TRF2 Interaction with Ku Heterotetramerization Interface Gives Insight into c-NHEJ Prevention at Human Telomeres

• TRF1 and TRF2 interact with Ku70 at functional telomeres
• TRF2, but not TRF1 or Rap1, interacts with Ku70 α5, a region required for NHEJ
• Ku70 α5 mediates Ku heterotetramerization in vivo
• Telomere NHEJ may be prevented by TRF2 blocking Ku heterotetramerization

SummaryTelomeres are protected from nonhomologous end-joining (NHEJ) to avoid deleterious chromosome fusions, yet they associate with the Ku heterodimer that is principal in the classical NHEJ (c-NHEJ) pathway. T-loops have been proposed to inhibit Ku’s association with telomeric ends, thus inhibiting c-NHEJ; however, deficiencies in the t-loop model suggest additional mechanisms are in effect. We demonstrate that TRF2 interacts with Ku at telomeres and via residues in Ku70 helix 5 (α5), which are vital for NHEJ. We show that Ku’s interaction with a TRF2 mutant that induces telomeric fusions is significantly impaired. Additionally, we demonstrate that Ku70 α5 is required for Ku self-association in live cells, which can bridge DNA ends. Together, these findings lead us to propose a model in which telomeres are directly protected from c-NHEJ via TRF2 impeding Ku’s ability to synapse telomere ends.

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