Tetraplex structure of fission yeast telomeric DNA and unfolding of the tetraplex on the interaction with telomeric DNA binding protein Pot1

To understand the regulation mechanism of fission yeast telomeric DNA, we analysed the structural properties of Gn: d(GnTTAC) (n=2-6) and 4Gn: d(GnTTAC)4 (n=3 and 4), and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). G4, G5 and G6 formed a parallel tetraplex in contrast with no tetraplex formation by G2 and G3. Also, 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The variety of tetraplex structures was governed by the number of consecutive guanines in a single copy and the number of repeats. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. The interaction with mutant Pot1DBD proteins revealed that the ability to unfold the antiparallel tetraplex was strongly correlated with the specific binding affinity for the single-stranded telomeric DNA. The result suggests that the decrease in the free single strand upon the complex formation with Pot1DBD may shift the equilibrium from the tetraplex to the single strand, which may cause the tetraplex unfolding. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.     

Mutational analyses of a single-stranded telomeric DNA binding domain of fission yeast pot1: conflict with X-ray crystallographic structure

To understand the telomere regulation mechanism in relation to cell aging and cancer, we examined the single-stranded telomeric DNA binding domain (ssDBD) of fission yeast telomere-binding protein Pot1 by constructing a series of deletion mutants. We found that Pot1(1-182) (amino acids 1-182) stably expressed in Escherichia coli without any degradation retained a stable folded structure and functional telomeric DNA binding activity, indicating that Pot1(1-182) corresponds to ssDBD. We investigated the amino acids of Pot1(1-182) involved in single-stranded telomeric DNA recognition by constructing a series of site-directed mutants. Although the previously reported X-ray crystallographic structure suggests that 12 amino acids contact the telomeric DNA, an electrophoretic mobility shift assay and isothermal titration calorimetry analyses of the binding ability of the site-directed mutants indicated that only five amino acids significantly contributed to telomeric DNA recognition. We conclude that the contribution to recognition is quite different in magnitude among the amino acids judged to contact the target by X-ray crystallographic structure.     

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.     

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.     

The yeast telomere-binding protein RAP1 binds to and promotes the formation of DNA quadruplexes in telomeric DNA.

The protein RAP1 is essential for the maintenance of the telomeres of Saccharomyces cerevisiae and binds in vitro to multiple sites found within the TG1-3 telomeric repeats. We show here that, in addition to its known binding activity for double-stranded DNA, RAP1 binds sequence-specifically to the GT-strands. This indicates that RAP1 is the protein that binds to the telomeric terminal GT-tails. Furthermore, we have found that RAP1 binds to and promotes the formation of G-tetrads, i.e. DNA quadruplexes, in GT-strand oligonucleotides at nanomolar concentrations. The formation of DNA quadruplexes appears to involve the intermolecular association of GT-strands. The minimal DNA-binding domain of RAP1 (DBD) binds only to double-stranded DNA, so that the novel DNA-binding activity we have found involves regions of the protein located outside of the DBD. The finding that a telomeric protein promotes the formation of G-tetrads argues for the use of DNA quadruplexes in telomere association.     

Effect of cationic comb-type copolymer on quadruplex folding of human telomeric DNA

Cationic comb-type copolymer (CCC) consisting of a polycationic backbone and abundant graft water-soluble chains exhibited considerable stabilization effect on DNA hybrids, such as double- and triple-stranded DNAs. Here, we describe the effect of CCC on antiparallel G-quadruplex folding of human telomeric DNA, d(GGGTTA)(n) in the presence of sodium ions. CCC did not significantly alter the circular dichroism (CD) spectra of d((GGGTTA)(3)GGG) and d((GGGTTA)(7)GGG) indicating that the CCC did not influence the antiparallel folding of the telomeric repeats. Hence, the ionic interaction of CCC with the DNA sequence did not interfere with specific interaction of the DNA with sodium ions to form G-quartets. Interestingly, CCC did not change the melting temperature of the d((GGGTTA)(3)GGG) suggesting negligible stabilizing effect of CCC on the antiparallel quadruplex structure.     

