Telomeric Repeat Binding Factor Research Articles

Chronic replicative stress induced by CCl4 in TRF1 knockout mice recapitulates the origin of large liver cell changes

Large liver cell changes (LLCC) are characterized by pleomorphic large nuclei frequently found in liver diseases as chronic viral hepatitis and liver cirrhosis. The origin of this lesion remains cryptic, but the presence of LLCC is correlated with an increased risk of hepatocellular carcinoma. Telomeric repeat binding factor 1 (TRF1) is part of the shelterin complex and is essential for telomere protection. Ablation of TRF1 induces telomere fragility and fusions and chromosomal instability. In this study, we addressed the role of TRF1 in liver regeneration generating a mouse model with conditional deletion of TRF1 in the liver. TRF1 deletion has no deleterious effects in liver and leads to increased ploidy of hepatocytes after 2/3 hepatectomy. Mice lacking TRF1 in the liver can survive for over one year without any evidence for altered liver function. Importantly, applying chronic replicative stress by frequent carbon tetrachloride (CCl4) injections, TRF1 deleted mice undergo ploidy changes consistent with endoreduplication and develop LLCC like lesions in the liver positive for p21, Cyclin D1 and PCNA as observed in humans. In summary, we provide mechanistic insight into the role of TRF1 in liver regeneration and provide a mouse model recapitulating the clinical features of LLCC.

Nanoparticle-mediated decrease of lamin B1 pools promotes a TRF protein-based adaptive response in cultured cells

In general, nanoparticle-based materials are promising candidates for use in biological systems for diagnostic and therapeutic approaches. However, these materials' actions at the molecular level remain poorly understood. Nanoparticle (silica, silver and diamond)-induced oxidative stress and activation of the NF-κB pathway lead to the depletion of lamin B1 pools, which, in turn, results in upregulation of telomeric repeat binding factor (TRF) protein expression and maintenance of telomere length. In cancer cells, the TRF-based response is independent of the p53 pathway. In fibroblasts with active p53/p21 signaling, the levels of p53 and p21 are elevated and stress-induced premature senescence is observed. These results suggest that nanoparticles promote a telomere-focused cell adaptive response.

Mir-23a induces telomere dysfunction and cellular senescence by inhibiting TRF2 expression.

Telomeric repeat binding factor 2 (TRF2) is essential for telomere maintenance and has been implicated in DNA damage response and aging. Telomere dysfunction induced by TRF2 inhibition can accelerate cellular senescence in human fibroblasts. While previous work has demonstrated that a variety of factors can regulate TRF2 expression transcriptionally and post-translationally, whether microRNAs (miRNAs) also participate in post-transcriptionally modulating TRF2 levels remains largely unknown. To better understand the regulatory pathways that control TRF2, we carried out a large-scale luciferase reporter screen using a miRNA expression library and identified four miRNAs that could target human TRF2 and significantly reduce the level of endogenous TRF2 proteins. In particular, our data revealed that miR-23a could directly target the 3′ untranslated region (3′UTR) of TRF2. Overexpression of miR-23a not only reduced telomere-bound TRF2 and increased telomere dysfunction-induced foci (TIFs), but also accelerated senescence of human fibroblast cells, which could be rescued by ectopically expressed TRF2. Our findings demonstrate that TRF2 is a specific target of miR-23a, and uncover a previously unknown role for miR-23a in telomere regulation and cellular senescence.

Human Rap1 modulates TRF2 attraction to telomeric DNA.

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.

In silico characterization and analysis of RTBP1 and NgTRF1 protein through MD simulation and molecular docking - A comparative study.

Gaining access to sequence and structure information of telomere binding proteins helps in understanding the essential biological processes involve in conserved sequence specific interaction between DNA and the proteins. Rice telomere binding protein (RTBP1) and Nicotiana glutinosa telomere repeat binding factor (NgTRF1) are helix turn helix motif type of proteins that plays role in telomeric DNA protection and length regulation. Both the proteins share same type of domain but till now there is very less communication on the in silico studies of these complete proteins.Here we intend to do a comparative study between two proteins through modeling of the complete proteins, physiochemical characterization, MD simulation and DNA-protein docking. I-TASSER and CLC protein work bench was performed to find out the protein 3D structure as well as the different parameters to characterize the proteins. MD simulation was completed by GROMOS forcefield of GROMACS for 10 ns of time stretch. The simulated 3D structures were docked with template DNA (3D DNA modeled through 3D-DART) of TTTAGGG conserved sequence motif using HADDOCK web server.Digging up all the facts about the proteins it was reveled that around 120 amino acids in the tail part was showing a good sequence similarity between the proteins. Molecular modeling, sequence characterization and secondary structure prediction also indicates the similarity between the protein's structure and sequence. The result of MD simulation highlights on the RMSD, RMSF, Rg, PCA and Energy plots which also conveys the similar type of motional behavior between them. The best complex formation for both the proteins in docking result also indicates for the first interaction site which is mainly the helix3 region of the DNA binding domain. The overall computational analysis reveals that RTBP1 and NgTRF1 proteins display good amount of similarity in their physicochemical properties, structure, dynamics and binding mode.

