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.     

Gbp1p, a protein with RNA recognition motifs, binds single-stranded telomeric DNA and changes its binding specificity upon dimerization

Gbp1p is a putative telomere-binding protein from Chlamydomonas reinhardtii that contains two RNA recognition motifs (RRMs) which are commonly found in heterogeneous nuclear ribonucleoproteins (hnRNPs). Previously we demonstrated that Gbp1p binds single-stranded DNA (ssDNA) containing the Chlamydomonas telomeric sequence but not the RNA containing the cognate sequence. Here we show that at lower protein concentrations Gbp1 can also bind an RNA containing the cognate sequence. We found that mutation of the two RRM motifs of Gbp1p to match the highly conserved region of hnRNP RRMs did not alter the affinity of Gbp1p for either RNA or DNA. The ability of Gbp1p to associate with either of these two nucleic acids is governed by the dimerization state of the protein. Monomeric Gbp1p associates with either ssDNA or RNA, showing a small binding preference for RNA. Dimeric Gbp1p has a strong preference for binding ssDNA and shows little affinity for RNA. To the best of our knowledge, this is the first example of a protein that qualitatively shifts its nucleic acid binding preference upon dimerization. The biological implications of a telomere-binding protein that is regulated by dimerization are discussed.     

Sequence-specific binding protein of single-stranded and unimolecular quadruplex telomeric DNA from rat hepatocytes

A rat liver nuclear protein, unimolecular quadruplex telomere-binding protein 25, (uqTBP25) is described that binds tightly and specifically single-stranded and unimolecular tetraplex forms of the vertebrate telomeric DNA sequence 5'-d(TTAGGG)n-3'. A near homogeneous uqTBP25 was purified by ammonium sulfate precipitation, chromatographic separation from other DNA binding proteins, and three steps of column chromatography. SDS-polyacrylamide gel electrophoresis and Superdex copyright 200 gel filtration disclosed for uqTBP25 subunit and native Mr values of 25.4 +/- 0.5 and 25.0 kDa, respectively. Sequences of uqTBP25 tryptic peptides were closely homologous, but not identical, to heterogeneous nuclear ribonucleoprotein A1, heterogeneous nuclear ribonucleoprotein A2/B1, and single-stranded DNA-binding proteins UP1 and HDP-1. Complexes of uqTBP25 with single-stranded or unimolecular quadruplex 5'-d(TTAGGG)4-3', respectively, had dissociation constants, Kd, of 2.2 or 13.4 nM. Relative to d(TTAGGG)4, complexes with 5'-r(UUAGGG)4-3', blunt-ended duplex telomeric DNA, or quadruplex telomeric DNA had >10 to >250-fold higher Kd values. Single base alterations within the d(TTAGGG) repeat increased the Kd of complexes with uqTBP25 by 9-215-fold. Association with uqTBP25 protected d(TTAGGG)4 against nuclease digestion, suggesting a potential role for the protein in telomeric DNA transactions.     

Sleep disorders in several pathologic states--endocrine diseases]

Mitochondrial telomere-binding protein (mtTBP) of Candida parapsilosis binds with high affinity to 5' single-stranded overhang of the linear mitochondrial DNA of this yeast (Tomáska, L'., Nosek, J., and Fukuhara, H. (1997) J. Biol. Chem. 272, 3049-3056). Here it is reported that mtTBP is phosphorylated by catalytic subunit of cAMP-dependent protein kinase in vitro. Phosphorylated mtTBP has dramatically reduced ability to bind telomeric oligonucleotide in the gel-mobility retardation assay without affecting the oligomerization of mtTBP in vitro. MtTBP is one of the few mitochondrial proteins and the first mitochondrial single-strand DNA binding proteins that was demonstrated to serve as a substrate for cAMP-dependent protein kinase.     

Construction and characterization of a quadruplex DNA selective single-chain autoantibody from a viable motheaten mouse hybridoma with homology to telomeric DNA binding proteins

An autoantibody produced by a hybridoma derived from a viable motheaten mouse was isolated and found to have moderately high binding affinity for nucleic acids and specific preference for quadruplex DNAs. Polymerase chain reaction primers were designed to link the cloned parental antibody variable region fragments together in a subcloning vector. This single-chain variable fragment construct was then subcloned into the T7 promoter-driven expression vector pET22b(+). The construct contains (N- to C-terminal) a pelB leader sequence, variable heavy chain, glycine-serine polylinker, variable light chain, and biotin mimic peptide "strep-tag" sequence (pelB-VH-linker-VL-strep-tag). The ca. 29 kDa protein was expressed, exported to the periplasmic space of NovaBlue (DE) Escherichia coli, and purified by streptavidin affinity chromatography by binding the fused strep-tag peptide. The specificity of the purified single-chain variable fragment (scFv) for quadruplex and duplex DNAs was evaluated by a radioimmunofilterbinding assay. It retained about 10-fold higher affinity for quadruplexes relative to duplex DNA, a reduction of ca. 4-fold from the relative preferences of the parent IgG. The complementary-determining regions contain sequences that are homologous to or conservatively divergent from the key DNA-binding helix-turn-helix-forming motifs of Myb/RAP1 family telomeric DNA-binding proteins (1-3). The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.     