Guanine tetraplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats

Secondary structures of the G-rich strand of human telomere DNA fragments G₃(TTAG₃)_n, n = 1–16, have been studied by means of circular dichroism spectroscopy and PAGE, in solutions of physiological potassium cation concentrations. It has been found that folding of these fragments into tetraplexes as well as tetraplex thermostabilities and enthalpy values depend on the number of TTAG₃ repeats. The suggested topologies include, e.g. antiparallel and parallel bimolecular tetraplexes, an intramolecular antiparallel tetraplex, a tetraplex consisting of three parallel chains and one antiparallel chain, a poorly stable parallel intramolecular tetraplex, and both parallel and antiparallel tetramolecular tetraplexes. G₃(TTAG₃)₃ folds into a single, stable and very compact intramolecular antiparallel tetraplex. With an increasing repeat number, the fragment tetraplexes surprisingly are ever less thermostable and their migration and enthalpy decrease indicate increasing irregularities or domain splitting in their arrangements. Reduced stability and different topology of lengthy telomeric tails could contribute to the stepwise telomere shortening process.     

Tethering telomeric double- and single-stranded DNA-binding proteins inhibits telomere elongation

Mammalian telomeres are composed of G-rich repetitive double-stranded (ds) DNA with a 3' single-stranded (ss) overhang and associated proteins that together maintain chromosome end stability. Complete replication of telomeric DNA requires de novo elongation of the ssDNA by the enzyme telomerase, with telomeric proteins playing a key role in regulating telomerase-mediated telomere replication. In regards to the protein component of mammalian telomeres, TRF1 and TRF2 bind to the dsDNA of telomeres, whereas POT1 binds to the ssDNA portion. These three proteins are linked through either direct interactions or by the proteins TIN2 and TPP1. To determine the biological consequence of connecting telomeric dsDNA to ssDNA through a multiprotein assembly, we compared the effect of expressing TRF1 and POT1 in trans versus in cis in the form of a fusion of these two proteins, on telomere length in telomerase-positive cells. When expressed in trans these two proteins induced extensive telomere elongation. Fusing TRF1 to POT1 abrogated this effect, inducing mild telomere shortening, and generated looped DNA structures, as assessed by electron microscopy, consistent with the protein forming a complex with dsDNA and ssDNA. We speculate that such a protein bridge between dsDNA and ssDNA may inhibit telomerase access, promoting telomere shortening.     

The telomeric protein Pot1 from Schizosaccharomyces pombe binds ssDNA in two modes with differing 3′ end availability

Telomere protection and length regulation are important processes for aging, cancer and several other diseases. At the heart of these processes lies the single-stranded DNA (ssDNA)-binding protein Pot1, a component of the telomere maintenance complex shelterin, which is present in species ranging from fission yeast to humans. Pot1 contains a dual OB-fold DNA-binding domain (DBD) that fully confers its high affinity for telomeric ssDNA. Studies of S. pombe Pot1-DBD and its individual OB-fold domains revealed a complex non-additive behavior of the two OB-folds in the context of the complete Pot1 protein. This behavior includes the use of multiple distinct binding modes and an ability to form higher order complexes. Here we use NMR and biochemical techniques to investigate the structural features of the complete Pot1-DBD. These experiments reveal one binding mode characterized by only subtle alternations to the individual OB-fold subdomain structures, resulting in an inaccessible 3′ end of the ssDNA. The second binding mode, which has equivalent affinity, interacts differently with the 3′ end, rendering it available for interaction with other proteins. These findings suggest a structural switch that contributes to telomere end-protection and length regulation.     

