Molecular mechanisms of DNA end-loop formation by TRF2

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, similar to1.8 (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.