Euplotes crassus has genes encoding telomere-binding proteins and telomere-binding protein homologs.

We have identified two 1.6 kb macronuclear DNA molecules from Euplotes crassus that hybridize to the alpha subunit of the Oxytricha telomere protein. We have shown that one of these molecules encodes the 51 kDa Euplotes telomere protein while the other appears to encode a homolog of the telomere protein. Although this homolog clearly differs in sequence from the Euplotes telomere protein, the two proteins share extensive amino acid sequence identity with each other and with the alpha subunit of the Oxytricha telomere protein. In all three proteins 35-36% of the amino acids are identical, while 54-56% are similar. The most extended regions of sequence conservation map within the N-terminal section; this section has been shown to comprise the DNA-binding domain in the Euplotes telomere protein. Our findings suggest that some of the conserved amino acids may be involved in DNA recognition and binding. The gene encoding the telomere protein homolog contains two introns; one of these introns is only 24 bp in length. This is the smallest mRNA intron reported to date.     

Sequence-specific and 3'-end selective single-strand DNA binding by the Oxytricha nova telomere end binding protein alpha subunit

Oxytricha nova telomere end binding protein (OnTEBP) specifically recognizes and caps single-strand (T(4)G(4))(2) telomeric DNA at the very 3'-ends of O. nova macronuclear chromosomes. The discovery of proteins homologous to the N-terminal domain of the OnTEBP alpha subunit in Euplotes crassus, Schizosaccharomyces pombe, and Homo sapiens suggests that related proteins are widely distributed in eukaryotes. Previously reported crystal structures of the ssDNA binding domain of the OnTEBP alpha subunit both uncomplexed and complexed with telomeric ssDNA have suggested specific mechanisms for sequence-specific and 3'-end selective recognition of the single-strand telomeric DNA. We now describe comparative binding studies of ssDNA recognition by the N-terminal domain of the OnTEBP alpha subunit. Addition of nucleotides to the 3'-end of the TTTTGGGG telomere repeat decreases the level of alpha binding by up to 7-fold, revealing a modest specificity for a 3'-terminus relative to an internal DNA binding site. Nucleotide substitutions at specific positions within the t(1)t(2)t(3)T(4)G(5)G(6)G(7)G(8) repeat show that base substitutions at some sites do not substantially decrease the binding affinity (<2-fold for lowercase letters), while substitutions at other sites dramatically reduce the binding affinity (>20-fold decrease for the uppercase bold letter). Comparison of the structural and binding data provides unique insights into the ways in which proteins recognize and bind single-stranded DNA.     

Binding linkage in a telomere DNA-protein complex at the ends of Oxytricha nova chromosomes

Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein-protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (K(D-DNA)=1.4 nM). Another fusion protein, constructed without the C-terminal protein-protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (K(D-DNA)=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA-protein stability to protein-protein contacts at a remote site may provide a trigger point for DNA-protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase.     

Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain

The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.     

DNA recognition and binding by the Euplotes telomere protein.

The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.     

Crystal structure of the N-terminal domain of Oxytricha nova telomere end-binding protein alpha subunit both uncomplexed and complexed with telomeric ssDNA.

Oxytricha nova telomere end-binding protein specifically recognizes and caps single strand (T(4)G(4))(n) telomeric DNA at the very 3'-ends of O. nova macronuclear chromosomes. Proteins homologous to the N-terminal domain of OnTEBP alpha subunit have now been identified in Oxytricha trifallax, Stylonychia mytilis, Euplotes crassus, Schizosaccharomyces pombe, and Homo sapiens, suggesting that this protein is widely distributed in eukaryotes. We describe here the crystal structures of the N-terminal single-stranded DNA (ssDNA)-binding domain of O. nova telomere end-binding protein alpha subunit both uncomplexed and complexed with single strand telomeric DNA. These structures show how the N-terminal domain of alpha alone, in the absence of the beta subunit and without alpha dimerization, can bind single-stranded telomeric DNA in a sequence-specific and 3'-end-specific manner. Furthermore, comparison of the uncomplexed and complexed forms of this protein shows that the ssDNA-binding site is largely pre-organized in the absence of ssDNA with modest, but interesting, rearrangements of amino acid side-chains that compose the ssDNA-binding site. The structures described here extend our understanding of structures of O. nova telomeric complexes by adding uncomplexed and complexed forms of monomeric alpha to previously described structures for (alpha 56/ssDNA)(2) dimer and alpha 56/beta 28/ssDNA ternary complexes. We believe that each of these four structures represent intermediates in an ordered assembly/disassembly pathway for O. nova telomeric complexes.     

Characterization of vascular endothelial cell growth factor interactions with the kinase insert domain-containing receptor tyrosine kinase. A real time kinetic study.

