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.     

Identification of human Rap1: implications for telomere evolution

It has been puzzling that mammalian telomeric proteins, including TRF1, TRF2, tankyrase, and TIN2 have no recognized orthologs in budding yeast. Here, we describe a human protein, hRap1, that is an ortholog of the yeast telomeric protein, scRap1p. hRap1 has three conserved sequence motifs in common with scRap1, is located at telomeres, and affects telomere length. However, while scRap1 binds telomeric DNA directly, hRap1 is recruited to telomeres by TRF2. Extending the comparison of telomeric proteins to fission yeast, we identify S. pombe Taz1 as a TRF ortholog, indicating that TRFs are conserved at eukaryotic telomeres. The data suggest that ancestral telomeres, like those of vertebrates, contained a TRF-like protein as well as Rap1. We propose that budding yeast preserved Rap1 at telomeres but lost the TRF component, possibly concomitant with a change in the telomeric repeat sequence.     

spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast.

Telomeres are essential for genome integrity. scRap1 (S. cerevisiae Rap1) directly binds to telomeric DNA and regulates telomere length and telomere position effect (TPE) by recruiting two different groups of proteins to its RCT (Rap1 C-terminal) domain. The first group, Rif1 and Rif2, regulates telomere length. The second group, Sir3 and Sir4, is involved in heterochromatin formation. On the other hand, human TRF1 and TRF2, as well as their fission yeast homolog, Taz1, directly bind to telomeric DNA and negatively regulate telomere length. Taz1 also plays important roles in TPE and meiosis. Human Rap1, the ortholog of scRap1, negatively regulates telomere length and appears to be recruited to telomeres by interacting with TRF2. Here, we describe two novel fission yeast proteins, spRap1 (S. pombe Rap1) and spRif1 (S. pombe Rif1), which are orthologous to scRap1 and scRif1, respectively. spRap1 and spRif1 are independently recruited to telomeres by interacting with Taz1. The rap1 mutant is severely defective in telomere length control, TPE, and telomere clustering toward the spindle pole body (SPB) at the premeiotic horsetail stage, indicating that spRap1 has critical roles in these telomere functions. The rif1 mutant also shows some defects in telomere length control and meiosis. Our results indicate that Taz1 provides binding sites for telomere regulators, spRap1 and spRif1, which perform the essential telomere functions. This study establishes the similarity of telomere organization in fission yeast and humans.     

Zinc finger protein overexpressed in colon carcinoma interacts with the telomeric protein hRap1

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We found an interaction between OZF and the telomeric hRap1 protein with a yeast two-hybrid screen. hRap1 (TERF2IP) is an ortholog of the yeast telomeric protein, scRap1 originally identified as a regulator of telomere length. In HeLa cells, it interacts with TRF2, a telomere repeat binding factor whose inactivation causes a dysregulation of telomere length and structure. Immunoprecipitation with anti-hRap1 antibodies in conditions that allow the purification of proteins associated with hRap1, demonstrated that OZF binds to hRap1 in HeLa cells. Using deletion mutants, we mapped the interacting domain of each protein. The three zinc fingers at the C-terminus of OZF interact with a region of hRap1 located downstream of the coil domain. It involves a stretch of at least 25 amino acids at the C-terminus of hRap1 that interact with TRF2. This suggests that OZF overexpression in tumours may alter the balance between hRap1 and other telomeric proteins and therefore that OZF function may be linked to telomere regulation.     

Counting of Rif1p and Rif2p on Saccharomyces cerevisiae telomeres regulates telomere length

Telomere length is negatively regulated by proteins of the telomeric DNA-protein complex. Rap1p in Saccharomyces cerevisiae binds the telomeric TG(1-3) repeat DNA, and the Rap1p C terminus interacts with Rif1p and Rif2p. We investigated how these three proteins negatively regulate telomere length. We show that direct tethering of each Rif protein to a telomere shortens that telomere proportionally to the number of tethered molecules, similar to previously reported counting of Rap1p. Surprisingly, Rif proteins could also regulate telomere length even when the Rap1p C terminus was absent, and tethered Rap1p counting was completely dependent on the Rif proteins. Thus, Rap1p counting is in fact Rif protein counting. In genetic settings that cause telomeres to be abnormally long, tethering even a single Rif2p molecule was sufficient for maximal effectiveness in preventing the telomere overelongation. We show that a heterologous protein oligomerization domain, the mammalian PDZ domain, when fused to Rap1p can confer telomere length control. We propose that a nucleation and spreading mechanism is involved in forming the higher-order telomere structure that regulates telomere length.     

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication

Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.     

Fission yeast Rap1 homolog is a telomere-specific silencing factor and interacts with Taz1p

Taz1p is the fission yeast orthologue of human TRF2, a telomeric repeat-binding protein. Delta(taz1) mutants are defective in telomeric silencing, telomere length control, and meiotic recombination events. A recent report demonstrated that the human Rap1p homolog (hRap1) is recruited to telomere by interaction with TRF2, arguing that the telomere control mechanism of higher eukaryotes is distinct from that of the budding yeast. Taz1p showed a significant similarity to human TRF2, but not with the budding yeast Rap1p (scRap1p). This suggests that Taz1p and TRF2 share common features in telomere regulation. To assess the roles of Taz1p in telomere-related functions in detail, we attempted to identify a protein(s) that interacts with Taz1p by using two-hybrid screening. Interestingly, the sequence analysis of a positive clone revealed a perfect match with a Rap1 homolog in S. pombe (spRap1), which showed a significant homology with scRap1p and hRap1p. Here we show that the spRap1 deficiency in haploid cells is viable, which results in increased telomere length regulation, disruption of telomere silencing, and aberrant meiosis (like the delta(taz1) mutant). This suggests that spRap1p might be recruited to the telomere by Taz1p and play crucial roles in telomere function. Interestingly, the delta(rap1) mutants in fission yeast are defective only for telomere silencing. Therefore, the role of spRap1p may be distinct from that of scRap1p, which is involved in the silencing at both the telomere and mating type locus. Our data, therefore, suggest that the regulation mechanisms of telomere in fission yeast resemble that of higher eukaryotic cells rather than the budding yeast.     