Defects in subventricular zone pigmented epithelium-derived factor niche signaling in the senescence-accelerated mouse prone-8.

We studied potential changes in the subventricular zone (SVZ) stem cell niche of the senescence-accelerated mouse prone-8 (SAM-P8) aging model. Bromodeoxyuridine (BrdU) assays with longtime survival revealed a lower number of label-retaining stem cells in the SAM-P8 SVZ compared with the SAM-Resistant 1 (SAM-R1) control strain. We also found that in SAM-P8 niche signaling is attenuated and the stem cell pool is less responsive to the self-renewal niche factor pigmented epithelium-derived factor (PEDF). Protein analysis demonstrated stable amounts of the PEDF ligand in the SAM-P8 SVZ niche; however, SAM-P8 stem cells present a significant expression decrease of patatin-like phospholipase domain containing 2, a receptor for PEDF (PNPLA2-PEDF) receptor, but not of laminin receptor (LR), a receptor for PEDF (LR-PEDF) receptor. We observed changes in self-renewal related genes (hairy and enhancer of split 1 (Hes1), hairy and enhancer of split 1 (Hes5), Sox2] and report that although these genes are down-regulated in SAM-P8, differentiation genes (Pax6) are up-regulated and neurogenesis is increased. Finally, sheltering mammalian telomere complexes might be also involved given a down-regulation of telomeric repeat binding factor 1 (Terf1) expression was observed in SAM-P8 at young age periods. Differences between these 2 models, SAM-P8 and SAM-R1 controls, have been previously detected at more advanced ages. We now describe alterations in the PEDF signaling pathway and stem cell self-renewal at a very young age, which could be involved in the premature senescence observed in the SAM-P8 model.

TRF2 acts as a transcriptional regulator in tumor angiogenesis

We recently showed that telomeric repeat-binding factor 2 (TRF2) regulates gene expression to promote angiogenesis. We found that TRF2 is highly expressed in tumor vessels and transcriptionally activates platelet-derived growth factor receptor β to promote endothelial cell angiogenic properties independently of its function in telomere protection. This work identifies TRF2 as a promising dual target for cancer therapy.

Inhibition of RAP1 Enhances Corneal Recovery Following Alkali Injury

Recently, RAP1 (Telomeric Repeat Binding Factor 2, Interacting Protein [TERF2IP]) was discovered as a modulator that selectively regulates nuclear factor light chain kappa enhancer of activated B cells (NFκB) signaling. The roles of RAP1 in regulation of inflammation and angiogenesis for corneal recovery following corneal injury remain poorly understood. The effects of RAP1 deletion on corneal resurfacing and neovascularization in a corneal alkali burn mouse model were examined. Corneal defects and neovascularization were induced in vivo by infliction of an alkali burn to the cornea with 1 N sodium hydroxide solution in RAP1 knock-out (RKO) and wild-type (RWT) mice. Corneal resurfacing was evaluated using slit-lamp biomicroscopy. Neovascularization following injury was evaluated by bright view biomicroscopy and immunofluorescence staining with the endothelial marker platelet endothelial cell adhesion molecule (PECAM). The cytokine profiles of corneal tissue involved in inflammation and neovascularization following injury was compared between RKO and RWT mice. Corneal epithelial cells were isolated for classic scratch wound healing assay and further testing with lipopolysaccharide stimulation. Resurfacing of the burned cornea was accelerated and angiogenesis was suppressed, faster recovery of corneal epithelial cells from classic scratch wound healing and superior tolerance of lipopolysaccharides challenge was observed in the RKO compared to RWT. Molecular investigation revealed that deletion of RAP1 reduced upregulation of inflammatory cytokine (IL1A), finely regulated the expression of angiogenic factor (VEGF), and antiangiogenic factor (PEDF), following injury for better corneal recovery. Deficiency of RAP1 facilitates corneal recovery after injury. Specificity of RAP1 inhibition may lead to design of specific inhibitors of NFκB in the treatment of ocular injuries.

