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.     

Molecular cloning and chromosomal localization of Chinese hamster telomeric protein chTRF1. Its potential role in chromosomal instability

Chinese hamster cells frequently have altered karyotypes. To investigate the basis of recent observations that karyotypic alterations are related to telomeric fusions, we asked whether these alterations are due to lack of telomere repeat binding factor/s. Further, Chinese hamster chromosomes contain large blocks of interstitial telomeric repeats, which are preferentially involved in chromosome breakage and exchange, rendering it an interesting model for such studies. Here, we report on the cloning and the chromosomal localization of the Chinese hamster telomere repeat binding factor, chTRF1. The sequence analysis revealed, similar to human TRF1 (hTRF1), an N-terminal acidic domain, a TRF1 specific DNA binding motif and a C-terminal Myb type domain. Unlike mouse TRF1 (mTRF1), chTRF1 shows 97.5% identity to hTRF1. chTRF1 gene was localized on the long arm of chromosome 5. In vitro translation of chTRF1 resulted in protein product similar in molecular weight to hTRF1. Immunostaining of Chinese hamster ovary cells (CHO) with anti-TRF1 antibody revealed punctate nuclear staining. At metaphase, antibodies failed to detect TRF1 on most of the chromosome ends and the interstitial telomeric repeat bands. These studies suggest that chTRF1 does not bind the interstitial telomeric repeats, and its presence at the metaphase chromosome ends is limited. The later could be a factor contributing to frequent karyotypic alterations observed in Chinese hamster cells.     

The telobox, a Myb-related telomeric DNA binding motif found in proteins from yeast, plants and human

The yeast TTAGGG binding factor 1 (Tbf1) was identified and cloned through its ability to interact with vertebrate telomeric repeats in vitro. We show here that a sequence of 60 amino acids located in its C-terminus is critical for DNA binding. This sequence exhibits homologies with Myb repeats and is conserved among five proteins from plants, two of which are known to bind telomeric-related sequences, and two proteins from human, including the telomeric repeat binding factor (TRF) and the predicted C-terminal polypeptide, called orf2, from a yet unknown protein. We demonstrate that the 111 C-terminal residues of TRF and the 64 orf2 residues are able to bind the human telomeric repeats specifically. We propose to call the particular Myb-related motif found in these proteins the 'telobox'. Antibodies directed against the Tbf1 telobox detect two proteins in nuclear and mitotic chromosome extracts from human cell lines. Moreover, both proteins bind specifically to telomeric repeats in vitro. TRF is likely to correspond to one of them. Based on their high affinity for the telomeric repeat, we predict that TRF and orf2 play an important role at human telomeres.     

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.     

Conserved sequence and structural motifs contribute to the DNA binding and cleavage activities of a geminivirus replication protein

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1-120) and oligomerization (amino acids 134-181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitro experiments demonstrated that a pair of predicted alpha-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions.     

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.     

Role of the telomeric DNA-binding protein TRF2 in the stability of human chromosome ends

A major issue in telomere research is to understand how the integrity of chromosome ends is preserved. A recent study shows that expression of a dominant-negative form of the human telomeric protein TRF2 increases the number of chromosome fusions in immortalized cells and decreases the quantity of G-rich telomeric DNA 3' overhang, the G tail. Consequently, TRF2 appears to control the structure of the very end of the chromosomal DNA molecule and to prevent recombination between two telomeres. Remarkably, the same study reveals a potential role of TRF2 in cell division control.     

Analysis of telomeric length in epithelial carcinoma of the ovary

BACKGROUND: As a consequence of the high cell division rate, telomeric repeat reduction in human tumor cells, giving rise to genetic instability, has recently been described. The aim of this study was to analyze by Southern blot telomeric length alterations in a retrospective group of patients with ovarian epithelial carcinoma. PATIENTS AND METHODS: Tumor and corresponding normal DNA were isolated from paraffin-embedded tissue of 16 patients with ovarian epithelial carcinoma. Telomeric Restriction Fragments (TRF) were studied by Southern blot and densitometric analysis. RESULTS: No telomere alterations were detected in 37.5% of patients (6/16). Of the remaining ten, 5 were found to have telomere reduction and five telomere elongation. No significant correlation was found between clinicopathological variables, response to chemotherapy, survival rate or time to progression, and telomere length alterations. CONCLUSIONS: In ovarian epithelial carcinoma telomere elongation may be a marker of the presence of immortal cells within the tumor, but telomere or the absence of telomeric alterations do not rule out the presence of these cells. Although TRF analysis can be performed in paraffin-embedded tissues, it is not the best indicator of telomerase activity and thus of tumor aggressiveness in early stages of this carcinoma.     

