RESUMO
In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.
Assuntos
Proteínas de Ligação a Telômeros , Telômero , Animais , Regulação da Expressão Gênica , Genoma , Mamíferos/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2. The structure provides a molecular basis for how dsRBMs recognize the shape, and also more surprisingly, the sequence of the dsRNA. The unexpected direct readout of the RNA primary sequence by dsRBMs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity at the R/G site of GluR-2. More generally, our findings suggest a solution to the sequence-specific paradox faced by many dsRBM-containing proteins that are involved in post-transcriptional regulation of gene expression.
Assuntos
Adenosina Desaminase/química , RNA de Cadeia Dupla/química , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Precursores de RNA/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA , Ratos , Receptores de AMPA/genética , Alinhamento de SequênciaRESUMO
Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.
Assuntos
Complexo Shelterina , Proteínas de Ligação a Telômeros , Proteína 2 de Ligação a Repetições Teloméricas , Dano ao DNA , Fosforilação , Proteômica , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , HumanosRESUMO
This Special Issue highlights the advantages of using combined approaches to explore chromatin molecular complexes [...].
Assuntos
Cromatina , Genoma , Cromatina/genética , Montagem e Desmontagem da CromatinaRESUMO
Telomeres form specialized chromatin that protects natural chromosome termini from being recognized as DNA double-strand breaks. Plants possess unusual blunt-ended telomeres that are unable to form t-loops or complex with single-strand DNA binding proteins, raising the question of the mechanism behind their protection. We have previously suggested that blunt-ended telomeres in Arabidopsis thaliana are protected by Ku, a DNA repair factor with a high affinity for DNA ends. In nonhomologous end joining, Ku loads onto broken DNA via a channel consisting of positively charged amino acids. Here, we demonstrate that while association of Ku with plant telomeres also depends on this channel, Ku's requirements for DNA binding differ between DNA repair and telomere protection. We show that a Ku complex proficient in DNA loading but impaired in translocation along DNA is able to protect blunt-ended telomeres but is deficient in DNA repair. This suggests that Ku physically sequesters blunt-ended telomeres within its DNA binding channel, shielding them from other DNA repair machineries.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , DNA de Plantas/genética , Autoantígeno Ku/metabolismo , Telômero/genética , Reparo do DNA/genética , Autoantígeno Ku/genéticaRESUMO
Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.
Assuntos
Prolina/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Serina/metabolismo , Sobrevivência Celular , Modelos Moleculares , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Telomeric repeat binding factor 2 (TRF2) folds human telomeres into loops to prevent unwanted DNA repair and chromosome end-joining. The N-terminal basic domain of TRF2 (B-domain) protects the telomeric displacement loop (D-loop) from cleavage by endonucleases. Repressor activator protein 1 (Rap1) binds TRF2 and improves telomeric DNA recognition. We found that the B-domain of TRF2 stabilized the D-loop and thus reduced unwinding by BLM and RPA, whereas the formation of the Rap1-TRF2 complex restored DNA unwinding. To understand how the B-domain of TRF2 affects DNA binding and D-loop processing, we analyzed DNA binding of full-length TRF2 and a truncated TRF2 construct lacking the B-domain. We quantified how the B-domain improves TRF2's interaction with DNA via enhanced long-range electrostatic interactions. We developed a structural envelope model of the B-domain bound on DNA. The model revealed that the B-domain is flexible in solution but becomes rigid upon binding to telomeric DNA. We proposed a mechanism for how the B-domain stabilizes the D-loop.
Assuntos
DNA/química , Proteínas de Ligação a Telômeros/metabolismo , Telômero/química , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , DNA/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos , Complexo Shelterina , Eletricidade Estática , Telômero/metabolismoRESUMO
The repetitive telomeric DNA at chromosome ends is protected from unwanted repair by telomere-associated proteins, which form the shelterin complex in mammals. Recent works have provided new insights into the mechanisms of how human shelterin assembles and recruits telomerase to telomeres. Inhibition of telomerase activity and telomerase recruitment to chromosome ends is a promising target for anticancer therapy. Here, we summarize results of quantitative assessments and newly emerged structural information along with the status of the most promising approaches to telomerase inhibition in cancer cells. We focus on the mechanism of shelterin assembly and the mechanisms of how shelterin affects telomerase recruitment to telomeres, addressing the conceptual dilemma of how shelterin allows telomerase action and regulates other essential processes. We evaluate how the identified critical interactions of telomerase and shelterin might be elucidated in future research of new anticancer strategies.
Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Ligação Proteica , Complexo Shelterina , Telomerase/antagonistas & inibidores , Telomerase/química , Proteínas de Ligação a Telômeros/químicaRESUMO
More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.
Assuntos
DNA/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Ligação Competitiva/efeitos dos fármacos , DNA/química , DNA/genética , Polarização de Fluorescência , Humanos , Cinética , Ligação Proteica/efeitos dos fármacos , Complexo Shelterina , Cloreto de Sódio/farmacologia , Espectrometria de Fluorescência , Eletricidade Estática , Ressonância de Plasmônio de Superfície , Telômero/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genéticaRESUMO
Double-stranded regions of the telomeres are recognized by proteins containing Myb-like domains conferring specificity toward telomeric repeats. Although biochemical and structural studies revealed basic molecular principles involved in DNA binding, relatively little is known about evolutionary pathways leading to various types of Myb domain-containing proteins in divergent species of eukaryotes. Recently we identified a novel type of telomere-binding protein YlTay1p from the yeast Yarrowia lipolytica containing two Myb domains (Myb1, Myb2) very similar to the Myb domain of mammalian TRF1 and TRF2. In this study we prepared mutant versions of YlTay1p lacking Myb1, Myb2, or both Myb domains and found that YlTay1p carrying either Myb domain exhibits preferential affinity to both Y. lipolytica (GGGTTAGTCA)(n) and human (TTAGGG)(n) telomeric sequences. Quantitative measurements of the protein binding to telomeric DNA revealed that the presence of both Myb domains is required for a high affinity of YlTay1p to either telomeric repeat. Additionally, we performed detailed thermodynamic analysis of the YlTay1p interaction with its cognate telomeric DNA, which is to our knowledge the first energetic description of a full-length telomeric-protein binding to DNA. Interestingly, when compared with human TRF1 and TRF2 proteins, YlTay1p exhibited higher affinity not only for Y. lipolytica telomeres but also for human telomeric sequences. The duplication of the Myb domain region in YlTay1p thus produces a synergistic effect on its affinity toward the cognate telomeric sequence, alleviating the need for homodimerization observed in TRF-like proteins possessing a single Myb domain.
Assuntos
Proteínas Fúngicas/química , Proteínas Proto-Oncogênicas c-myb/química , Proteína 1 de Ligação a Repetições Teloméricas/química , Yarrowia/metabolismo , Sequência de Aminoácidos , Anisotropia , Biofísica/métodos , Calorimetria/métodos , Mapeamento Cromossômico , Evolução Molecular , Proteínas Fúngicas/metabolismo , Humanos , Cinética , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Telômero/ultraestrutura , TermodinâmicaRESUMO
Novel approach to functionalized polycyclic aromatic hydrocarbons (PAHs) is presented. Incorporation of cyclic nitrone framework into the structure of PAHs was studied with respect to their anti-proliferative activities and interaction with double stranded DNA. Theoretical docking studies and UV titration methods were used for preliminary evaluation of binding of new PAH derivatives to DNA structure.
Assuntos
DNA/metabolismo , Desenho de Fármacos , Óxidos de Nitrogênio/química , Hidrocarbonetos Policíclicos Aromáticos/síntese química , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/química , Células HCT116 , Humanos , Substâncias Intercalantes/síntese química , Substâncias Intercalantes/química , Substâncias Intercalantes/toxicidade , Células K562 , Células MCF-7 , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Hidrocarbonetos Policíclicos Aromáticos/química , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Relação Estrutura-AtividadeRESUMO
The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map suggests that REST forms a torus that can accommodate DNA double-helix in the central hole. Additionally, we quantitatively described REST binding to the canonical DNA sequence of the neuron-restrictive silencer element. We developed protocols for the expression and purification of full-length REST and the shortened variant REST-N62 produced by alternative splicing. We tested the mutual interaction of full-length REST and the splicing variant REST-N62. Revealed structure-function relationships of master neuronal repressor REST will allow finding new biological ways of prevention and treatment of neurodegenerative disorders and diseases.
RESUMO
Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the ß-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.
Assuntos
Proteínas Nucleares/química , Oligonucleotídeos/química , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SoluçõesRESUMO
Transcription is often the first biosynthetic event of viral infection. Viruses produce preferentially viral transcriptional regulators (vTRs) essential for expressing viral genes and regulating essential host cell proteins to enable viral genome replication. As vTRs are unique viral proteins that promote the transcription of viral nucleic acid, vTRs interact with host proteins to suppress detection and immune reactions to viral infection. Thus, vTRs are promising therapeutic targets that are sequentially and structurally distinct from host cell proteins. Here, we review vTRs of three human oncoviruses: HBx of hepatitis B virus, HBZ of human T-lymphotropic virus type 1, and Rta of Epstein-Barr virus. We present three cunningly exciting and dangerous transcription strategies that make viral infections so efficient. We use available structural and functional knowledge to critically examine the potential of vTRs as new antiviral-anticancer therapy targets. For each oncovirus, we describe (i) the strategy of viral genome transcription; (ii) vTRs' structure and binding partners essential for transcription regulation; and (iii) advantages and challenges of vTR targeting in antiviral therapies. We discuss the implications of vTR regulation for oncogenesis and perspectives on developing novel antiviral and anticancer strategies.
RESUMO
Proteins that bind telomeric DNA modulate the structure of chromosome ends and control telomere function and maintenance. It has been shown that AtTRB (Arabidopsis thaliana telomere-repeat-binding factor) proteins from the SMH (single-Myb-histone) family selectively bind double-stranded telomeric DNA and interact with the telomeric protein AtPOT1b (A. thaliana protection of telomeres 1b), which is involved in telomere capping. In the present study, we performed the first quantitative DNA-binding study of this plant-specific family of proteins. Interactions of full-length proteins AtTRB1 and AtTRB3 with telomeric DNA were analysed by electrophoretic mobility-shift assay, fluorescence anisotropy and surface plasmon resonance to reveal their binding stoichiometry and kinetics. Kinetic analyses at different salt conditions enabled us to estimate the electrostatic component of binding and explain different affinities of the two proteins to telomeric DNA. On the basis of available data, a putative model explaining the binding stoichiometry and the protein arrangement on telomeric DNA is presented.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Histonas/metabolismo , Telômero/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ensaio de Desvio de Mobilidade Eletroforética , Polarização de Fluorescência , Histonas/genética , Cinética , Modelos Biológicos , Ligação Proteica/genética , Ressonância de Plasmônio de Superfície , Telômero/genéticaRESUMO
Human telomeric repeat binding factors TRF1 and TRF2 along with TIN2 form the core of the shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1-TIN2-TRF2 complex formation in solution at physiological conditions. Fluorescence cross-correlation spectroscopy was used to describe the complex assembly by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substitutes TRF2 on TIN2. We assessed also the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1-TIN2-TRF2 core complex.
Assuntos
Complexo Shelterina , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Complexo Shelterina/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/genéticaRESUMO
Telomeres are nucleoprotein structures ensuring the stability of eukaryotic chromosome ends. Two protein families, TRFL (TFL-Like) and SMH (Single-Myb-Histone), containing a specific telobox motif in their Myb domain, have been identified as potential candidates involved in a functional nucleoprotein structure analogous to human "shelterin" at plant telomeres. We analyze the DNA-protein interaction of the full-length and truncated variants of AtTRB1, a SMH-family member with a typical structure: N-terminal Myb domain, central H1/5 domain and C-terminal coiled-coil. We show that preferential interaction of AtTRB1 with double-stranded telomeric DNA is mediated by the Myb domain, while the H1/5 domain is involved in non-specific DNA-protein interaction and in the multimerization of AtTRB1.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Telômero/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Clonagem Molecular , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Histonas/metabolismo , Humanos , Proteínas Oncogênicas v-myb/metabolismo , Ligação ProteicaRESUMO
We analyzed DNA duplexes modified at central guanine residues by monofunctional Ru(II) arene complexes [(eta(6)-arene)Ru(II)(en)(Cl)](+) (arene = tetrahydroanthracene or p-cymene, Ru-THA or Ru-CYM, respectively). These two complexes were chosen as representatives of two different classes of Ru(II) arene compounds for which initial studies revealed different binding modes: one that may involve DNA intercalation (tricyclic-ring Ru-THA) and the other (mono-ring Ru-CYM) that may not. Ru-THA is approximately 20 times more toxic to cancer cells than Ru-CYM. The adducts of Ru-THA and Ru-CYM have contrasting effects on the conformation, thermodynamic stability, and polymerization of DNA in vitro. In addition, the adducts of Ru-CYM are removed from DNA more efficiently than those of Ru-THA. Interestingly, the mammalian nucleotide excision repair system has low efficiency for excision of ruthenium adducts compared to cisplatin intrastrand crosslinks.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Compostos Organometálicos/química , Rutênio/química , Sequência de Bases , Calorimetria , DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Relação Estrutura-AtividadeRESUMO
Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.