The yeast Ku heterodimer is essential for protection of the telomere against nucleolytic and recombinational activities

The Ku heterodimer, conserved in a wide range of eukaryotes, plays a multiplicity of roles in yeast. First, binding of Ku, which is composed of a 70 kDa (Hdf1p) and an 80 kDa (Hdf2p) subunit [1-3], to double-strand breaks promotes non-homologous end-to-end joining of DNA [3]. Second, Ku appears to participate in DNA replication, regulating both the number of rounds of replication permissible within the cell cycle and the structure of the initiation complex [3,4]. Furthermore, mutations in HDF1 or HDF2 rapidly reduce telomeric poly (TG1-3) tract size [1-3], hinting also at a possible telomeric function of Ku. We show here that the two subunits of the Ku heterodimer play a key role in maintaining the integrity of telomere structure. Mutations in either Ku subunit increased the single-strandedness of the telomere in a cell-cycle-independent fashion, unlike wild-type cells which form 3' poly(TG1-3) overhangs exclusively in late S phase [5]. In addition, mutations enhanced the instability of elongated telomeres to degradation and recombination. Both Ku subunits genetically interacted with the putative single-stranded telomere-binding protein Cdc13p. We propose that Ku protects the telomere against nucleases and recombinases.     

Intervening to reduce anxiety for women with mild dyskaryosis: do we know what works and why?

Telomeric repeat sequences (TRS) have been identified close to, but not at, the genome termini of several lymphotropic herpesviruses, including human herpesviruses 6 and 7 (HHV-6, HHV-7). The functional significance of these motifs remains uncertain. Since telomeric sequences can mediate stable retention of episomal DNA in yeast, we have tested whether the TRS motifs from HHV-6 might mediate a similar function in human cells. Several candidate sequences were assessed for their ability to provide nuclear retention to an autonomously replicating vector in rapidly dividing human tissue culture cells, including HHV-6 TRS DNA, as well as telomeric DNA from human cells and sequences from Epstein-Barr virus (EBV). However, only a vector carrying the EBV-derived retention mechanism showed a significant level of nuclear retention. Neither the HHV-6 TRS motifs, nor human telomeric sequences, mediated nuclear retention of episomal DNA in human cells.     

Telomere-binding protein from the nuclear envelope of oocytes of the frog Rana temporaria

The identification of telomere-binding protein on the nuclear envelope (NE) of the frog oocyte is reported here. Nuclei and the NE were isolated manually and extracted, and the extracts were used to search for DNA-protein specific interactions. Fragment of the Tetrahymena telomere sequence (T2G4)130 from the plasmid YAC4 was used as a labeled probe. DNA-protein complexes revealed by gel shift assay were cut out of the gel and injected into a guinea pig. The antibodies obtained have common antigenic determinants with keratins; this was shown on Western-blot under conditions of competitive binding. Antibodies were purified on an affinity column with keratins attached and used in the following work. Antibodies recognize one protein with molecular weight 70 kD among the NE proteins; no signal was obtained to proteins isolated from the inner part of the oocyte, but two polypeptides of 70 and 120 kD were detected in the proteins from frog liver nuclei. The 70-kD protein is situated on the NE in a distinct pattern that looks like a network, probably of double-dots as was shown by immunofluorescence of the NE. Electron-microscope immunogold staining showed that the protein is localized in the outer surface of the oocyte NE within cup-like structures attached to the membrane. Combined in situ hybridization using the mammalian telomeric DNA sequence (T2AG3)135 and immunocytochemistry using antibodies showed them to be colocalized in the frog blood cells, so telomere sequence coexists with the protein in the interphase nuclei. Most of the telomeres are fused in highly differentiated blood cells though a double dot signal is visible. The signal appears to be localized in the outer part of the nuclei. The existence of membrane telomere-binding protein allows a discussion of the attachment of the telemeters to the membrane.     

Rap1 protein regulates telomere turnover in yeast

Telomere length is maintained through a dynamic balance between addition and loss of the terminal telomeric DNA. Normal telomere length regulation requires telomerase as well as a telomeric protein-DNA complex. Previous work has provided evidence that in the budding yeasts Kluyveromyces lactis and Saccharomyces cerevisiae, the telomeric double-stranded DNA binding protein Rap1p negatively regulates telomere length, in part by nucleating, by its C-terminal tail, a higher-order DNA binding protein complex that presumably limits access of telomerase to the chromosome end. Here we show that in K. lactis, truncating the Rap1p C-terminal tail (Rap1p-DeltaC mutant) accelerates telomeric repeat turnover in the distal region of the telomere. In addition, combining the rap1-DeltaC mutation with a telomerase template mutation (ter1-kpn), which directs the addition of mutated telomeric DNA repeats to telomeres, synergistically caused an immediate loss of telomere length regulation. Capping of the unregulated telomeres of these double mutants with functionally wild-type repeats restored telomere length control. We propose that the rate of terminal telomere turnover is controlled by Rap1p specifically through its interactions with the most distal telomeric repeats.     