The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence

Telomeric DNA can form duplex regions or single-stranded loops that bind multiple proteins, preventing it from being processed as a DNA repair intermediate. The bases within these regions are susceptible to damage; however, mechanisms for the repair of telomere damage are as yet poorly understood. We have examined the effect of three thymine (T) analogs including uracil (U), 5-fluorouracil (5FU) and 5-hydroxymethyluracil (5hmU) on DNA–protein interactions and DNA repair within the GGTTAC telomeric sequence. The replacement of T with U or 5FU interferes with Pot1 (Pot1pN protein of Schizosaccharomyces pombe) binding. Surprisingly, 5hmU substitution only modestly diminishes Pot1 binding suggesting that hydrophobicity of the T-methyl group likely plays a minor role in protein binding. In the GGTTAC sequence, all three analogs can be cleaved by DNA glycosylases; however, glycosylase activity is blocked if Pot1 binds. An abasic site at the G or T positions is cleaved by the endonuclease APE1 when in a duplex but not when single-stranded. Abasic site formation thermally destabilizes the duplex that could push a damaged DNA segment into a single-stranded loop. The inability to enzymatically cleave abasic sites in single-stranded telomere regions would block completion of the base excision repair cycle potentially causing telomere attrition.     

Tetrahelical forms of the fragile X syndrome expanded sequence d(CGG)(n) are destabilized by two heterogeneous nuclear ribonucleoprotein-related telomeric DNA-binding proteins

Formations of hairpin and tetrahelical structures by the trinucleotide repeat sequence d(CGG)(n) might contribute to its expansion in fragile X syndrome. Here we show that tetraplex structures of d(CGG)(n) are destabilized by two mammalian heterogeneous nuclear ribonucleoprotein-related tetraplex telomeric DNA-binding and -stabilizing proteins, quadruplex telomeric DNA-binding protein 42 (qTBP42) (Sarig, G., Weisman-Shomer, P., Erlitzki, R., and Fry, M. (1997) J. Biol. Chem. 272, 4474-4482) and unimolecular quadruplex telomeric DNA-binding protein 25 (uqTBP25) (Erlitzki, R., and Fry, M. (1997) J. Biol. Chem. 272, 15881-15890). Blunt-ended and 3'-tailed or 3'- and 5'-tailed bimolecular tetraplex structures of d(CGG)(n) and guanine-sparse 20-/46-mer partial DNA duplex were progressively destabilized by increasing amounts of qTBP42 or uqTBP25 in time-dependent and ATP- or Mg(2+)-independent reactions. By contrast, tetraplex structures of telomeric and IgG sequences or guanine-rich double-stranded DNA resisted destabilization by qTBP42 or uqTBP25. Increased stability of tetraplex d(CGG)(n) in the presence of K(+) or Na(+) ions or at lowered reaction temperature diminished the destabilizing activity of uqTBP25. The contrasting stabilization of tetraplex telomeric DNA and destabilization of tetraplex d(CGG)(n) by qTBP42 and uqTBP25 suggested that sequence or structural differences between these tetraplexes might serve as cues for the differential stabilizing/destabilizing activities.     

Structural basis for telomeric single-stranded DNA recognition by yeast Cdc13

The essential budding yeast telomere-binding protein Cdc13 is required for telomere replication and end protection. Cdc13 specifically binds telomeric, single-stranded DNA (ssDNA) 3' overhangs with high affinity using an OB-fold domain. We have determined the high-resolution solution structure of the Cdc13 DNA-binding domain (DBD) complexed with a cognate telomeric ssDNA. The ssDNA wraps around one entire face of the Cdc13-DBD OB-fold in an extended, irregular conformation. Recognition of the ssDNA bases occurs primarily through aromatic, basic, and hydrophobic amino acid residues, the majority of which are evolutionarily conserved among budding yeast species and contribute significantly to the energetics of binding. Contacting five of 11 ssDNA nucleotides, the large, ordered beta2-beta3 loop is crucial for complex formation and is a unique elaboration on the binding mode commonly observed in OB-fold proteins. The sequence-specific Cdc13-DBD/ssDNA complex presents a complementary counterpoint to the interactions observed in the Oxytricha nova telomere end-binding and Schizosaccharomyces pombe Pot1 complexes. Analysis of the Cdc13-DBD/ssDNA complex indicates that molecular recognition of extended single-stranded nucleic acids may proceed via a folding-type mechanism rather than resulting from specific patterns of hydrogen bonds. The structure reported here provides a foundation for understanding the mechanism by which Cdc13 recognizes GT-rich heterogeneous sequences with both unusually strong affinity and high specificity.     