The kinase insert domain-containing receptor (KDR) tyrosine kinase mediates calcium mobilization in endothelial cells and plays a key role during physiological and pathological angiogenesis. To provide a detailed understanding of how KDR is activated, we analyzed the kinetics of ligand-receptor interaction using BIAcore. Both predimerized (KDR-Fc) and monomeric (KDR-cbu) receptors were examined with vascular endothelial cell growth factor (VEGF) homodimers and VEGF/placental growth factor (PlGF) heterodimers. VEGF binds to KDR-Fc with ka = 3.6 +/- 0.07e6, kd = 1.34 +/- 0.19e-4, and KD = 37.1 +/- 4.9 pM. These values are similar to those displayed by monomeric KDR where ka = 5.23 +/- 1.4e6, kd = 2.74 +/- 0.76e-4, and KD = 51.7 +/- 5.8 pM were apparent. In contrast, VEGF/PlGF bound to KDR-Fc with ka = 7.3 +/- 1.6e4, kd = 4.4 +/- 1. 2e-4, and KD = 6 +/- 1.2 nM. Thus, the heterodimer displays a 160-fold reduced KD for binding to predimerized KDR, which is mainly a consequence of a 50-fold reduction in ka. We were unable to detect association between VEGF/PlGF and monomeric KDR. However, nanomolar concentrations of VEGF/PlGF were able to elicit weak calcium mobilization in endothelial cells. This latter observation may indicate partial predimerization of KDR on the cell surface or facilitation of binding due to accessory receptors.     

The N-terminal domain of the Agrobacterium tumefaciens telomere resolvase,TelA, regulates its DNA cleavage and rejoining activities

Linear replicons can be found in a minority of prokaryotic organisms, including Borrelia species and Agrobacterium tumefaciens. The problem with replicating the lagging strand end of linear DNAs is circumvented in these organisms by the presence of covalently closed DNA hairpin telomeres at the DNA termini. Telomere resolvases are enzymes responsible for generating these hairpin telomeres from a dimeric replication intermediate through a two-step DNA cleavage and rejoining reaction referred to as telomere resolution. It was previously shown that the agrobacterial telomere resolvase, TelA, possesses ssDNA annealing activity in addition to telomere resolution activity. The annealing activity derives, chiefly, from the N-terminal domain. This domain is dispensable for telomere resolution. In this study, we used activity analyses of an N-terminal domain deletion mutant, domain add back experiments, and protein–protein interaction studies and we report that the N-terminal domain of TelA is involved in inhibitory interactions with the remainder of TelA that are relieved by the binding of divalent metal ions. We also found that the regulation of telomere resolution by the N-terminal domain of TelA extends to suppression of inappropriate enzymatic activity, including hairpin telomere fusion (reaction reversal) and recombination between replicated telomeres to form a Holliday junction.     

Interactions between telomerase and primase physically link the telomere and chromosome replication machinery

In the ciliate Euplotes crassus, millions of new telomeres are synthesized by telomerase and polymerase alpha-primase during macronuclear development in mated cells. Concomitant with de novo telomere formation, telomerase assembles into higher-order complexes of 550 kDa, 1,600 kDa, and 5 MDa. We show here that telomerase is physically associated with the lagging-strand replication machinery in these complexes. Antibodies against DNA primase precipitated telomerase activity from all three complexes from mated cells but not the 280-kDa telomerase complex from vegetatively growing cells. Moreover, when telomerase was affinity purified, primase copurified with enzyme from mated cells but not with the 280-kDa vegetative complex. Thus, the association of telomerase and primase is developmentally regulated. Intriguingly, PCNA (proliferating cell nuclear antigen) was also found in the 5-MDa complex from mated cells. We therefore speculate that this complex is a complete telomere synthesis machine, while the smaller complexes are assembly intermediates. The physical association of telomerase and primase explains the coordinate regulation of telomeric G- and C-strand synthesis and the efficiency of telomere addition in E. crassus.     

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.     

Telomere processing in Euplotes.

In Euplotes crassus millions of telomeres are synthesized during the sexual phase of the life cycle. Since these newly synthesized telomeres are longer than normal macronuclear telomeres, they must be trimmed to the mature size. We have examined the timing and mechanism of this trimming step. We have shown that a sudden decrease in telomere length takes place at a specific time during macronuclear development. The decrease in telomere length is not caused by incomplete replication of the most terminal DNA sequences; rather it is the result of an active processing event that occurs independently of DNA replication. The developmentally regulated telomere shortening that takes place in Euplotes is reminiscent of the sudden reductions in telomere length which have been observed in other eukaryotes.     