RAP46 is a negative regulator of glucocorticoid receptor action and hormone-induced apoptosis

RAP46 was first identified by its ability to bind the glucocorticoid receptor. It has since been reported to bind several cellular proteins, including the anti-apoptotic protein Bcl-2, but the biological significance of these interactions is unknown. Here we show that RAP46 binds the hinge region of the glucocorticoid receptor and inhibits DNA binding and transactivation by the receptor. We further show that overexpression of RAP46 in mouse thymoma S49.1 cells inhibits glucocorticoid-induced apoptosis. Conversely, glucocorticoid-induced apoptosis and transactivation were enhanced after treating S49.1 cells with the immunosuppressant rapamycin, which down-regulates cellular levels of BAG-1, the mouse homolog of RAP46. The effect of rapamycin can, however, be overcome by overexpression of RAP46. These results together identify RAP46 as a protein that controls glucocorticoid-induced apoptosis through its negative regulatory action on the transactivation property of the glucocorticoid receptor.     

The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA

TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA.     

Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I

In vivo, nucleosomes are formed rapidly on newly synthesized DNA after polymerase passage. Previously, a protein complex from human cells, termed chromatin assembly factor-I (CAF-I), was isolated that assembles nucleosomes preferentially onto SV40 DNA templates that undergo replication in vitro. Using a similar assay, we now report the purification of CAF-I from the budding yeast Saccharomyces cerevisiae. Amino acid sequence data from purified yeast CAF-I led to identification of the genes encoding each subunit in the yeast genome data base. The CAC1 and CAC2 (chromatin assembly complex) genes encode proteins similar to the p150 and p60 subunits of human CAF-I, respectively. The gene encoding the p50 subunit of yeast CAF-I (CAC3) is similar to the human p48 CAF-I subunit and was identified previously as MSI1, a member of a highly conserved subfamily of WD repeat proteins implicated in histone function in several organisms. Thus, CAF-I has been conserved functionally and structurally from yeast to human cells. Genes encoding the CAF-I subunits (collectively referred to as CAC genes) are not essential for cell viability. However, deletion of any CAC gene causes an increase in sensitivity to ultraviolet radiation, without significantly increasing sensitivity to gamma rays. This is consistent with previous biochemical data demonstrating the ability of CAF-I to assemble nucleosomes on templates undergoing nucleotide excision repair. Deletion of CAC genes also strongly reduces silencing of genes adjacent to telomeric DNA; the CAC1 gene is identical to RLF2 (Rap1p localization factor-2), a gene required for the normal distribution of the telomere-binding Rap1p protein within the nucleus. Together, these data suggest that CAF-I plays a role in generating chromatin structures in vivo.     

The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.     

Structural basis of DNA recognition and mechanism of quadruplex formation by the beta subunit of the Oxytricha telomere binding protein

Interactions of the beta subunit of the Oxytricha nova telomere binding protein with the telomeric DNA sequences, d(T4G4)2 and dT6(T4G4)2, have been investigated in vitro using Raman and fluorescence spectroscopies. Raman difference spectra show that the beta subunit binds to both d(T4G4)2 and dT6(T4G4)2 but promotes the formation of a parallel-stranded quadruplex only in dT6(T4G4)2, thus demonstrating the importance of the telomeric 5' tail for in vitro recognition and guanine quadruplex formation. While d(T4G4)2 is not a suitable substrate for quadruplex promotion by the beta subunit, the Raman spectra reveal other structural rearrangements of this DNA strand upon beta subunit binding, including changes in guanine glycosyl torsion angles from syn to anti and disruption of carbonyl hydrogen-bonding interactions. The conformation of d(T4G4)2 in the beta:d(T4G4)2 complex is suggested as a plausible intermediate along the pathway to formation of the parallel-stranded guanine quadruplex. Fluorescence band shifts indicate that at least one of the two tryptophans of the beta subunit is shielded from solvent as a consequence of DNA binding in both the beta:dT6(T4G4)2 and beta:d(T4G4)2 complexes. However, the Raman spectra of these complexes suggest no significant changes in the beta subunit secondary structure attendant with DNA binding. A model for beta subunit binding by Oxytricha telomeric DNA sequences and a mechanism for quadruplex formation are proposed. A key feature of this model is the use of a telomeric hairpin secondary structure as the recognition motif.     

Tankyrase, a poly(ADP-ribose) polymerase at human telomeres

Tankyrase, a protein with homology to ankyrins and to the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP), was identified and localized to human telomeres. Tankyrase binds to the telomeric protein TRF1 (telomeric repeat binding factor-1), a negative regulator of telomere length maintenance. Like ankyrins, tankyrase contains 24 ankyrin repeats in a domain responsible for its interaction with TRF1. Recombinant tankyrase was found to have PARP activity in vitro, with both TRF1 and tankyrase functioning as acceptors for adenosine diphosphate (ADP)-ribosylation. ADP-ribosylation of TRF1 diminished its ability to bind to telomeric DNA in vitro, suggesting that telomere function in human cells is regulated by poly(ADP-ribosyl)ation.