A Protein from Tetrahymena thermophila That Specifically Binds Parallel-Stranded G4-DNA

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a singlestranded 3 overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomerebinding proteins. Although proteins that bind singlestranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants.Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-offlight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomerebinding protein 1), binds specifically the singlestranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplastlocalized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.

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confidence: 99%

Interaction of an Arabidopsis RNA-binding Protein with Plant Single-stranded Telomeric DNA Modulates Telomerase Activity

Kwon

Chung

2004

“…analog of the nuclear telomerase) and proteins that play a role in the stabilization, maintenance, and proper segregation of the linear mtDNA. For nuclear genomes, specific telomere-binding proteins (TBPs) 1 have been identified in protozoa (11)(12)(13)(14)(15)(16), slime molds (17,18), yeasts (19 -26), algae (27), plants (28), and vertebrates (29 -32). These proteins presumably protect the telomere DNA from nucleolytic degradation, mediate telomeretelomere and telomere-nuclear matrix interactions, promote the formation of a typical telomeric structure, and regulate the accessibility of telomeres to the replication machinery (33).…”

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confidence: 99%

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

Tomáška¹,

Nosek

Fukuhara

1997

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...

“…Many different proteins with specificity for binding to G4-DNA have been identified (17)(18)(19)(20)(21)(22). The identification of a G4-DNA-specific nuclease from yeast as the SEP1/KEM1 protein, and the meiotic block at the 4N stage for KEM1-null cells, supports the hypothesis that G4-DNA is involved in meiosis (23, 24, 9).…”

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confidence: 81%

“…The thermodynamic parameters of parrellel-stranded G4-DNA are indicative of its stability with the free energy of formation equal to Ϫ21 kcal/mol and the transition temperature above 82°C (13). DNA sequences able to form G4-DNA have also been found at sites of spontaneous gene rearrangements, point mutations and, along with triplex DNA, have been implicated in causing DNA mutations (9, 14 -16).Many different proteins with specificity for binding to G4-DNA have been identified (17)(18)(19)(20)(21)(22). The identification of a G4-DNA-specific nuclease from yeast as the SEP1/KEM1 protein, and the meiotic block at the 4N stage for KEM1-null cells, supports the hypothesis that G4-DNA is involved in meiosis (23, 24, 9).…”

mentioning

confidence: 99%

The Identification and Characterization of a G4-DNA Resolvase Activity

Harrington¹,

Lan²,

Akman

1997

There is increasing evidence that four-stranded Hoogsteen-bonded DNA structures, G4-DNA, play an important role in cellular processes such as meiosis and recombination. The Hoogsteen-bonded G4-DNA is thermodynamically more stable than duplex DNA, and many guanine-rich genomic DNA sequences with the ability to form G4-DNA have been identified. A protein-dependent activity that resolves G4-DNA into single-stranded DNA has been identified in human placental tissue. The resolvase activity was purified from any apparent nuclease activity and is dependent on NTP hydrolysis and MgCl 2 . Resolvase activity is optimal with 5 mM MgCl 2 . The V max /K m of ATP is 0.055%/min/ M, higher than the V max /K m of the other dNTPs. The products of the resolvase reaction are unmodified single-stranded DNA. The resolvase is not a duplex DNA helicase or a topoisomerase II activity and does not unwind Hoogsteenbonded triplex DNA. Resolvase is a novel activity that unwinds stable G4-DNA structures using a dNTPdependent mechanism producing unmodified singlestranded DNA. Potential in vivo roles for this G4-DNA resolvase activity are discussed.Guanine-rich DNA sequences that form G4-DNA are found in a number of evolutionarily conserved genomic regions such as telomeres, dimerization domains of retroviruses, and the insulin gene promoter (1-5). The four-stranded structure requires a monovalent cation to form, and the DNA strands can run in either a parallel or anti-parallel orientation (6 -8). G4-DNA contains Hoogsteen bonds between the guanine residues forming square planar guanine quartets (9). X-ray crystal diffraction and two-dimensional nuclear magnetic resonance show the sugar backbone can exist in many variations (10 -12). Guanine quartets have unusual stacking energy and high stability. The ability of the O-6 of guanine to form a coordination complex with either Na ϩ or K ϩ in guanine quartets is thought to stabilize telomeres (6 -8). The thermodynamic parameters of parrellel-stranded G4-DNA are indicative of its stability with the free energy of formation equal to Ϫ21 kcal/mol and the transition temperature above 82°C (13). DNA sequences able to form G4-DNA have also been found at sites of spontaneous gene rearrangements, point mutations and, along with triplex DNA, have been implicated in causing DNA mutations (9, 14 -16).Many different proteins with specificity for binding to G4-DNA have been identified (17)(18)(19)(20)(21)(22). The identification of a G4-DNA-specific nuclease from yeast as the SEP1/KEM1 protein, and the meiotic block at the 4N stage for KEM1-null cells, supports the hypothesis that G4-DNA is involved in meiosis (23, 24, 9). More recently, two yeast gene products with specific activity for G4-DNA were cloned and sequenced, G4p1 and G4p2 (19, 20). G4p1 is a homodimer of the gene encoding a novel protein with a domain homologous to the bacterial methionyl-tRNA synthetase dimerization domains, and G4p2 is encoded by a gene identical to genes that appear to function in protein kinase-controlled signal transduction,...