A DNA Spiegelmer to staphylococcal enterotoxin B

Bacterial staphylococcal enterotoxin B is involved in several severe disease patterns and it was therefore used as a target for the generation of biologically stable mirror-image oligonucleotide ligands, so called Spiegelmers. The toxin is a 28 kDa protein consisting of 239 amino acids. Since the full-length protein is not accessible to chemical peptide synthesis, a stable domain of 25 amino acids was identified as a suitable selection target. DNA in vitro selection experiments were carried out against the equivalent mirror-image D-peptide domain resulting in high affinity D-DNA aptamers. As expected, the corresponding enantiomeric L-DNA Spiegelmer showed comparable binding characteristics to the L-peptide domain. Moreover, the Spiegelmer bound the whole protein target with only slightly reduced affinity. Dissociation constants of both peptide-oligonucleotide complexes were measured in the range of 200 nM, whereas the Spiegelmer binding to the full-length protein was determined at approximately 420 nM. These data demonstrate the possibility to identify Spiegelmers against large protein targets by a domain approach.     

Structural reorganization and the cooperative binding of single-stranded telomere DNA in Sterkiella nova

In Sterkiella nova, alpha and beta telomere proteins bind cooperatively with single-stranded DNA to form a ternary alpha.beta.DNA complex. Association of telomere protein subunits is DNA-dependent, and alpha-beta association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, alpha and DNA first form a stable alpha.DNA complex followed by the addition of beta in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different, with DeltaCp = -305 +/- 3 cal mol(-1) K(-1) for alpha binding with DNA and DeltaCp = -2010 +/- 20 cal mol(-1) K(-1) for the addition of beta to complete the alpha.beta.DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed alpha.beta complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of beta. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length beta or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of beta.     

Domain structure and DNA binding regions of beta protein from bacteriophage lambda

beta protein from bacteriophage lambda promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. beta protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of beta protein in the presence and absence of DNA using limited proteolysis. Residues 1-130 form an N-terminal "core" domain that is resistant to proteases in the absence of DNA, residues 131-177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178-261 of beta protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of beta protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of beta protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of beta protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1-177 of beta protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA.     

Cdc13 delivers separate complexes to the telomere for end protection and replication

In Saccharomyces cerevisiae, the telomere binding protein Cdc13 mediates telomere replication by recruiting telomerase, and also performs an essential function in chromosome end protection. We show here that delivery of the Stn1 protein to the telomere, by fusing the DNA binding domain of Cdc13 (DBD(CDC13)) to Stn1, is sufficient to rescue the lethality of a cdc13 null strain and, hence, provide end protection. Telomere replication is still defective in this strain, but can be restored by delivering telomerase to the telomere as a DBD(CDC13)-telomerase fusion. These results establish Stn1 as the primary effector of chromosome end protection, whereas the principal function of Cdc13 is to provide a loading platform to recruit complexes that provide end protection and telomere replication.     

TPP1 as a versatile player at the ends of chromosomes

Telomeres, the ends of linear eukaryotic chromosomes, are tandem DNA repeats and capped by various telomeric proteins. These nucleoprotein complexes protect telomeres from DNA damage response （DDR）, recombination, and end-to-end fusions, ensuring genome stability. The human telosome/shelterin complex is one of the best-studied telomere-associated protein complexes, made up of six core telomeric proteins TRF1, TRF2, TIN2, RAPI, POT1, and TPPI. TPP1, also known as adrenocortical dysplasia protein homolog （ACD）, is a putative mammalian homolog of TEBP-β and belongs to the oligonucleotide binding （OB）-fold-containing protein family. Three functional domains have been identified within TPP1, the N-terminal OB fold, the POT1 binding recruitment domain （RD）, and the carboxyl-terminal TIN2-interacting domain （TID）. TPP1 can interact with both POT1 and TIN2 to maintain telomere structure, and mediate telomerase recruitment for telomere elongation. These features have indicated TPP1 play an essential role in telomere maintenance. Here, we will review important findings that highlight the functional significance of TPP1, with a focus on its interaction with other telosome components and the telomerase. We will also discuss potential implications in disease therapies.     

Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains

Borrelia burgdorferi, a causative agent of Lyme disease, has a highly unusual segmented genome composed of both circular molecules and linear DNA replicons terminated by covalently closed hairpin ends or telomeres. Replication intermediates of the linear molecules are processed into hairpin telomeres via the activity of ResT, a telomere resolvase. We report here the results of limited proteolysis and mass spectroscopy to identify two main structural domains in ResT, separated by a chymotrypsin cleavage site between residues 163 and 164 of the 449 amino acid protein. The two domains have been overexpressed and purified. DNA electrophoretic mobility shift assays revealed that the C-terminal domain (ResT(164-449)) displays sequence-specific DNA binding to the box 3,4,5 region of the telomere, while the N-terminal domain (ResT(1-163)) exhibits sequence-independent DNA binding activity. Further analysis by DNase I footprinting supports a model for telomere resolution in which the hairpin binding module of the N-terminal domain is delivered to the box 1,2 region of the telomere through its tethering to ResT(164-449). Conversely, ResT(1-164) may play an important regulatory role by modulating both sequence-specific DNA binding activity and catalysis by the C-terminal domain.