Tay1 protein, a novel telomere binding factor from Yarrowia lipolytica

Inspection of the complete genome of the yeast Yarrowia lipolytica for the presence of genes encoding homologues of known telomere-binding proteins surprisingly revealed no counterparts of typical yeast Myb domain-containing telomeric factors including Rap1 or Taz1. Instead, we identified a gene, YALIOD10923g, encoding a protein containing two Myb domains, exhibiting a high degree of similarity to the Myb domain of human telomeric proteins TRF1 and TRF2 and homologous to an essential fission yeast protein Mug152 whose expression is elevated during meiosis. The protein, which we named Tay1p (telomere-associated in Yarrowia lipolytica 1), was purified for biochemical studies. Using a model Y. lipolytica telomere, we demonstrate that the protein preferentially binds to Y. lipolytica telomeric tracts. Tay1p binds along the telomeric tract as dimers and larger oligomers, and it is able to remodel the telomeric DNA into both looped structures and synaptic complexes of two model telomere DNAs. The ability of Tay1p to induce dimerization of telomeres in vitro goes in line with its oligomeric nature, where each oligomer can employ several Myb domains to form intermolecular telomere clusters. We also provide experimental evidence that Tay1p may be associated with Y. lipolytica telomeres in vivo. Together with its homologues from Schizosaccharomyces pombe and several basidiomycetous fungi (Sánchez-Alonso, P., and Guzman, P. (2008) Fungal Genet. Biol. 45, S54-S62), Tay1p constitutes a novel family of putative telomeric factors whose analysis may be instrumental in understanding the function and evolution of double-stranded DNA telomeric proteins.     

The human Rap1 protein complex and modulation of telomere length

Proper maintenance of telomere length and structure is necessary for normal proliferation of mammalian cells. Mammalian telomere length is regulated by a number of proteins including human repressor activator protein (hRap1), a known association factor of TRF2. To further delineate hRap1 function and its associated proteins, we affinity-purified and identified the hRap1 protein complex through mass spectrometry analysis. In addition to TRF2, we found DNA repair proteins Rad50, Mre11, PARP1 (poly(ADP-ribose) polymerase), and Ku86/Ku70 to be in this telomeric complex. We demonstrated by deletional analysis that Rad-50/Mre-11 and Ku86 were recruited to hRap1 independent of TRF2. PARP1, however, most likely interacted with hRap1 through TRF2. Interestingly, knockdown of endogenous hRap1 expression by small hairpin interference RNA resulted in longer telomeres. In addition, overexpression of full-length and mutant hRap1 that lacked the BRCA1 C-terminal domain functioned as dominant negatives and extended telomeres. Deletion of a novel linker domain of hRap1 (residues 199-223), however, abolished the dominant negative effect of hRap1 overexpression. These results indicate that hRap1 negatively regulates telomere length in vivo and suggest that the linker region of hRap1 may modulate the recruitment of negative regulators of telomere length.     

Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules

We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.     

Telomere binding of the Rap1 protein is required for meiosis in fission yeast.

Telomeres are essential for chromosome integrity, protecting the ends of eukaryotic linear chromosomes during cell proliferation. Telomeres also function in meiosis; a characteristic clustering of telomeres beneath the nuclear membrane is observed during meiotic prophase in many organisms from yeasts to plants and humans, and the role of the telomeres in meiotic pairing and the recombination of homologous chromosomes has been demonstrated in the fission yeast Schizosaccharomyces pombe and in the budding yeast Saccharomyces cerevisiae. Here we report that S. pombe Rap1 is a telomeric protein essential for meiosis. While Rap1 is conserved in budding yeast and humans, schemes for telomere binding vary among species: human RAP1 binds to the telomere through interaction with the telomere binding protein TRF2; S. cerevisiae Rap1, however, binds telomeric DNA directly, and no orthologs of TRF proteins have been identified in this organism. In S. pombe, unlike in S. cerevisiae, an ortholog of human TRF has been identified. This ortholog, Taz1, binds directly to telomere repeats [18] and is necessary for telomere clustering in meiotic prophase. Our results demonstrate that S. pombe Rap1 binds to telomeres through interaction with Taz1, similar to human Rap1-TRF2, and that Taz1-mediated telomere localization of Rap1 is necessary for telomere clustering and for the successful completion of meiosis. Moreover, in taz1-disrupted cells, molecular fusion of Rap1 with the Taz1 DNA binding domain recovers telomere clustering and largely complements defects in meiosis, indicating that telomere localization of Rap1 is a key requirement for meiosis.     

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.     

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。     

TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres

Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.     

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.     

Identification of the Functional Domains of the Telomere Protein Rap1 in Schizosaccharomyces pombe

The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1 ⁺ have revealed that the long N-terminal region (1–456 a.a. [amino acids]) of Rap1 (full length: 693 a.a.) is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457–693 a.a.) containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus) and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.