Characterization of t-loop formation by TRF2.

The telomeric t-loop structure is thought to hide chromosome ends from DNA damage signaling and repair pathways that can act on DNA ends. Previously, it was shown that t-loop formation requires the shelterin component TRF2, which represses ATM signaling and Non-Homologous End Joining (NHEJ). Here we establish that TRF2 alone, in the absence of other shelterin proteins can remodel telomeres into t-loops. Mouse and human TRF2 are expressed as two isoforms, only one of which had been studied to date. Here we characterize the expression of the TRF2 isoforms and show that they are equal in their ability to protect telomeres and form t-loops. Both isoforms are expressed throughout the cell cycle and t-loop frequencies appeared unaffected by cell cycle stage. To characterize the mechanism of t-loop formation, we analyzed the TRF2 Topless mutant which is deficient in a DNA-wrapping activity implicated in t-loop formation. We confirm that TRF2 Topless is defective in t-loop formation and repression of ATM but report that it is also defective in repression of NHEJ and shows diminished telomere localization. Therefore, the role of topological changes enforced by TRF2 remains unclear. Finally, we tested whether t-loop formation, which includes a strand-invasion step, is dependent on the Rad51 recombinase. Rad51 deletion did not affect t-loop frequencies and did not induce robust telomere damage, indicating that Rad51 is not required for this aspect of telomere protection. Therefore, alternative models for how TRF2 forms t-loops should be explored.