Characterization and developmental expression of single-stranded telomeric DNA-binding proteins from mung bean (Vigna radiata)

We have identified and characterized protein factors from mung bean (Vigna radiata) nuclear extracts that specifically bind the single-stranded G-rich telomeric DNA repeats. Nuclear extracts were prepared from three different types of plant tissue, radicle, hypocotyl, and root, in order to examine changes in the expression patterns of telomere-binding proteins during the development of mung bean. At least three types of specific complexes (A, B, and C) were detected by gel retardation assays with synthetic telomere and nuclear extract from radicle tissue, whereas the two major faster-migrating complexes (A and B) were formed with nuclear extracts from hypocotyl and root tissues. Gel retardation assays also revealed differences in relative amount of each complex forming activity in radicle, hypocotyl, and root nuclear extracts. These data suggest that the expression of telomere-binding proteins is developmentally regulated in plants, and that the factor involved in the formation of complex C may be required during the early stages of development. The binding factors have properties of proteins and are hence designated as mung bean G-rich telomere-binding proteins (MGBP). MGBPs bind DNA substrates with three or more single-stranded TTTAGGG repeats, while none of them show binding affinity to either double-stranded or single-stranded C-rich telomeric DNA. These proteins have a lower affinity to human telomeric sequences than to plant telomeric sequences and do not exhibit a significant binding activity to Tetrahymena telomeric sequence or mutated plant telomeric sequences, indicating that their binding activities are specific to plant telomere. Furthermore, RNase treatment of the nuclear extracts did not affect the complex formation activities. This result indicates that the single-stranded telomere-binding activities may be attributed to a simple protein but not a ribonucleoprotein. The ability of MGBPs to bind specifically the single-stranded TTTAGGG repeats may suggest their in vivo functions in the chromosome ends of plants.     

DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe

Eukaryotic chromosome ends are protected from illicit DNA joining by protein-DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA.     

Sequence-specific DNA recognition by the Myb-like domain of plant telomeric protein RTBP1

We have identified a rice gene encoding a DNA-binding protein that specifically recognizes the telomeric repeat sequence TTTAGGG found in plants. This gene, which we refer to as RTBP1 (rice telomere-binding protein 1), encodes a polypeptide with a predicted molecular mass of 70 kDa. RTBP1 is ubiquitously expressed in various organs and binds DNA with two or more duplex TTTAGGG repeats. The predicted protein sequence includes a single domain at the C terminus with extensive homology to Myb-like DNA binding motif. The Myb-like domain of RTBP1 is very closely related to that of other telomere-binding proteins, including TRF1, TRF2, Taz1p, and Tbf1p, indicating that DNA-binding domains of telomere-binding proteins are well conserved among evolutionarily distant species. To obtain precise information on the sequence of the DNA binding site recognized by RTBP1, we analyzed the sequence-specific binding properties of the isolated Myb-like domain of RTBP1. The isolated Myb-like domain was capable of sequence-specific DNA binding as a homodimer. Gel retardation analysis with a series of mutated telomere probes revealed that the internal GGGTTT sequence in the two-telomere repeats is critical for binding of Myb-like domain of RTBP1, which is consistent with the model of the TRF1.DNA complex showing that base-specific contacts are made within the sequence GGGTTA. To the best of our knowledge, RTBP1 is the first cloned gene in which the product is able to bind double-stranded telomeric DNA in plants. Because the Myb-like domain appears to be a significant motif for a large class of proteins that bind the duplex telomeric DNA, RTBP1 may play important roles in plant telomere function in vivo.     

Identification of centromeric and telomeric DNA‐binding proteins in rice

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G‐rich telomere repeats, respectively. However, the protein components are not fully understood. DNA‐binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA‐specific binding proteins, affinity pull‐down technique was applied in this study to isolate rice centromeric and telomeric DNA‐binding proteins. Fifty‐five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO‐binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA‐binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull‐down technique is effective in the isolation of sequence‐specific binding proteins and will be applicable in future studies of centromere and telomere proteins.     

Identification and characterization of a putative telomere end-binding protein from Tetrahymena thermophila.