Telomere Repeat-Binding Factor 2 Is Responsible for the Telomere Attachment to the Nuclear Membrane.

Telomeres are nucleoprotein structures that specify ends of eukaryotic chromosomes. They enable complete DNA replication, protect chromosomes from end-to-end fusions, and help organize chromatin structure. These functions are mediated by special telomeric proteins. TRF2 (telomeric repeat-binding factor 2) is an essential component of shelterin, a telomere-binding protein complex. TRF2 induces formation of a special structure of telomeric DNA, counteracts activation of double-strand break response pathway and ataxia telangiectasia mutated kinase pathway at telomeres. Some line of evidence implicates TRF2 in interactions with the nuclear envelope (NE). TRF2 is tightly bound to the nuclear membrane in frog oocytes nucleus, and it was found colocalized with NE or its remnants in mouse cells. Computer analysis of TRF2 amino acid sequence has shown that TRF2 possesses motifs, which resemble rod domain characteristic of intermediate filament proteins. These observations suggest that TRF2 is a good candidate for the attachment of telomeres to the NE in somatic cells.

Trastuzumab increases the sensitivity of HER2-amplified human gastric cancer cells to oxaliplatin and cisplatin by affecting the expression of telomere-associated proteins.

HER2 amplification occurs in ~20% of gastric cancer (GC) cases; however, in gastric and gastroesophageal junction cancer with HER2 gene amplification, trastuzumab in combination with cisplatin (DDP)-based chemotherapy has been reported to improve the oncological outcome. The aim of the present study was to evaluate the combined antitumor efficacy of trastuzumab and various platinum agents in GC cells and to elucidate mechanisms that may be involved in the interaction between trastuzumab and the platinum agents. The in vitro chemosensitivity of the GC cells to platinum agents was evaluated using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit. Treatment with 1.0μg/ml trastuzumab for 48 h significantly increased the sensitivity of NCI-N87 cells with HER2 amplification to oxaliplatin (Oxa) and DDP. This chemosensitivity was most prominent in the NCI-N87 cells, in which the half maximal inhibitory concentration of Oxa and DDP was decreased to ~3.29 and 6.91 times, respectively. The apoptotic effect of the platinum agents was evaluated by double-staining the GC cells with Annexin V-fluorescein isothiocyanate and propodium iodide. Consistent with the chemosensitivity analysis, apoptotic analysis indicated that trastuzumab significantly increased Oxa- and DDP-induced apoptosis in the NCI-N87 cells. Furthermore, the mRNA expression levels of various telomere-associated genes was determined by performing quantitative reverse transcription-polymerase chain reactions in a number of GC cell lines, and revealed that trastuzumab (alone and in combination with DDP) may downregulate the mRNA expression levels of the TPP1, TRF1, TRF2, TRF2IP and POT1 genes. However, western blot analysis demonstrated that trastuzumab (alone and in combination with DDP) may significantly downregulate the protein expression levels of telomeric repeat binding factor 2, protection of telomere 1 and TPP1 (formerly known as TINT1, PTOP and PIP). The results of the present study indicate a potential role of low-dose trastuzumab administration for increasing Oxa and DDP sensitivity in HER2-amplified GC cells, possibly via the downregulation of telomere-associated gene expression.

Abstract IA20: A telomere-dependent DNA damage checkpoint induced by prolonged mitotic arrest

Abstract Telomere shortening and disruption of telomeric components are pathways that induce telomere deprotection. Here we describe another pathway, where prolonged mitotic arrest induces damage signals at telomeres in human cells. Exposure to microtubule drugs, kinesin inhibitors, proteasome inhibitors or the disruption of proper chromosome cohesion resulted in the formation of damage-foci at telomeres. Induction of mitotic telomere deprotection coincided with dissociation of TRF2 (Telomere Repeat binding Factor 2) from telomeres, telomeric 3′-overhang degradation and ATM (Ataxia Telangiectasia Mutated) activation, and could be suppressed by TRF2 overexpression or inhibition of Aurora B kinase. Normal cells that escape from prolonged mitotic arrest halted in the following G1 phase, whereas cells lacking p53 continued to cycle and became aneuploid. We propose a telomere dependent mitotic duration monitoring system that reacts to improper progression through mitosis and will discuss the molecular details and implications of this pathway. Citation Format: Makoto T. Hayashi, Jan Karlseder. A telomere-dependent DNA damage checkpoint induced by prolonged mitotic arrest. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA20. doi:10.1158/1538-7445.CANSUSC14-IA20

