SUMMARY The shelterin protein protects telomeres against activation of the DNA damage checkpoint and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps ~90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired.

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.

Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/ PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/ Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo's conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/ Apollo À/À ATM À/À MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5 0 -3 0 exonuclease function of SNM1B/Apollo in the generation of 3 0 single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway. The EMBO Journal (2010) IntroductionMammalian telomeres consist of TTAGGG repetitive sequences that terminate in a 3 0 single-stranded (ss) G-rich overhang. Telomeres are bound and stabilized by a number of telomere-specific-binding proteins that form a core complex termed shelterin that protects telomeres from inappropriately activating the DNA damage response (DDR) (Palm and de Lange, 2008). Three sequence-specific DNA-binding proteins are recruited to chromosomal ends: the duplex telomerebinding proteins TRF1 and TRF2/RAP1, and the ss TTAGGG repeat-binding protein POT1. These proteins are interconnected by the adapter proteins TIN2 and TPP1. Telomeres rendered dysfunctional by the removal of TRF2/RAP1 activate ATM and are repaired by the non-homologous endjoining (NHEJ) pathway, whereas removal of the POT1-TPP1 complex activates NHEJ-mediated repair that requires ATR (Wu et al, 2006;Denchi and de Lange, 2007;Guo et al, 2007;Deng et al, 2009).Emerging evidence suggests that the core shelterin complex is insufficient for complete chromosomal end protection. Rather, accessory proteins that interact with the shelterin complex are also essential for telomere stability. One such protein is SNM1B/Apollo, a member of a small gene family that also includes SNM1A and SNMIC/Artemis. All three proteins share sequence similarity to the yeast interstrand crosslink (ICL) repair protein PSO2/SNM1 (Dronkert et al, 2000). These proteins are characterized by a conserved metallo-b-lactamase-fold and an appended b-CPSF-ArtemisSnm1-Pso2 (CASP) domain that together imparts 5 0 exonuclease function (Callebaut et al, 2002;Poinsignon et al, 2004;Lenain et al, 2006). SNM1A localizes to ionizing radiation (IR)-induced DNA breaks (Richie et al...