Telomeric DNA of Tetrahymena thermophila consists of a long stretch of (TTGGGG)n double-stranded repeats with a single-stranded (TTGGGG)2 3' overhang at the end of the chromosome. We have identified and characterized a protein that specifically binds to a synthetic telomeric substrate consisting of duplex DNA and the 3' telomeric repeat overhang. This protein is called TEP (telomere end-binding protein). A change from G to A in the third position of the TTGGGG overhang repeat converts the substrate to a human telomere analog and reduces the binding affinity approximately threefold. Changing two G's to C's in the TTGGGG repeats totally abolishes binding. However, permutation of the Tetrahymena repeat sequence has only a minor effect on binding. A duplex structure adjacent to the 3' overhang is required for binding, although the duplex need not contain telomeric repeats. TEP does not bind to G-quartet DNA, which is formed by many G-rich sequences. TEP has a greatly reduced affinity for RNA substrates. The copy number of TEP is at least 2 x 10(4) per cell, and it is present under different conditions of cell growth and development, although its level varies. UV cross-linking experiments show that TEP has an apparent molecular mass of approximately 65 kDa. Unlike other telomere end-binding proteins, TEP is sensitive to high salt concentrations.     

A Chlamydomonas protein that binds single-stranded G-strand telomere DNA.

We have identified a protein in Chlamydomonas reinhardtii cell extracts that specifically binds the single-stranded (ss) Chlamydomonas G-strand telomere sequence (TTTTAGGG)n. This protein, called G-strand binding protein (GBP), binds DNA with two or more ss TTTTAGGG repeats. A single polypeptide (M(r) 34 kDa) in Chlamydomonas extracts binds (TTTTAGGG)n, and a cDNA encoding this G-strand binding protein was identified by its expression of a G-strand binding activity. The cDNA (GBP1) sequence predicts a protein product (Gbp1p) that includes two domains with extensive homology to RNA recognition motifs (RRMs) and a region rich in glycine, alanine and arginine. Antibody raised against a peptide within Gbp1p reacted with both the 34 kDa polypeptide and bound G-strand DNA-protein complexes in gel retardation assays, indicating that GBP1 encodes GBP. Unlike vertebrate heteronuclear ribonucleoproteins, GBP does not bind the cognate telomere RNA sequence UUUUAGGG in gel retardation, North-Western or competition assays. Thus, GBP is a new type of candidate telomere binding protein that binds, in vitro, to ss G-strand telomere DNA, the primer for telomerase, and has domains that have homology to RNA binding domains in other proteins.     

Characterization of the DNA binding features of Saccharomyces castellii Cdc13p

The principal function of Saccharomyces cerevisiae Cdc13p is to provide a loading platform to recruit complexes that provide end protection and telomere replication. We isolated the Saccharomyces castellii Cdc13p homolog (scasCdc13p) and characterized the in vitro DNA binding features of the purified recombinant scasCdc13p. The full-length scasCdc13p binds specifically to G-rich single-stranded telomeric DNA, and not to double-stranded DNA or the C-rich strand. Moreover, the minimal binding site for scasCdc13p is the octamer 5'-GTGTCTGG-3' of the S.castellii telomeric sequence. The scasCdc13p displayed a high affinity binding, where four individual nucleotide residues were found to be of most importance for the sequence specificity. Nonetheless, scasCdc13p binds the telomeric repeats from various other species, including the human. In spite of considerable divergence in telomere repeat length and sequence between these species, a conserved Cdc13p binding motif was detected. Among the budding yeasts this conserved Cdc13p binding site overlaps the Rap1p binding site. Together, these data implicate scasCdc13p as a telomere end-binding protein with a potential role in the regulation of telomere maintenance in vivo. Moreover, the results suggest that Rap1p and Cdc13p act together to preserve the conserved core present within the otherwise highly divergent btelomeric sequences among a wide variety of yeasts.     

Coordinated Interactions of Multiple POT1-TPP1 Proteins with Telomere DNA

Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging the DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated double-stranded sequence 5′-TTAGGG-3′ in vertebrates, followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding, we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of a G-quadruplex secondary structure, whereas the altered telomere sequences were devoid of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared with the wild-type telomere sequence. Finally, we show that a 48-nucleotide DNA with a telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.     

POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition.     

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.     

Interaction of an Arabidopsis RNA-binding protein with plant single-stranded telomeric DNA modulates telomerase activity

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a single-stranded 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 telomere-binding proteins. Although proteins that bind single-stranded 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-of-flight 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 telomere-binding protein 1), binds specifically the single-stranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplast-localized 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.     

