Goldfarb, A. M.; Sasi, N. K.; Dilgen, T. C.; Cai, S. W.; Myler, L. R.; de Lange, T.

Telomeres often occur in the t-loop structure, formed through the base-pairing of the three prime telomeric overhang is base-paired with more internal telomeric DNA. T-loops are proposed to avert ATM kinase signaling and classical non-homologous end joining (cNHEJ) by sequestering the telomere end. The shelterin subunit TRF2 is required for t-loop formation but how it generates t-loops is not known and the t-loop model for telomere protection remains untested. We show that TRF2 binds telomeric DNA as a tetramer using two dimerization domains and that tetramerization is critical for telomere protection. Furthermore, TRF1, the TRF2 paralog that binds DNA as a dimer and does not form t-loops, gained the ability to form t-loops in vivo upon its tetramerization through the addition of a second dimerization domain. We propose that TRF2 tetramers loop telomeric DNA, resulting in torsional stress and opening of the helix, which is a requirement for t-loop formation. Importantly, t-loops formed by tetrameric TRF1 were sufficient to avert ATM signaling and blocked most cNHEJ at telomeres, demonstrating that t-loops per se protect telomeres from these threats.