16 research outputs found

    A single-molecule view of telomerase regulation at telomeres

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    Telomerase plays a key role in the immortalization of cancer cells by maintaining telomeres length. Using single-molecule imaging of telomerase RNA molecules in cancer cells, we recently reported novel insights into the role of Cajal bodies in telomerase biogenesis and the regulation of telomerase recruitment to telomeres

    Does a Sentinel or a Subset of Short Telomeres Determine Replicative Senescence?

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    The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with γH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence

    Taking apart Rap1

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    The involvement of the Mre11/Rad50/Nbs1 complex in the generation of G-overhangs at human telomeres

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    A central function of telomeres is to prevent chromosome ends from being recognized as DNA double-strand breaks (DSBs). Several proteins involved in processing DSBs associate with telomeres, but the roles of these factors at telomeres are largely unknown. To investigate whether the Mre11/Rad50/Nbs1 (MRN) complex is involved in the generation of proper 3′ G-overhangs at human telomere ends, we used RNA interference to decrease expression of MRN and analysed their effects. Reduction of MRN resulted in a transient shortening of G-overhang length in telomerase-positive cells. The terminal nucleotides of both C- and G-rich strands remain unaltered in Mre11-diminished cells, indicating that MRN is not responsible for specifying the final end-processing event. The reduction in overhang length was not seen in telomerase-negative cells, but was observed after the expression of exogenous telomerase, which suggested that the MRN complex might be involved in the recruitment or action of telomerase

    ATM and ATR Signaling Regulate the Recruitment of Human Telomerase to Telomeres

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    The yeast homologs of the ATM and ATR DNA damage response kinases play key roles in telomerase-mediated telomere maintenance, but the role of ATM/ATR in the mammalian telomerase pathway has been less clear. Here, we demonstrate the requirement for ATM and ATR in the localization of telomerase to telomeres and telomere elongation in immortal human cells. Stalled replication forks increased telomerase recruitment in an ATR-dependent manner. Furthermore, increased telomerase recruitment was observed upon phosphorylation of the shelterin component TRF1 at an ATM/ATR target site (S367). This phosphorylation leads to loss of TRF1 from telomeres and may therefore increase replication fork stalling. ATM and ATR depletion reduced assembly of the telomerase complex, and ATM was required for telomere elongation in cells expressing POT1ΔOB, an allele of POT1 that disrupts telomere-length homeostasis. These data establish that human telomerase recruitment and telomere elongation are modulated by DNA-damage-transducing kinases

    Nontelomeric Role for Rap1 in Regulating Metabolism and Protecting against Obesity

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    The mammalian telomere-binding protein Rap1 was recently found to have additional nontelomeric functions, acting as a transcriptional cofactor and a regulator of the NF-κB pathway. Here, we assess the effect of disrupting mouse Rap1 in vivo and report on its unanticipated role in metabolic regulation and body-weight homeostasis. Rap1 inhibition causes dysregulation in hepatic as well as adipose function, leading to glucose intolerance, insulin resistance, liver steatosis, and excess fat accumulation. Furthermore, Rap1 appears to play a pivotal role in the transcriptional cascade that controls adipocyte differentiation in vitro. Using a separation-of-function allele, we show that the metabolic function of Rap1 is independent of its recruitment to TTAGGG binding elements found at telomeres and at other interstitial loci. In conclusion, our study underscores an additional function for the most conserved telomere-binding protein, forging a link between telomere biology and metabolic signaling
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