Heterogeneous nuclear ribonucleoprotein A3 binds single-stranded telomeric DNA and inhibits telomerase extension in vitro

Telomeres are dynamic DNA-protein complexes at the end of linear chromosomes. Maintenance of functional telomeres is required for chromosome stability, and to avoid the activation of DNA damage response pathway and cell cycle arrest. Telomere-binding proteins play crucial roles in the maintenance of functional telomeres. In this study, we employed affinity pull-down and proteomic approach to search for novel proteins that interact with the single-stranded telomeric DNA. The proteins identified by two-dimensional gel electrophoresis were further characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF-TOF tandem MS. Among the five identified proteins, we report here the biochemical properties of a novel protein, hnRNP A3. The purified hnRNP A3 bound specifically to G-rich strand, but not to C-rich strand or double-stranded telomeric DNA. The RRM1 (RNA recognition motif 1) domain, but not RRM2, of hnRNP A3 is sufficient to confer specific binding to the telomeric sequence. In addition, we present evidence that hnRNP A3 can inhibit telomerase extension in vitro. These biochemical properties of hnRNP A3 suggest that hnRNP A3 can participate in telomere regulation in vivo.     

An evolutionary change in telomere sequence motif within the plant section Asparagales had significance for telomere nucleoprotein complexes

In association with a phylogenetic tree of Asparagales, our previous results showed that a distinct clade included plant species where the ancestral, Arabidopsis-type of telomeric repeats (TTTAGGG)n had been partially, or fully, replaced by the human-type telomeric sequence (TTAGGG)n. Telomerases of these species synthesize human repeats with a high error rate in vitro. Here we further characterize the structure of telomeres in these plants by analyzing the overall arrangement of major and minor variants of telomeric repeats using fluorescence in situ hybridization on extended DNA strand(s). Whilst the telomeric array is predominantly composed of the human variant of the repeat, the ancestral, Arabidopsis-type of telomeric repeats was ubiquitously observed at one of the ends and/or at intercalary positions of extended telomeric DNAs. Another variant of the repeat typical of Tetrahymena was observed interspersed in about 20% of telomerics. Micrococcal nuclease digestions indicated that Asparagales plants with a human-type of telomere have telomeric DNA organised into nucleosomes. However, unexpectedly, the periodicity of the nucleosomes is not significantly shorter than bulk chromatin as is typical of telomeric chromatin. Using electrophoretic mobility shift assays we detected in Asparagales plants with a human type of telomere a 40-kDa protein that forms complexes with both Arabidopsis- and human-type G-rich telomeric strands. However, the protein shows a higher affinity to the ancestral Arabidopsis-type sequence. Two further proteins were found, a 25-kDa protein that binds specifically to the ancestral sequence and a 15-kDa protein that binds to the human-type telomeric repeat. We discuss how the organisation of the telomere repeats in Asparagales may have arisen and stabilised the new telomere at the point of mutation.     

Characterization of single stranded telomeric DNA-binding proteins in cultured soybean (Glycine max) cells

We have identified and characterized a protein factor in soybean (Glycine max) nuclear extracts that binds to plant single stranded telomeric DNA repeats. A single DNA-protein complex was detected in gel retardation assays using synthetic telomeres and nuclear extracts. The protein forming this complex was designated soy-bean (Glycine max) single stranded telomeric DNA-binding protein (Gm-STBP). Gm-STBP binds to single stranded telomeric DNA containing more than two repeats. It does not bind to Tetrahymena, human or mutated plant telomere sequences, and its binding activity is not affected by RNase treatment. Gm-STBP activity gradually decreased after suspension cultures entered stationary phase. A slower migrating band was formed with extracts of earlier and later phases of soybean suspension cultures. Our findings suggest that binding of Gm-STBP to plant single stranded telomeric DNA may play a role in the proper functioning of telomeres during development.     

