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, telomerase1,2,3. Shelterin includes the heterodimeric POT1–TPP1 protein, which binds the telomeric single-stranded DNA tail4,5,6,7,8,9. 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)9,10,11,12,13,14. 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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We thank T. de Lange, M. Terns and S. Langer for suggestions and sharing protocols; T. Nahreini for maintenance of the departmental tissue culture facility; J. Friedman and G. Voeltz for help with confocal microscopy; and A. Berman, S. Borah and M. Nakashima for critical reading of the manuscript. T.R.C. is an investigator of the Howard Hughes Medical Institute (HHMI). J.N. was an HHMI fellow of the Helen Hay Whitney Foundation during a major part of this study and is supported by the National Cancer Institute of the National Institutes of Health under award number K99CA167644. This work was supported in part by US National Institutes of Health grant R01GM29090 to L.A.L. and R01GM099705 to T.R.C.
This file contains Supplementary Figures 1-13, a Supplementary Discussion and additional references. Supplementary Figures 2-6 show in vitro DNA-binding and direct telomerase assays with TPP1-OB mutants, Supplementary Figure 7 shows an investigation of telomerase components contributing to TPP1-OB binding, and Supplementary Figures 8-13 show the development, validation, and utilization of the HeLa-based system used to address the biological phenotypes (chromosome end protection, telomere maintenance, and telomerase recruitment) of the TEL patch mutants of TPP1. The Supplementary Discussion describes how telomerase recruitment in budding yeast compares to that in humans and S. pombe.