to Kodak X-Omat film at −70 °C between intensifying screens. DNA samples were prepared as described previously (16Nosek J. Fukuhara H. J. Bacteriol. 1994; 176: 5622-5630Crossref PubMed Google Scholar). Briefly, yeast cells were lysed in agarose blocks by sequential treatment with zymolyase and proteinase K. Samples were electrophoretically separated in a 0.8% agarose gel in 0.5× TBE (45 mm Tris borate, 1 mm EDTA) buffer in a Pulsaphor apparatus (LKB) in contour-clamped homogeneous electric field configuration. Pulse switching involved three steps of linear interpolation as follows: (i) 10 to 200 s for 48 h, (ii) 200 to 400 s for 16 h, (iii) 400 to 600 s for 48 h, at 100 V and 9 °C throughout. C. parapsilosisgenomic DNA was isolated using a protocol described for S. cerevisiae (17Phillippsen P. Stotz A. Scherf C. Methods Enzymol. 1991; 194: 169-182Crossref PubMed Scopus (269) Google Scholar). Restriction and DNA modification enzymes were from New England Biolabs and used according to manufacturer instructions. Southern and Northern blotting, DNA hybridization, DNA cloning, and sequencing were performed essentially as described in Sambrook et al. (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Intact cells of S. cerevisiae were transformed by standard lithium acetate/ssDNA/polyethylene glycol protocol (19Gietz R.D. Schiestl R.H. Methods Mol. Cell. Biol. 1995; 5: 255-269Google Scholar). Previously we reported the purification of a 15-kDa protein designated mtTBP that selectively bound to an affinity matrix containing the terminal 51 nucleotides of the 5′ overhang of mitochondrial telomere from C. parapsilosis (7Tomás̆ka L'. Nosek J. Fukuhara H. J. Biol. Chem. 1997; 272: 3049-3056Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The amino acid sequence of three peptides were determined (HAEIVQWGK, YSLAVNK, and LDKFEDP), and the corresponding DNA sequence of the peptide HAEIVQWGK was used to design a degenerated oligonucleotide primer for polymerase chain reaction. The entire coding sequence of mtTBP was isolated using two strategies: (i) amplification of the 3′ end of the cDNA by reverse transcription-PCR followed by (ii) amplification of the 5′ end by a linker ligation-mediated PCR approach. The full-length PCR product was obtained, and its DNA sequence was verified by hybridization to total genomic DNA digested with several restriction enzymes (data not shown). The sequences of several PCR products were determined, and the computer sequence analysis identified a 399-bp open reading frame containing regions corresponding to all three peptides (Fig.1 A). The gene coding for mtTBP was assigned MTP1 (mitochondrialtelomere protein 1). A GenBank™ data base search revealed that mtTBP displays significant homology to a family of bacterial and mitochondrial SSB proteins. Its amino acid sequence exhibits 33.8% identity and 62.4% homology to the mitochondrial SSB protein encoded by the S. cerevisiaenuclear gene RIM1 (Fig. 1 B). Although theRIM1 gene contains an intron, we were unable to detect any intervening sequences in the C. parapsilosis gene when we compared the sequence of MTP1 PCR products amplified from both a cDNA and genomic DNA template. To rule out the possibility that MTP1 is encoded by the mitochondrial genome, we examined restriction enzyme-digested mtDNA and pulsed field gel electrophoresis-separated C. parapsilosischromosomes, respectively, by Southern blot analysis. Results indicated that MTP1 is localized on the 2.2 Mbp chromosome, thereby confirming its nuclear localization (Fig.2). In addition, the sequence analysis predicted putative mitochondrial import signal at the N terminus of deduced MTP1 protein product. To determine whether C. parapsilosis does not contain homologous sequences that might encode additional mitochondrial SSB protein(s), we performed the Southern hybridization of restriction enzyme-digested genomic DNA withMTP1 probe at both high and low stringency conditions. The results revealed that MTP1 represents a single copy gene (data not shown). To study mtTBP in vitro,we used an E. coli expression system to produce sufficient quantities of recombinant protein as described under “Experimental Procedures.” Using the pGEX-2T vector, mtTBP lacking the first seven amino acid residues was fused to glutathione S-transferase