Abstract 13060: Abrogation of miR-195 Improves Function in Aged Heart by Preventing Telomere Shortening and Mitochondrial Dysfunction

Background: Recently, we discovered a distinct microRNAs profile in young and old MSCs by microarray which led us to hypothesize that senescence-associated microRNA(miR)-195 plays a critical role in stem cell aging through telomerase reverse transcriptase (tert) deactivation and determined whether direct inhibition of miR-195 in aged myocardium restores juvenile characteristics and improves cardiac function. Methods and Results: To examine the mechanistic participation of miR-195 in cardiac aging and function, we directly injected lentivral miR-195 inhibitor into the normal mice heart through open chest surgery. Interestingly, we observed a significant telomere re-lengthening in myocardium injected with anti-miR-195 as examined by Q-FISH analysis. In addition, heart tissues treated with miR-195 inhibitor markedly restored the expression of anti-aging factors such as Tert, telomeric repeat-binding factor 2 (Trf2) and Sirt1, whereas expression of pro-aging markers including p53 and p16 were reduced. Additionally, we found that mitochondrial activity was significantly improved in miR-195 knocked down heart as examined by cytochrome c oxidase activity assay. To investigate whether abrogation of miR-195 in myocardium improves cardiac function, we performed echocardiography at 4 weeks after injection. The cardiac function was markedly improved in miR-195 knocked down myocardium as evaluated by cardiac performance index, a useful marker for heart failure (0.24±0.05 vs 0.44±0.13, p<0.05, n=6), whereas there was no significant change in systolic function . Furthermore, histological analysis showed that transfection of miR-195 inhibitor markedly reduced fibrosis as well as expression of connective tissue growth factor (CTGF), a fibrogenic master switch, in the aged myocardium as compared to scramble transfected heart. Conclusions: These results demonstrate that abrogation of miR-195 improves cardiac function through restoration of anti-aging factors as well as amelioration of mitochondrial dysfunction. Suppression of miR-195 in aging myocardium is a novel therapeutic strategy for treatment against age-dependent cardiovascular diseases.

TRF2 and lamin A/C interact to facilitate the functional organization of chromosome ends.

Telomeres protect the ends of linear genomes, and the gradual loss of telomeres is associated with cellular ageing. Telomere protection involves the insertion of the 3′ overhang facilitated by telomere repeat-binding factor 2 (TRF2) into telomeric DNA, forming t-loops. We present evidence suggesting that t-loops can also form at interstitial telomeric sequences in a TRF2-dependent manner, forming an interstitial t-loop (ITL). We demonstrate that TRF2 association with interstitial telomeric sequences is stabilized by co-localization with A-type lamins (lamin A/C). We also find that lamin A/C interacts with TRF2 and that reduction in levels of lamin A/C or mutations in LMNA that cause an autosomal dominant premature ageing disorder—Hutchinson Gilford Progeria Syndrome (HGPS)—lead to reduced ITL formation and telomere loss. We propose that cellular and organismal ageing are intertwined through the effects of the interaction between TRF2 and lamin A/C on chromosome structure.

Genome-wide analysis of in vivo TRF1 binding to chromatin restricts its location exclusively to telomeric repeats

Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that protect them from degradation, end-to-end fusions, and fragility. In mammals, telomeres are composed of TTAGGG tandem repeats bound by a protein complex called shelterin, which has fundamental roles in the regulation of telomere protection and length. The telomeric repeat binding factor 1 (TERF1 or TRF1) is one of the components of shelterin and has been shown to be essential for telomere protection. Telomeric repeats can also be found throughout the genome, as Internal or Interstitial Telomeric Sequences (ITSs). Some of the components of shelterin have been described to bind to ITSs as well as other extra-telomeric regions, which in the case of RAP1 exert a key role in transcriptional regulation. Here, we set to address whether TRF1 can be found at extra-telomeric sites both under normal conditions and upon induction of telomere shortening. In particular, we performed a ChIP-sequencing technique to map TRF1 binding sites in MEFs wild-type and deficient for the telomerase RNA component (Terc−/−), with increasingly short telomeres. Our findings indicate that TRF1 is exclusively located at telomeres both under normal conditions, as well as under extreme telomere shortening. These results indicate that in mice not all members of shelterin have extra-telomeric roles as it was described for RAP1.