TRF2 protects human telomeres from end-to-end fusions

The mechanism by which telomeres prevent end-to-end fusion has remained elusive. Here, we show that the human telomeric protein TRF2 plays a key role in the protective activity of telomeres. A dominant negative allele of TRF2 induced end-to-end chromosome fusions detectable in metaphase and anaphase cells. Telomeric DNA persisted at the fusions, demonstrating that TTAGGG repeats per se are not sufficient for telomere integrity. Molecular analysis suggested that the fusions represented ligation of telomeres that have lost their single-stranded G-tails. Therefore, TRF2 may protect chromosome ends by maintaining the correct structure at telomere termini. In addition, expression of mutant forms of TRF2 induced a growth arrest with characteristics of senescence. The results raise the possibility that chromosome end fusions and senescence in primary human cells may be caused by loss by TRF2 from shortened telomeres.     

Isolation and characterization of a monoclonal anti-quadruplex DNA antibody from autoimmune "viable motheaten" mice

A cell line that produces an autoantibody specific for DNA quadruplex structures has been isolated and cloned from a hybridoma library derived from 3-month-old nonimmunized autoimmune, immunodeficient "viable motheaten" mice. This antibody has been tested extensively in vitro and found to bind specifically to DNA quadruplex structures formed by two biologically relevant sequence motifs. Scatchard and nonlinear regression analyses using both one- and two-site models were used to derive association constants for the antibody-DNA binding reactions. In both cases, quadruplexes had higher association constants than triplex and duplex molecules. The anti-quadruplex antibody binds to the quadruplex formed by the promoter-region-derived oligonucleotide d(CGCG4GCG) (Ka = 3.3 x 10(6) M-1), and has enhanced affinity for telomere-derived quadruplexes formed by the oligonucleotides d(TG4) and d(T2G4T2G4T2G4T2G4) (Ka = 5.38 x 10(6) and 1.66 x 10(7) M-1, respectively). The antibody binds both types of quadruplexes but has preferential affinity for the parallel four-stranded structure. In vitro radioimmunofilter binding experiments demonstrated that purified anti-DNA quadruplex antibodies from anti-quadruplex antibody-producing tissue culture supernatants have at least 10-fold higher affinity for quadruplexes than for triplex and duplex DNA structures of similar base composition and length. The antibody binds intramolecular DNA triplexes formed by d(G4T3G4T3C4) and d(C4T3G4T3G4), and the duplex d(CGCGCGCGCG)2 with an affinities of 6. 76 x 10(5), 5.59 x 10(5), and 8.26 x 10(5) M-1, respectively. Competition experiments showed that melted quadruplexes are not effective competitors for antibody binding when compared to native structures, confirming that the quadruplex is bound structure-specifically. To our knowledge, this is the first immunological reagent known to specifically recognize quadruplex structures. Subsequent sequence analysis demonstrates homologies between the antibody complementarity determining regions and sequences from Myb family telomere binding proteins, which are hypothesized to control cell aging via telomeric DNA interactions. The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.     

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.     

A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Rap1 protein binds with high affinity to sites within the poly(C(1-3)A) tracts at telomeres, where it plays a role in both telomere length regulation and the initiation of telomeric silencing. Rap1p initiates silencing at telomeres by interacting through its carboxy-terminal domain with Sir3p and Sir4p, both of which are required for repression. This same domain of Rap1p also negatively regulates telomere elongation, through an unknown mechanism. We have identified a new Rap1-interacting factor (Rif2p) that plays a role in telomere length regulation. Rif2p has considerable functional similarities with a Rap1p-interacting factor (Rif1p) identified previously. Mutations in RIF1 or RIF2 (unlike mutations in the silencing genes SIR3 and SIR4) result in moderate telomere elongation and improved telomeric silencing. However, deletion of both RIF1 and RIF2 in the same cell results in a dramatic increase in telomere length, similar to that seen with a carboxy-terminal truncation of Rap1p. In addition, overexpression of either RIF1 or RIF2 decreases telomere length, and co-overexpression of these proteins can reverse the telomere elongation effect of overexpression of the Rap1p carboxyl terminus. Finally, we show that Rif1p and Rif2p can interact with each other in vivo. These results suggest that telomere length regulation is mediated by a protein complex consisting of Rif1p and Rif2p, each of which has distinct regulatory functions. One role of Rap1p in telomere length regulation is to recruit these proteins to the telomeres.     

Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure

The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins.