Human Ku70/80 associates physically with telomerase through interaction with hTERT

Telomere length maintenance, an activity essential for chromosome stability and genome integrity, is regulated by telomerase- and telomere-associated factors. The DNA repair protein Ku (a heterodimer of Ku70 and Ku80 subunits) associates with mammalian telomeres and contributes to telomere maintenance. Here, we analyzed the physical association of Ku with human telomerase both in vivo and in vitro. Antibodies specific to human Ku proteins precipitated human telomerase in extracts from tumor cells, as well as from telomerase-immortalized normal cells, regardless of the presence of DNA-dependent protein kinase catalytic subunit. The same Ku antibodies also precipitated in vitro reconstituted telomerase, suggesting that this association does not require telomeric DNA. Moreover, Ku associated with the in vitro translated catalytic subunit of telomerase (hTERT) in the absence of telomerase RNA (hTR) or telomeric DNA. The results presented here are the first to report that Ku associates with hTERT, and this interaction may function to regulate the access of telomerase to telomeric DNA ends.     

Stable association of hsp90 and p23, but Not hsp70, with active human telomerase

The ribonucleoprotein telomerase holoenzyme is minimally composed of a catalytic subunit, hTERT, and its associated template RNA component, hTR. We have previously found two additional components of the telomerase holoenzyme, the chaperones p23 and heat shock protein (hsp) 90, both of which are required for efficient telomerase assembly in vitro and in vivo. Both hsp90 and p23 bind specifically to hTERT and influence its proper assembly with the template RNA, hTR. We report here that the hsp70 chaperone also associates with hTERT in the absence of hTR and dissociates when telomerase is folded into its active state, similar to what occurs with other chaperone targets. Our data also indicate that hsp90 and p23 remain associated with functional telomerase complexes, which differs from other hsp90-folded enzymes that require only a transient hsp90.p23 binding. Our data suggest that components of the hsp90 chaperone complex, while required for telomerase assembly, remain associated with active enzyme, which may ultimately provide critical insight into the biochemical properties of telomerase assembly.     

Cell cycle-dependent regulation of yeast telomerase by Ku

The heterodimeric Ku complex affects telomere structure in diverse organisms. We report here that in the absence of Ku, the catalytic subunit of telomerase, Est2p, was not telomere-associated in G1 phase, and its association in late S phase was decreased. The telomere association of Est1p, a telomerase component that binds telomeres only in late S phase, was also reduced in the absence of Ku. The effects of Ku on telomerase binding require a 48-nucleotide (nt) stem-loop region of TLC1 telomerase RNA. Ku interacts with TLC1 RNA via this 48-nt region throughout the cell cycle, but this interaction was reduced after telomere replication. These data support a model in which Ku recruits telomerase to telomeres in G1 phase when telomerase is inactive and promotes telomerase-mediated telomere lengthening in late S phase.     

RNA recognition by the DNA end-binding Ku heterodimer

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem–loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem–loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.     

hnRNP A1 may interact simultaneously with telomeric DNA and the human telomerase RNA in vitro

The hnRNP A1 protein and a shortened derivative (UP1) promote telomere elongation in mammalian cells. In support of a direct role for A1 in telomere biogenesis, we have shown that the recombinant UP1 protein binds to telomeric DNA sequences in vitro, and pulls down telomerase activity from a cell extract. Here we show that A1/UP1 can interact directly with the RNA component of human telomerase (hTR). A portion of A1/UP1 that contains RNA recognition motif 2 (RRM2) is sufficient for an interaction with the first 208 nt of hTR. Given that the portion of A1/UP1 that contains RRM1 is sufficient for binding to a telomeric DNA oligonucleotide, we have tested whether A1/UP1 can interact simultaneously with both nucleic acids. Using a chromatography assay, we find that A1/UP1 bound to hTR can interact with telomeric DNA. Notably, these interactions are sufficiently robust to withstand incubation in a cell extract. Our results suggest that hnRNP A1 may help recruit telomerase to the ends of chromosomes.     

Insulin-like growth factor binding protein-6 (IGFBP-6) interacts with DNA-end binding protein Ku80 to regulate cell fate

Insulin-like growth factor binding protein-6 (IGFBP-6) is a growth inhibitory protein that regulates the availability of insulin-like growth factors (IGFs). We recently reported that IGFBP-6 exerts intracellular actions via its translocation to the nucleus. We now show that IGFBP-6 co-purifies by tandem-affinity with nuclear proteins involved in DNA stability and repair such as Ku80, Ku70, histone H2B and importin-α. Furthermore, this report shows that IGFBP-6 and Ku80 interact specifically using two active binding sites for Ku80 in IGFBP-6. One of the binding sites [196RKR199], as part of the NLS-sequence in IGFBP-6 also binds importin-α which may selectively compete with Ku80 regulating its trafficking to the nucleus. Moreover, IGFBP-6 co-localized with Ku80 based on a cell cycle pattern. Overexpression of IGFBP-6 increased the nuclear Ku80 in mitotic cells and reduced it post-mitosis. It is known that if highly expressed IGFBP-6 induces apoptosis and in our model, the down-regulation of Ku80 by specific siRNAs enhanced the apoptotic effect caused by the IGFBP-6 overexpression. This study demonstrates that IGFBP-6 alters cell survival by potentially regulating the availability of Ku80 for the DNA-repair process. This action represents a novel mechanism by which growth inhibitory proteins such as IGFBP-6 regulate cell fate.     

Synergistic Activity of Deguelin and Fludarabine in Cells from Chronic Lymphocytic Leukemia Patients and in the New Zealand Black Murine Model

B-cell chronic lymphocytic leukemia (CLL) remains an incurable disease, and despite the improvement achieved by therapeutic regimes developed over the last years still a subset of patients face a rather poor prognosis and will eventually relapse and become refractory to therapy. The natural rotenoid deguelin has been shown to induce apoptosis in several cancer cells and cell lines, including primary human CLL cells, and to act as a chemopreventive agent in animal models of induced carcinogenesis. In this work, we show that deguelin induces apoptosis in vitro in primary human CLL cells and in CLL-like cells from the New Zealand Black (NZB) mouse strain. In both of them, deguelin dowregulates AKT, NFκB and several downstream antiapoptotic proteins (XIAP, cIAP, BCL2, BCL-X_L and survivin), activating the mitochondrial pathway of apoptosis. Moreover, deguelin inhibits stromal cell-mediated c-Myc upregulation and resistance to fludarabine, increasing fludarabine induced DNA damage. We further show that deguelin has activity in vivo against NZB CLL-like cells in an experimental model of CLL in young NZB mice transplanted with spleen cells from aged NZB mice with lymphoproliferation. Moreover, the combination of deguelin and fludarabine in this model prolonged the survival of transplanted mice at doses of both compounds that were ineffective when administered individually. These results suggest deguelin could have potential for the treatment of human CLL.     

Different role of functional domains of hTR in DNA binding to telomere and telomerase reconstruction

Even if template sequence of hTR played an essential role in telomere binding, a 326 nucleotide fragment of hTR containing template, pseudoknot, and CR4-5 domains is critical for both binding with telomeric DNA and reconstitution of telomerase activity. A functional study with antisense oligonucleotides suggested that targeted disruption of the template region efficiently abrogated both telomeric DNA binding and telomerase activity, whereas disruption of the CR4-5 region induced only loss of telomerase activity. hTR interacts with telomeric DNA via structural region composed of the template, pseudoknot, and CR4-5 domains, however, each structural domain plays a distinct role in telomere binding and telomerase activity reconstitution.     

Mutually exclusive binding of telomerase RNA and DNA by Ku alters telomerase recruitment model

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in?vitro and in?vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PAPERCLIP:     

An enhanced H/ACA RNP assembly mechanism for human telomerase RNA

The integral telomerase RNA subunit templates the synthesis of telomeric repeats. The biological accumulation of human telomerase RNA (hTR) requires hTR H/ACA domain assembly with the same proteins that assemble on other human H/ACA RNAs. Despite this shared RNP composition, hTR accumulation is particularly sensitized to disruption by disease-linked H/ACA protein variants. We show that contrary to expectation, hTR-specific sequence requirements for biological accumulation do not act at an hTR-specific step of H/ACA RNP biogenesis; instead, they enhance hTR binding to the shared, chaperone-bound scaffold of H/ACA core proteins that mediates initial RNP assembly. We recapitulate physiological H/ACA RNP assembly with a preassembled NAF1/dyskerin/NOP10/NHP2 scaffold purified from cell extract and demonstrate that distributed sequence features of the hTR 3' hairpin synergize to improve scaffold binding. Our findings reveal that the hTR H/ACA domain is distinguished from other human H/ACA RNAs not by a distinct set of RNA-protein interactions but by an increased efficiency of RNP assembly. Our findings suggest a unifying mechanism for human telomerase deficiencies associated with H/ACA protein variants.