The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity

1] University of Colorado BioFrontiers Institute, Boulder, Colorado 80309, USA [2] Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Nature (Impact Factor: 41.46). 10/2012; 492(7428). DOI: 10.1038/nature11648
Source: PubMed


Human chromosome ends are capped by shelterin, a protein complex that protects the natural ends from being recognized as sites of DNA damage and also regulates the telomere-replicating enzyme, telomerase. Shelterin includes the heterodimeric POT1-TPP1 protein, which binds the telomeric single-stranded DNA tail. TPP1 has been implicated both in recruiting telomerase to telomeres and in stimulating telomerase processivity (the addition of multiple DNA repeats after a single primer-binding event). Determining the mechanisms of these activities has been difficult, especially because genetic perturbations also tend to affect the essential chromosome end-protection function of TPP1 (refs 15, 16, 17). Here we identify separation-of-function mutants of human TPP1 that retain full telomere-capping function in vitro and in vivo, yet are defective in binding human telomerase. The seven separation-of-function mutations map to a patch of amino acids on the surface of TPP1, the TEL patch, that both recruits telomerase to telomeres and promotes high-processivity DNA synthesis, indicating that these two activities are manifestations of the same molecular interaction. Given that the interaction between telomerase and TPP1 is required for telomerase function in vivo, the TEL patch of TPP1 provides a new target for anticancer drug development.

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    • "TRF2 represses ataxia-telangiectasia mutated (ATM)-mediated DDR and non-homologous end joining, while POT1 inhibits DNA-damage kinase ataxia-telangiectasia and Rad3 related (ATR) (Denchi and de Lange, 2007; Karlseder et al., 2004). Contrastingly, the POT1-TPP1 complex positively regulates TL, likely due to the role of TPP1 in the recruitment of telomerase (Nandakumar et al., 2012; Wang et al., 2007). While a clear role of RAP1 has not been established, it has been shown to be involved in homologous recombination and inhibition of the DDR, and is recruited to telomeres via TRF2 (Sfeir et al., 2010). "
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    ABSTRACT: Telomeres are the heterochromatic repeat regions at the ends of eukaryotic chromosomes, whose length is considered to be a determinant of biological ageing. Normal ageing itself is associated with telomere shortening. Here, critically short telomeres trigger senescence and eventually cell death. This shortening rate may be further increased by inflammation and oxidative stress and thus affect the ageing process. Apart from shortened or dysfunctional telomeres, cells undergoing senescence are also associated with hyperactivity of the transcription factor NF-κB and overexpression of inflammatory cytokines such as TNF-α, IL-6, and IFN-γ in circulating macrophages. Interestingly, telomerase, a reverse transcriptase that elongates telomeres, is involved in modulating NF-κB activity. Furthermore, inflammation and oxidative stress are implicated as pre-disease mechanisms for chronic diseases of ageing such as neurodegenerative diseases, cardiovascular disease, and cancer. To date, inflammation and telomere shortening have mostly been studied individually in terms of ageing and the associated disease phenotype. However, the interdependent nature of the two demands a more synergistic approach in understanding the ageing process itself and for developing new therapeutic approaches. In this review, we aim to summarize the intricate association between the various inflammatory molecules and telomeres that together contribute to the ageing process and related diseases.
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    • "TPP1 and POT1 also have roles in mediating telomere-length regulation. A surface on the N-terminal oligonucleotide/oligosaccharide-binding (OB) domain of TPP1 termed the TEL patch activates telomerase by stimulating telomerase processivity and providing a direct binding site for telomerase recruitment to telomeres ; mutation of the TEL patch can lead to telomere shortening syndromes characterized by bone marrow failure (Abreu et al., 2010; Nandakumar et al., 2012; Zhong et al., 2012; Kocak et al., 2014; Guo et al., 2014; Dalby et al., 2015). Additionally, mutation analyses at sites independent of the TEL patch have implicated TPP1 as part of a telomere-length-dependent feedback loop that regulates telomere-length homeostasis (Sexton et al., 2014). "
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    ABSTRACT: 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.
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    • "Recombinant human wild-type and mutant TPP1-N constructs, composed of the OB and POT1 binding domains, were overexpressed in BL21-DE3 cells and purified as previously described [36]. Recombinant human full-length POT1 was overexpressed and purified from insect cells as previously described [62]. "
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    ABSTRACT: Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer as well as telomere-shortening syndromes. Recent studies have shown that the TEL-patch - a cluster of amino acids on the surface of the shelterin component TPP1 - is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment - binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action. Copyright © 2015. Published by Elsevier Ltd.
    Preview · Article · Jan 2015 · Journal of Molecular Biology
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