Identification of the determinants for the specific recognition of single-strand telomeric DNA by Cdc13

The single-strand overhang present at telomeres plays a critical role in mediating both the capping and telomerase regulation functions of telomeres. The telomere end-binding proteins, Cdc13 in Saccharomyces cerevisiae, Pot1 in higher eukaryotes, and TEBP in the ciliated protozoan Oxytricha nova, exhibit sequence-specific binding to their respective single-strand overhangs. S. cerevisiae telomeres are composed of a heterogeneous mixture of GT-rich telomeric sequence, unlike in higher eukaryotes which have a simple repeat that is maintained with high fidelity. In yeast, the telomeric overhang is recognized by the essential protein Cdc13, which coordinates end-capping and telomerase activities at the telomere. The Cdc13 DNA-binding domain (Cdc13-DBD) binds these telomere sequences with high affinity (3 pM) and sequence specificity. To better understand the basis for this remarkable recognition, we have investigated the binding of the Cdc13-DBD to a series of altered DNA substrates. Although an 11-mer of GT-rich sequence is required for full binding affinity, only three of these 11 bases are recognized with high specificity. This specificity differs from that observed in the other known telomere end-binding proteins, but is well suited to the specific role of Cdc13 at yeast telomeres. These studies expand our understanding of telomere recognition by the Cdc13-DBD and of the unique molecular recognition properties of ssDNA binding.     

RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity

T loops and telomeric G-quadruplex (G4) DNA structures pose a potential threat to genome stability and must be dismantled to permit efficient telomere replication. Here we implicate the helicase RTEL1 in the removal of telomeric DNA secondary structures, which is essential for preventing telomere fragility and loss. In the absence of RTEL1, T loops are inappropriately resolved by the SLX4 nuclease complex, resulting in loss of the telomere as a circle. Depleting SLX4 or blocking DNA replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(-/-) cells but failed to suppress telomere fragility. Conversely, stabilization of telomeric G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient cells but had no impact on TC formation or telomere loss. We propose that RTEL1 performs two distinct functions at telomeres: it disassembles T loops and also counteracts telomeric G4-DNA structures, which together ensure the dynamics and stability of the telomere.     

Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection

The POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryotes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells. Here, we report the crystal structure at a resolution of 1.73 A of the N-terminal half of human POT1 (hPOT1) protein bound to a telomeric single-stranded DNA (ssDNA) decamer, TTAGGGTTAG, the minimum tight-binding sequence indicated by in vitro binding assays. The structure reveals that hPOT1 contains two oligonucleotide/ oligosaccharide-binding (OB) folds; the N-terminal OB fold binds the first six nucleotides, resembling the structure of the S. pombe Pot1pN-ssDNA complex, whereas the second OB fold binds and protects the 3' end of the ssDNA. These results provide an atomic-resolution model for chromosome end-capping.     

The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro

Human TIN2 interacts with the telomeric-DNA-binding protein TRF1, suppresses telomere elongation in telomerase-positive cells, and may control telomere length by modulating telomere structure. To test the latter idea, we developed an in vitro assay, using biotinylated telomeric DNA probes and streptavidin–agarose, to quantify the ability of TRF1 and TIN2 to stimulate interactions of telomeric DNA tracts with each other (probe clustering). This assay revealed that TRF1 alone had weak probe-clustering activity, but TIN2 stimulated activity fivefold to tenfold. A dominant-negative TIN2 mutant protein that increased telomere length in vivo disrupted probe clusters formed by TRF1 and TIN2, suggesting that the ability to stimulate telomeric DNA interactions is important for telomere-length regulation. Unlike TRF1, TIN2 did not form homodimers. We propose that TIN2 alters the conformation of TRF1, which favours a tertiary telomeric structure that hinders telomerase from gaining access to telomeres.     

The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA.

TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA.     

TRF2 controls telomeric nucleosome organization in a cell cycle phase-dependent manner

Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization.