Identification of a Putative Mitochondrial Telomere-binding Protein of the Yeast Candida parapsilosis

Abstract

Terminal segments (telomeres) of linear mitochondrial DNA (mtDNA) molecules of the yeast Candida parapsilosis consist of large sequence units repeated in tandem. The extreme ends of mtDNA terminate with a 5 single-stranded overhang of about 110 nucleotides. We identified and purified a mitochondrial telomere-binding protein (mtTBP) that specifically recognizes a synthetic oligonucleotide derived from the extreme end of this linear mtDNA. MtTBP is highly resistant to protease and heat treatments, and it protects the telomeric probe from degradation by various DNA-modifying enzymes. Resistance of the complex to bacterial alkaline phosphatase suggests that mtTBP binds the very end of the molecule. We purified mtTBP to near homogeneity using DNA affinity chromatography based on the telomeric oligonucleotide covalently bound to Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fractions revealed the presence of a protein with an apparent molecular mass of ϳ15 kDa. UV cross-linking and gel filtration chromatography experiments suggested that native mtTBP is probably a homo-oligomer. MtTBP of C. parapsilosis is the first identified protein that specifically binds to telomeres of linear mitochondrial DNA.Although circularity is accepted as the most common form of mitochondrial DNA (mtDNA), there are numerous examples of organisms with linear mtDNA, raising questions about their evolutionary origin and mode of replication (1). Mitochondrial genomes of many yeast species are represented by linear DNA molecules with defined terminal structures (2-5). Two different types of yeast linear mtDNA have been described. Type 1 (Pichia pijperi, Pichia jadinii, Williopsis mrakii) possesses inverted terminal repeats with a covalently closed singlestranded hairpin at each ends (6). In contrast, terminal structures of type 2 linear mtDNA (Candida parapsilosis, Candida salmanticensis, Pichia philodendra) have inverted terminal repeats consisting of units repeated in tandem. The variable number of repeat units generates a population of mtDNA molecules of heterogeneous size. The largest fraction of mtDNA molecules of C. parapsilosis is represented by the shortest molecules, which contain only one incomplete tandem repeat unit. Regardless of the number of repetitions, both ends of the mtDNA molecule terminate at a unique position in the last repeat unit sequence thus leaving an open structure with a 5Ј single-stranded extension of about 110 nucleotides (7). This is in contrast to typical nuclear telomeres of most eukaryotes consisting of simple arrays of tandem G-rich repeats that run 5Ј to 3Ј at the chromosome ends and protrude as 3Ј overhangs (8). The structure of the mitochondrial telomeres of C. parapsilosis is more reminiscent of those of Tetrahymena mtDNA, although the telomeric tandem repeat unit of the latter is considerably shorter (9, 10). The role of terminal structures in the replication and maintenance of C. parapsilosis mtDNA is not known. Their unique organization raised many intri...

Linear versus circular mitochondrial genomes: intraspecies variability of mitochondrial genome architecture in Candida parapsilosis

“…they have to (i) ensure the complete replication of the linear genophore, (ii) mask the ends from DNA repair machinery and (iii) protect them from exonucleolytic degradation and/or endto-end fusions. Identification of a specific mitochondrial telomere-binding protein (mtTBP) (Tomaska et al, 1997;Nosek et al, 1999) and mitochondrial telomeric loop (t-loop) structures similar to the t-loops present at the ends of mammalian chromosomes (Griffith et al, 1999) substantiates the analogy. Observation of extragenomic telomeric minicircles in mitochondria of several yeast species (Tomaska et al, 2000) uncovered yet another general theme in telomere biology.…”

Amplification of Telomeric Arrays via Rolling-circle Mechanism

“…As mitochondrial telomeres seem to fulfill analogous biological functions and display all the essential features of their nuclear counterparts, i.e. they consist of arrays of tandem repeats and possess a protruding single-stranded overhang (16) that is capped by specific telomere-binding protein (33,34) and telomeric loops (27), mitochondria harboring linear mitochondrial genomes may represent a molecular fossil (35) of the original telomere replication strategy (36). Telomeric loops seem to represent a general, evolutionary conserved property of terminal tandem arrays and may explain several telomererelated phenomena such as capping function, masking the ends from DNA repair machinery providing a solution to the endreplication problem, and telomere rapid deletion (7,8).…”

Mitochondrial Telomere-binding Protein from Candida parapsilosis Suggests an Evolutionary Adaptation of a Nonspecific Single-stranded DNA-binding Protein

“…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 5Ј 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 (7). The properties of mtTBP suggested that it may play an important role in the stabilization and/or replication of linear mtDNA of C. parapsilosis.…”