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Ted removal of TPP1 (and POT1a/b) from telomeres of TPP1F/F mouse embryo fibroblasts (MEFs). This hyper-resection is counteracted by 53BP1 and Rif120, which accumulate in response to ATR signaling at telomeres lacking POT1a (Fig. 1a). Like 53BP1, Shieldin limited hyper-resection at telomeres lacking TPP1: TPP1-/- cells lacking either Rev7 or Shld2 showed telomere hyper-resection (Fig. 1b-d, Extended Information Fig. 1a-c). As CST is essential21,22, we employed shRNAs to discover the role of CST in telomere hyperresection. Depletion of Stn1 or Ctc1 enhanced the telomeric overhang signal in cells lacking TPP1 (Fig. 1b-d; Extended Information Fig. 1d; Extended Information Fig. 2) and tests with E. coli ExoI confirmed that the signal derived from a three overhang (Extended Information Fig. 1e, f). Importantly, Stn1 or Ctc1 knockdown didn’t have an effect on the resection at telomeres when TPP1 was deleted from Rev7-deficient cells (Fig. 1b-d; Extended Data Fig. 2). Moreover, Stn1 knockdown had no effect on telomere hyper-resection when either 53BP1 or Rif1 had been absent or when cells contained an allele of 53BP1 that will not recruit Rif123 (Extended Data Fig. 3). These information suggest that CST acts in a 53BP1-, Rif1-, and Shieldin-dependent manner to limit the formation of ssDNA at dysfunctional telomeres. To figure out regardless of whether CST also counteracted resection at web sites of ATM signaling, we utilized conditional deletion of TRF2 (Fig. 1e). Telomeres lacking TRF2 undergo c-NHEJ-mediated fusion246. In DNA ligase IV (Lig4)-deficient cells exactly where such telomere JF549 custom synthesis fusions are prevented26, telomeres lacking TRF2 undergo 5 finish resection that is certainly exacerbated by loss of 53BP1 or Rif18,19 (Fig. 1e). Similarly, the 5 end resection was improved by Rev7- or Shld2-deficiency (Fig. 1f-h; Extended Data Fig. 4). When Stn1 was depleted from cells lacking TRF2, resection at telomeres was substantially improved (Fig. 1f-h) and this effect was epistatic with Rev7 (Fig. 1f-h). Apraclonidine Purity & Documentation Therefore, CST counteracts resection within a Shieldin-dependent manner inside the context of ATM signaling. We next determined no matter whether CST localized to broken telomeres inside a 53BP1- and Shieldindependent manner. Myc-tagged Ctc1 was detectable at telomeres with functional shelterin, whereas in POT1b-deficient cells which show extended telomeric three overhangs but no DNA damage signaling27 Ctc1 localization at telomeres was minimal (Fig. 2a,b). WhenAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptNature. Author manuscript; out there in PMC 2019 January 18.Mirman et al.PageATR was activated by deletion of TPP1 (Fig. 2a; correct panel), Ctc1 was once again detectable at telomeres (Fig. 2a, b), despite the absence of POT1b. Recruitment of Ctc1 to dysfunctional telomeres depended on ATR signaling, 53BP1, and Shieldin (Fig. 2b,c). Similarly, Cremediated deletion in the single human POT1 protein from conditional POT1 KO HT1080 cells28 led to telomeric accumulation of Stn1 that needed ATR kinase (Fig. 2d-f). Hence, CST localizes to damaged telomeres within a Shieldin-dependent manner. Co-IP experiments showed that Shieldin components could associate with CST (Fig. 2g; Extended Information Fig. 5a). Within a yeast 2-hybrid assay, Ctc1 robustly interacted with Shld1, and Stn1 did so with Shld3 (Fig. 2h and Extended Information Fig. 5b). Weaker interactions have been detectable in between Ten1 and Shld3; Stn1 and Shld1, Shld2, and Rev7; and Ctc1 and Rev7. Hence, Shieldin binds CST by means of many direct interactions. Ionizing radiation (IR)-induced DSBs in human cells showed Stn1 co-loc.