Molecular mechanisms of DNA end‐loop formation by TRF2

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Abstract
In the telomere region of human chromosomes, the (TTAGGG)n sequence stretches over several kilobases and forms a distinct higher‐order structure with various proteins. Telomere repeat binding factors (TRFs) bind specifically to this sequence and play critical roles in the maintenance of telomere structure and function. Here, we prepared a series of linear DNA carrying a stretch of telomeric sequence ((TTAGGG)n, ∼1.8 (kb) with different end‐structures and observed their higher‐order complexes with TRFs by atomic force microscopy. TRF2 molecules exclusively bound to the telomeric DNA region at several different places simultaneously mainly as a dimer, and often mediated DNA loop formation by forming a tetramer at the root. These multiple‐binding, multimerization and DNA loop formation by TRF2 were observed regardless of the DNA‐end structure (blunt, 3′‐overhanging, telomeric, non‐telomeric). However, when the DNA end carried the telomeric‐3′‐overhanging region, the loop was frequently formed at the end of the DNA. Namely, the TRF2‐mediated DNA loop formation is independent of the end‐structure and the 3′‐overhanging TTAGGG sequence is responsible for the stabilization of the loop. TRF1 also bound to the telomeric DNA as a dimer, but did not mediate DNA loop formation by itself. These results provide a new insight into the molecular mechanism of DNA end‐loop formation by TRFs.