Impact of Shelterin Complex on Telomere Accessibility

ABSTRACT Shelterin plays critical roles in maintaining and protecting telomeres by regulating access of various physiological agents to telomeric DNA. We present single molecule measurements investigating the impact of the POT1 and a four-component shelterin complex on the accessibility of human telomeric DNA overhangs with physiologically relevant lengths (28-150 nt), which to our knowledge is the first direct approach to measure this effect on such telomeric constructs. To quantify telomere accessibility, we monitored transient binding events of a short peptide nucleic acid (PNA) probe that is complementary to telomeric overhangs using FRET-PAINT. Although POT1 has a mild G-quadruplex unfolding activity, it reduced accessibility of the PNA probe by ∼2.5 fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin reduced the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the region between single and double stranded telomere, which is otherwise the most accessible part of the overhang, by a synergistic cooperation of shelterin components located on single and double stranded telomere.