containing a thrombin cleavage site. Treatment with protease releases a soluble mtTBP protein containing one additional glycine residue at its N terminus. To assess the specificity of the recombinant mtTBP for the telomeric sequence, an oligonucleotide identical to the terminal 31 nucleotides (TEL31) from the 5′ end of mitochondrial telomere was used as a probe in a gel-retardation assay (Fig.3). Mixing of TEL31 with purified recombinant mtTBP resulted in a DNA-protein complex that migrated slower than the free probe alone (Fig. 3, lanes 1 and2). A 3- to 300-fold molar excess of unlabeled TEL31 quantitatively competed with the labeled probe for complex formation (Fig. 3, lanes 3–5). In contrast, oligonucleotide OLI31 (see Table I) derived from another part of C. parapsilosis mtDNA did not compete with labeled TEL31 as effectively as homologous competitor under identical conditions (Fig. 3, lanes 6–8). These data demonstrate that the preference of recombinant mtTBP for the telomeric sequence parallels the behavior of the natural mtTBP isolated from C. parapsilosis mitochondria (7Tomás̆ka L'. Nosek J. Fukuhara H. J. Biol. Chem. 1997; 272: 3049-3056Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Recombinant mtTBP was used to raise the rabbit polyclonal antisera, which ultimately recognized a single 15-kDa protein under denaturing conditions in cell extracts prepared from C. parapsilosis. No similar size protein has been detected in several other yeast species from various genera including Candida,Williopsis, Pichia, Saccharomyces, andKluyveromyces. It was shown previously that mtTBP forms homo-oligomeric complexes in its native state. Using two chemical cross-linkers, glutaraldehyde andbis(sulfosuccinimidyl)suberate, we now demonstrate that recombinant mtTBP also undergoes homo-oligomerization in vitro. Dimers were the predominant oligomeric form in the presence of either cross-linker (Fig. 4). To compare the DNA binding properties of mtTBP with a homologous protein from a yeast with circular mitochondrial DNA, we performed a gel retardation experiment using mitochondrial protein extracts fromC. parapsilosis and S. cerevisiae, respectively. Both extracts contained ssDNA binding activity. It was shown previously that the factor responsible for the ssDNA-protein complex formation is mtTBP in C. parapsilosis (7Tomás̆ka L'. Nosek J. Fukuhara H. J. Biol. Chem. 1997; 272: 3049-3056Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and Rim1p in S. cerevisiae (20Van Dyck E. Foury F. Stillman B. Brill S.J. EMBO J. 1992; 11: 3421-3430Crossref PubMed Scopus (147) Google Scholar), respectively. However, in contrast to C. parapsilosis, 300-fold excess of OLI31 competed quantitatively with the labeled TEL31 probe for complex formation in mitochondrial lysates of S. cerevisiae (Fig.5). This suggests that, in addition to general ssDNA binding activity, mtTBP gained a preference for a telomeric sequence. Next we tested whether mtTBP was capable of binding natural mitochondrial telomeres in a reconstitution experiment. (Fig.6). Recombinant mtTBP was incubated withBglII-digested mtDNA of C. parapsilosis, and the DNA-protein complexes were immunoprecipitated with anti-mtTBP and Protein A-Sepharose. DNA-mtTBP complexes were eluted from the beads, and the fractions were examined by Southern blot analysis following hybridization to either a specific 738-bp EcoRI fragment representing the telomere repeat unit or to a control 2.9-kilobaseBglII fragment derived from an internal region of mtDNA. The results shown in Fig. 6 demonstrate that anti-mtTBP antiserum immunoprecipitated mtTBP complexed to terminal mtDNA fragments, producing a ladder pattern typical of telomeric DNA (5Nosek J. Dinouël N. Kovác̆ L. Fukuhara H. Mol." @default.
• W2056075515 created "2016-06-24" @default.
• W2056075515 creator A5012452039 @default.
• W2056075515 creator A5041903432 @default.
• W2056075515 creator A5045052742 @default.
• W2056075515 creator A5055180595 @default.
• W2056075515 date "1999-03-01" @default.
• W2056075515 modified "2023-10-18" @default.
• W2056075515 title "Mitochondrial Telomere-binding Protein from Candida parapsilosis Suggests an Evolutionary Adaptation of a Nonspecific Single-stranded DNA-binding Protein" @default.
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