Specific association of a small protein with the telomeric DNA-protein complex during the onset of leaf senescence in Arabidopsis thaliana

Telomeres and their changes in length throughout the life span of cells have been intensively investigated in different organisms. Telomere length is assumed to control replicative senescence in mammalian cells. However, only very few data are available on the developmental dynamics of plant telomeres. Here, changes of telomere length and DNA-protein structure of Arabidopsis thaliana telomeres were analysed in different stages of development, with the main focus resting on the transition from pre-senescent to senescent leaves. The lengths of the telomeres, ranging from ca. 2.0 to 6.5 kb, do not significantly change during plant development indicating that telomere length is not involved in differentiation and replicative senescence nor in post-mitotic senescence of A. thaliana. In dedifferentiated cultured cells a slight increase in length can be determined. The nucleoprotein structure of the telomeric DNA was investigated by gel mobility shift assays, with synthetic oligonucleotides and nuclear protein extracts derived from four defined stages of post-mitotic leaf senescence. In all four stages, a highly salt-resistant DNA-protein complex was formed with the double-stranded as well as with the single-stranded G-rich telomeric DNA. An additional DNA-protein complex was identified in nuclear protein extracts isolated from plants in the transition stage from pre-senescence to senescence. The protein components of the DNA-protein complexes were analysed on native PAGE and SDS-PAGE gels. A protein of 67 kDa (ATBP1) bound to the telomeric DNA in all developmental stages. An additional protein of merely 22 kDa (ATBP2) was associated via protein-protein interaction with ATBP1 to form a higher-order complex exclusively during the onset of senescence. DNA interaction of this higher-order protein complex seems to be restricted to double-stranded telomeric DNA. The defined period of ATBP1/ATBP2 complex formation with the telomeric DNA probably indicates that ATBP2 is involved in the onset of post-mitotic leaf senescence by either disturbing an established or establishing an additional function exhibited by the telomeres in the interphase nuclei.     

Characterization of nucleoprotein complexes in plants with human-type telomere motifs.

A conserved feature of telomeres is the 3'-overhang of their G-rich strand. These G-overhangs function as substrates for telomerase-mediated strand extension, and are critical for end-protection of telomeres. These functions and their regulations are mediated by specific G-overhang binding proteins. In species of the plant order Asparagales, telomere motifs have diverged from a type typical of the plant Arabidopsis thaliana (TTTAGGG)(n) to a type typical of human (TTAGGG)(n). Presumably, this change in motif had an impact on the structure of the telomere and/or the binding of telomeric proteins, including the G-overhang binding proteins. Therefore, we analyse here nucleoprotein complexes formed by protein extracts from plants possessing human-type telomeres (Muscari armeniacum and Scilla peruviana). Proteins were characterized that bind to the G-rich strand of both telomere motifs, or to the ancestral Arabidopsis-type motif alone, but none bound to double-stranded or C-rich complementary strand telomere motifs. We demonstrate the size, sequence-specificity and thermostability of these DNA-binding proteins. We also analysed the formation of complexes from renatured protein fractions after SDS-PAGE (sodium-dodecyl-sulphate polyacrylamide-gel-electrophoresis). We discuss the evolutionary consequences of protein binding flexibility, to act on both ancestral and present telomeric sequences. Of particular interest is that the ancestral repeat, which is thought not to form the telomere, binds the proteins most strongly. These data are discussed in line with other known plant telomere-binding proteins and with the complex nature of the telomere in Asparagales carrying a human-type motif.     

Chromosome-specific distribution of nucleotide substitutions in telomeric repeats of rice (Oryza sativa L.)

Examination of the genomic sequence of the telomere region makes it possible to understand the evolution of the structure of chromosomal ends. We compared the genomic sequences of 14 chromosomal ends of rice, Oryza sativa, L., on the basis of the variation in TTTAGGG repeats. In the proximal telomere repeats, nucleotide substitution occurred more frequently than in the more distal repeats. The most significant diversity was observed at the 1st, 2nd, or 3rd position of TTTAGGG, suggesting that T has been a target of mutation preferentially. Copies of ATTAGGG, CTTAGGG, GTTAGGG, TTCAGGG, TTGAGGG, or TATAGGG were arrayed in tandem, or the same subtypes were located close to each other. The substituted variants were accumulated in chromosomes 2L, 3L, 7L, and 10S but not in the ends of the other chromosomes. In contrast, deletion variants, almost all of which were TTTAGGG to TTAGGG, were dispersed over approximately 4.9% of the sequenced telomere repeats. In summary, the rice proximal telomeric arrays were composed of blocks of at least 6 types of substituted variants and the canonical sequence in a chromosome-specific manner. These results suggest that the variants might arise from the rapid expansion of a single mutation rather than from the gradual accumulation of random mutations.