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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    • "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.
    Cell Reports 11/2015; 13(8). DOI:10.1016/j.celrep.2015.10.041 · 8.36 Impact Factor
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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.
    Journal of Molecular Biology 01/2015; 427(6). DOI:10.1016/j.jmb.2015.01.008 · 4.33 Impact Factor
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    • "ed Biosystems ) . The reaction mixtures were incubated at 30˚C for 30 min and then PCR - amplified ( 40 cycles of 95˚C for 15 s and 60˚C for 60 s ) using an ABI StepOne Plus real - time PCR system ( Applied Biosystems ) . Telomerase activity primer extension assays were performed as described previously ( Wang et al . , 2007 ; Xin et al . , 2007 ; Nandakumar et al . , 2012 ) . Briefly , 293T cells transiently expressing 36 - FLAG - hTERT and hTERC were immunopurified using anti - FLAG beads . A synthetic telomeric DNA primer was then extended in the presence of a - 32 P - dGTP and unlabeled dATP and TTP . Telomerase activity and processivity were normalized to the amount of hTERT as determined by western "
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    ABSTRACT: Most human cancers depend on the telomerase to maintain telomeres; however, about 10% of cancers are telomerase negative and utilize the Alternative Lengthening of Telomeres (ALT) mechanism. Mutations in the DAXX gene have been found frequently in both telomerase-positive and ALT cells, and how DAXX mutations contribute to cancers remains unclear. We report here that endogenous DAXX can localize to Cajal bodies, associate with the telomerase, and regulate telomerase targeting to telomeres. Furthermore, disease mutations that are located in different regions of DAXX differentially impacted its ability to interact with its binding partners, and its targeting to Cajal bodies and telomeres. In addition, DAXX inhibition by RNAi led to reduced telomerase targeting to telomeres and telomere shortening. These findings collectively support a DAXX-centric pathway for telomere maintenance, where DAXX interaction with the telomerase regulates telomerase assembly in Cajal bodies and telomerase targeting to telomeres.
    Journal of Cell Science 11/2014; 128(2). DOI:10.1242/jcs.159467 · 5.43 Impact Factor
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