The Telomeric Protein TRF2 Regulates Angiogenesis by Binding and Activating the PDGFRβ Promoter

Telomeric repeat binding factor 2 (TRF2), which plays a central role in telomere capping, is frequently increased in human tumors. We reveal here that TRF2 is expressed in the vasculature of most human cancer types, where it colocalizes with the Wilms' tumor suppressor WT1. We further show that TRF2 is a transcriptional target of WT1 and is required for proliferation, migration, and tube formation of endothelial cells. These angiogenic effects of TRF2 are uncoupled from its function in telomere capping. Instead, TRF2 binds and transactivates the promoter of the angiogenic tyrosine kinase platelet-derived growth factor receptor β (PDGFRβ). These findings reveal an unexpected role of TRF2 in neoangiogenesis and delineate a distinct function of TRF2 as a transcriptional regulator.

A switch-like dynamic mechanism for the initiation of replicative senescence

Telomeres are specialized structures protecting chromosomes against genome instability. Telomeres shorten with cell division, and replicative senescence is induced when telomeres are badly eroded. Whereas TRF2 (telomeric-repeat binding factor 2), ATM (ataxia telangiectasia mutated) and p53 have been identified involved in senescence induction, how it is triggered remains unclear. Here, we propose an integrated model associating telomere loss with senescence trigger. We characterize the dynamics of telomere shorting and the p53-centered regulatory network. We show that senescence is initiated in a switch-like manner when both the shortest telomere becomes uncapped and the TRF2-ATM-p53-Siah1 positive feedback loop is switched on. This work provides a coherent picture of senescence induction in terms of telomere shortening and p53 activation.

DNA and RNA quadruplex-binding proteins.

Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

Molecular inhibition of telomerase recruitment using designer peptides: an in silico approach

Telomere holds special mechanism for solving end repair problems and maintaining genomic stability. Protection of telomeres 1 (POT1) which belongs to shelterin family is identified as a key protein that recruits telomerase by interacting with telomere repeat binding factors (TRB1-3). Since, deciphering the mechanism through which POT assembles telomerase is of great interest, computational approaches have been undertaken to understand the mechanism in a well- developed model system – Arabidopsis thaliana. As a first step, an untraditional approach was mediated to locate the active site residues on modeled AtPOT1b protein by interaction studies using molecular docking. To keep in trend with the recent developments, peptide construction and validation was promoted as the next step via molecular dynamics simulation studies. Finally, the validated peptides based on propensity score was evaluated for its efficacy as a potent inhibitor for POT and TRB1-3 interactions. The best peptide, namely, (1-2-d) out of 30 designed peptides, was proved to be vital inhibitor by weakening the interacting complexes.

DNA-PKcs-interacting protein KIP binding to TRF2 is required for the maintenance of functional telomeres.

Human telomeres associate with shelterin, a six-protein complex that protects chromosome ends from being recognized as sites of DNA damage. The shelterin subunit TRF2 (telomeric repeat-binding factor 2) protects telomeres by facilitating their organization into the protective capping structure. We have reported previously that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit)-interacting protein KIP associates with telomerase through an interaction with hTERT (human telomerase reverse transcriptase). In the present study, we identify KIP as a novel interacting partner of TRF2. KIP is able to interact with both TRF2 and DNA-PKcs at telomeres. Because KIP is required for the association between TRF2 and DNA-PKcs, the interplay of these three proteins may provide a mechanism for the recruitment of DNA-PKcs to telomeres. We also show that KIP binding to TRF2 enhances the telomere-binding activity of TRF2, suggesting that KIP acts as a positive regulator of TRF2 function. Furthermore, depletion of KIP induces DNA-damage response foci at telomeres, thereby leading to induction of growth arrest, cellular senescence and altered cell cycle distribution. Collectively, our findings suggest that KIP, in addition to its association with catalytically active telomerase, plays important roles in the maintenance of functional telomeres and the regulation of telomere-associated DNA-damage response. Thus KIP represents a new pathway for modulating telomerase and telomere function in cancer.

TRF1 and TRF2 differentially modulate Rad51-mediated telomeric and nontelomeric displacement loop formation in vitro.

A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and nontelomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90-nucleotide telomeric and nontelomeric substrates into telomeric and nontelomeric plasmid templates. We report that preincubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation upon preincubation with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated nontelomeric D-loop formation, possibly explaining how TRF2 promotes nontelomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication.