Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2.     

POT1 and TRF2 cooperate to maintain telomeric integrity

Mammalian telomeric DNA contains duplex TTAGGG repeats and single-stranded overhangs. POT1 (protection of telomeres 1) is a telomere-specific single-stranded DNA-binding protein, highly conserved in eukaryotes. The biological function of human POT1 is not well understood. In the present study, we demonstrate that POT1 plays a key role in telomeric end protection. The reduction of POT1 by RNA interference led to the loss of telomeric single-stranded overhangs and induced apoptosis, chromosomal instability, and senescence in cells. POT1 and TRF2 interacted with each other to form a complex with telomeric DNA. A dominant negative TRF2, TRF2(DeltaBDeltaM), bound to POT1 and prevented it from binding to telomeres. POT1 overexpression protected against TRF2(DeltaBDeltaM)-induced loss of telomeric single-stranded overhangs, chromosomal instability, and senescence. These results demonstrate that POT1 and TRF2 share in part in the same pathway for telomere capping and suggest that POT1 binds to the telomeric single-stranded DNA in the D-loop and cooperates with TRF2 in t-loop maintenance.     

TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection.     

TRF1 binds a bipartite telomeric site with extreme spatial flexibility.

TRF1 is a key player in telomere length regulation. Because length control was proposed to depend on the architecture of telomeres, we studied how TRF1 binds telomeric TTAGGG repeat DNA and alters its conformation. Although the single Myb-type helix-turn-helix motif of a TRF1 monomer can interact with telomeric DNA, TRF1 predominantly binds as a homodimer. Systematic Evolution of Ligands by Exponential enrichment (SELEX) with dimeric TRF1 revealed a bipartite telomeric recognition site with extreme spatial variability. Optimal sites have two copies of a 5'-YTAGGGTTR-3' half-site positioned without constraint on distance or orientation. Analysis of binding affinities and DNase I footprinting showed that both half-sites are simultaneously contacted by the TRF1 dimer, and electron microscopy revealed looping of the intervening DNA. We propose that a flexible segment in TRF1 allows the two Myb domains of the homodimer to interact independently with variably positioned half-sites. This unusual DNA binding mode is directly relevant to the proposed architectural role of TRF1.     

Structure,dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD⁺ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.     

How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures

Human telomeres consist of tandem arrays of TTAGGG sequence repeats that are specifically bound by two proteins, TRF1 and TRF2. They bind to DNA as preformed homodimers and have the same architecture in which the DNA-binding domains (Dbds) form independent structural units. Despite these similarities, TRF1 and TRF2 have different functions at telomeres. The X-ray crystal structures of both TRF1- and TRF2-Dbds in complex with telomeric DNA (2.0 and 1.8 angstroms resolution, respectively) show that they recognize the same TAGGGTT binding site by means of homeodomains, as does the yeast telomeric protein Rap1p. Two of the three G-C base pairs that characterize telomeric repeats are recognized specifically and an unusually large number of water molecules mediate protein-DNA interactions. The binding of the TRF2-Dbd to the DNA double helix shows no distortions that would account for the promotion of t-loops in which TRF2 has been implicated.     

端粒结合蛋白TRF2的研究进展

端粒DNA结合蛋白TRF2(TTAGGG repeat binding factor-2)以二聚体形式通过Myb结构域与端粒重复序列TTAGGG结合,并与TRF1、TIN2、Rap1、TINT1及POT1蛋白组成Shelterin蛋白复合物,协同在端粒动态平衡维持过程中起关键作用,进而影响整个基因组的稳定性。此外,TRF2在细胞DNA损伤应答过程中可能发挥重要作用。本文将对TRF2结构和功能研究的最新进展进行综述。     

Platination of telomeric DNA by cisplatin disrupts recognition by TRF2 and TRF1

Telomeres, the nucleoprotein complexes located at the ends of chromosomes, are involved in chromosome protection and genome stability. Telomeric repeat binding factor 1 (TRF1) and telomeric repeat binding factor 2 (TRF2) are the two telomeric proteins that bind to duplex telomeric DNA through interactions between their C-terminal domain and several guanines of the telomeric tract. Since the antitumour drug cisplatin binds preferentially to two adjacent guanines, we have investigated whether cisplatin adducts could affect the binding of TRF1 and TRF2 to telomeric DNA and the property of TRF2 to stimulate telomeric invasion, a process that is thought to participate in the formation of the t-loop. We show that the binding of TRF1 and TRF2 to telomeric sequences selectively modified by one GG chelate of cisplatin is markedly affected by cisplatin but that the effect is more drastic for TRF2 than for TRF1 (3–5-fold more sensitivity for TRF2 than for TRF1). We also report that platinum adducts cause a decrease in TRF2-dependent stimulation of telomeric invasion in vitro. Finally, in accordance with in vitro data, analysis of telomeric composition after cisplatin treatment reveals that 60% of TRF2 dissociate from telomeres.     

Structure, subnuclear distribution, and nuclear matrix association of the mammalian telomeric complex

Mammalian telomeres are composed of long arrays of TTAGGG repeats complexed with the TTAGGG repeat binding factor, TRF. Biochemical and ultrastructural data presented here show that the telomeric DNA and TRF colocalize in individual, condensed structures in the nuclear matrix. Telomeric TTAGGG repeats were found to carry an array of nuclear matrix attachment sites occurring at a frequency of at least one per kb. The nuclear matrix association of the telomeric arrays extended over large domains of up to 20-30 kb, encompassing the entire length of most mammalian telomeres. TRF protein and telomeric DNA cofractionated in nuclear matrix preparations and colocalized in discrete, condensed sites throughout the nuclear volume. FISH analysis indicated that TRF is an integral component of the telomeric complex and that the presence of TRF on telomeric DNA correlates with the compact configuration of telomeres and their association with the nuclear matrix. Biochemical fractionation of TRF and telomeric DNA did not reveal an interaction with the nuclear lamina. Furthermore, ultrastructural analysis indicated that the mammalian telomeric complex occupied sites throughout the nuclear volume, arguing against a role for the nuclear envelope in telomere function during interphase. These results are consistent with the view that mammalian telomeres form nuclear matrix- associated, TRF-containing higher order complexes at dispersed sites throughout the nuclear volume.     

AKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53^-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication.     

TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1’s 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼9–17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼2.8–3.6 κ_BT greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This ‘tag-team proofreading’ represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.     

Sequence-specific DNA recognition by the myb-like domain of the human telomere binding protein TRF1: a model for the protein-DNA complex.

Telomeres consist of tandem arrays of short G-rich sequence motifs packaged by specific DNA binding proteins. In humans the double-stranded telomeric TTAGGG repeats are specifically bound by TRF1 and TRF2. Although telomere binding proteins from evolutionarily distant species are not sequence homologues, they share a Myb-like DNA binding motif. Here we have used gel retardation, primer extension and DNase I footprinting analyses to define the binding site of the isolated Myb-like domain of TRF1 and present a three-dimensional model for its interaction with human telomeric DNA. Our results suggest that the Myb-like domain of TRF1 recognizes a binding site centred on the sequence GGGTTA and that its DNA binding mode is similar to that of the homeodomain-like motifs of the yeast telomere binding protein RAP1. The implications of these findings for recognition of telomeric DNA in general are discussed.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

NMR structure of the hRap1 Myb motif reveals a canonical three-helix bundle lacking the positive surface charge typical of Myb DNA-binding domains.

Mammalian telomeres are composed of long tandem arrays of double-stranded telomeric TTAGGG repeats associated with the telomeric DNA-binding proteins, TRF1 and TRF2. TRF1 and TRF2 contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In the budding yeast, telomeric DNA is associated with scRap1p, which has a central DNA-binding domain that contains two structurally related Myb domains connected by a long linker, an N-terminal BRCT domain, and a C-terminal RCT domain. Recently, the human ortholog of scRap1p (hRap1) was identified and shown to contain a BRCT domain and an RCT domain similar to scRap1p. However, hRap1 contained only one recognizable Myb motif in the center of the protein. Furthermore, while scRap1p binds telomeric DNA directly, hRap1 has no DNA-binding ability. Instead, hRap1 is tethered to telomeres by TRF2. Here, we have determined the solution structure of the Myb domain of hRap1 by NMR. It contains three helices maintained by a hydrophobic core. The architecture of the hRap1 Myb domain is very close to that of each of the Myb domains from TRF1, scRap1p and c-Myb. However, the electrostatic potential surface of the hRap1 Myb domain is distinguished from that of the other Myb domains. Each of the minimal DNA-binding domains, containing one Myb domain in TRF1 and two Myb domains in scRap1p and c-Myb, exhibits a positively charged broad surface that contacts closely the negatively charged backbone of DNA. By contrast, the hRap1 Myb domain shows no distinct positive surface, explaining its lack of DNA-binding activity. The hRap1 Myb domain may be a member of a second class of Myb motifs that lacks DNA-binding activity but may interact instead with other proteins. Other possible members of this class are the c-Myb R1 Myb domain and the Myb domains of ADA2 and Adf1. Thus, while the folds of all Myb domains resemble each other closely, the function of each Myb domain depends on the amino acid residues that are located on the surface of each protein.     

A higher-order entity formed by the flexible assembly of RAP1 with TRF2

Telomere integrity is essential to maintain genome stability, and telomeric dysfunctions are associated with cancer and aging pathologies. In human, the shelterin complex binds TTAGGG DNA repeats and provides capping to chromosome ends. Within shelterin, RAP1 is recruited through its interaction with TRF2, and TRF2 is required for telomere protection through a network of nucleic acid and protein interactions. RAP1 is one of the most conserved shelterin proteins although one unresolved question is how its interaction may influence TRF2 properties and regulate its capacity to bind multiple proteins. Through a combination of biochemical, biophysical and structural approaches, we unveiled a unique mode of assembly between RAP1 and TRF2. The complete interaction scheme between the full-length proteins involves a complex biphasic interaction of RAP1 that directly affects the binding properties of the assembly. These results reveal how a non-DNA binding protein can influence the properties of a DNA-binding partner by mutual conformational adjustments.     

Interaction of human Ku70 with TRF2

Ku, a heterodimer of 70- and 80-kDa subunits, plays a general role in the metabolism of DNA ends in eukaryotic cells, including double-strand DNA break repair, V(D)J recombination, and maintenance of telomeres. We have utilized the yeast two-hybrid system to identify Ku70-interacting proteins other than Ku80. Two reactive clones were found to encode the dimerization domain of TRF2, a mammalian telomeric protein that binds to duplex TTAGGG repeats at chromosome ends. This interaction was confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells overexpressing TRF2. The transfected TFR2 colocalized with Ku70.     

The human telomeric protein TRF1 specifically recognizes nucleosomal binding sites and alters nucleosome structure

Telomeres are dynamic nucleoprotein structures that cap the ends of eukaryotic chromosomes. In humans, the long (TTAGGG)(n) double-stranded telomeric DNA repeats are bound specifically by the two related proteins TRF1 and TRF2, and are organized in nucleosomes. Whereas the role of TRF1 and TRF2 in telomeric function has been studied extensively, little is known about the involvement of telomeric nucleosomes in telomere structures or how chromatin formation may affect binding of the TRFs. Here, we address the question of whether TRF1 is able to bind to telomeric binding sites in a nucleosomal context. We show that TRF1 is able to specifically recognize telomeric binding sites located within nucleosomes, forming a ternary complex. The formation of this complex is strongly dependent on the orientation of binding sites on the nucleosome surface, rather than on the location of the binding sites with respect to the nucleosome dyad. Strikingly, TRF1 binding causes alterations in nucleosome structure without dissociation of histone subunits. These results indicate that nucleosomes contribute to the establishment of a telomeric capping complex, whose structure and dynamics can be modulated by the binding of telomeric factors.     

Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment

Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization. A telomere-binding protein complex from human sperm (hSTBP) has been isolated by detergent treatment and was partially purified. hSTBP specifically binds double-stranded telomeric DNA and does not contain known somatic telomere proteins TRF1, TRF2, and Ku. Surprisingly, the essential component of this complex has been identified as a specific variant of histone H2B. Indirect immunofluorescence shows punctate localization of H2B in sperm nuclei, which in part coincides with telomeric DNA localization established by fluorescent in situ hybridization. Anti-H2B antibodies block interactions of hSTBP with telomere DNA, and spH2B forms specific complex with this DNA in vitro, indicating that this protein plays a role in telomere DNA recognition. We propose that hSTBP participates in the membrane attachment of telomeres that may be important for ordered chromosome withdrawal after fertilization.     

TRF1 inhibits telomere C-strand DNA synthesis in vitro

Human TTAGGG repeat-binding factor 1 (TRF1) is involved in the regulation of telomere length in vivo, but the mechanism of regulation remains largely undefined. We have developed an in vitro system for assessing the effect of TRF1 on DNA synthesis using purified proteins and synthetic DNA substrates. Results reveal that TRF1, when bound to telomeric duplex DNA, inhibits DNA synthesis catalyzed by DNA polymerase alpha/primase (pol alpha). Inhibition required that TRF1 be bound to duplex telomeric DNA as no effect of TRF1 was observed on nontelomeric, random DNA substrates. Inhibition was shown to be dependent on TRF1 concentration and the length of the telomeric duplex region of the DNA substrate. When bound in cis to telomeric duplex DNA, TRF1 was also capable of inhibiting pol alpha-catalyzed DNA synthesis on nontelomeric DNA sequences from positions both upstream and downstream of the extending polymerase. Inhibition of DNA synthesis was shown to be specific for TRF1 but not necessarily for the DNA polymerase used in the extension reaction. In a series of control experiments, we assessed T7 DNA polymerase-catalyzed synthesis on a DNA template containing tandem gal4 operators. In these experiments, the addition of the purified Gal4-DNA binding domain (Gal4-DBD) protein has no effect on the ability of T7 polymerase to copy the DNA template. Interestingly, TRF1 inhibition was observed on telomeric DNA substrates using T7 DNA polymerase. These results suggest that TRF1, when bound to duplex telomeric DNA, serves to block extension by DNA polymerases. These results are discussed with respect to the role of TRF1 in telomere length regulation.