POT1/TPP1 and Telomeric G-Quadruplexes Synergistically Block RPA's Access to Telomeres

Abstract

Human telomeres terminate with a G-overhang, which can be misrecognized as a DNA damage site by RPA. In cells, POT1/TPP1 protects telomeres against RPA binding. The G-overhang folds into different G-quadruplex (GQ) conformations. The role of GQ formation in protection of telomeric overhangs remains unclear. Using single-molecule FRET, we show that the ability of POT1/TPP1 to compete against RPA binding to telomeres is significantly enhanced by GQ formation. In the absence of POT1, RPA efficiently unfolds single-stranded telomeric GQs and binds to the unfolded DNA. POT1 and POT1/TPP1 stably load on to a 3'-TTAG sequence adjacent to a folded GQ. As a result of this loading two possible pathways emerge: 1) A fraction of GQs are unfolded at a level that depends on their folding conformation. The unfolded GQs are bound by POT1 or POT1/TPP1 which provides protection against RPA binding. 2) The GQs that remain folded after POT1 or POT1/TPP1 loading are greatly stabilized against RPA mediated unfolding, which is the dominant pathway of telomere protection against RPA binding. TPP1 enhances POT1's ability to compete against RPA in both pathways. Thus, the synergistic effect of GQ formation and POT1/TPP1 binding protects telomeric overhangs against DNA damage signals.