G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding

Significance This paper uses a single-molecule imaging approach based on energy transfer to study how telomeric DNA is protected against the DNA damage-signaling protein replication protein A (RPA). Telomeres terminate with a single-stranded overhang, which is protected by protection of telomere (POT1) and POT1-interacting protein 1 (TPP1) against RPA. Telomeric overhangs have a guanine-rich sequence, forming a four-stranded G-quadruplex structure. Using model telomeric DNA, we studied the competition between POT1/TPP1 and RPA to access telomeric G-quadruplexes in vitro. We showed that G-quadruplex formation of telomeres significantly enhances the ability of POT1/TPP1 to block RPA’s access to telomeres. The results suggest that secondary structure of the telomeric overhang plays an important role in suppressing the DNA damage signals in telomeres. Human telomeres terminate with a single-stranded 3′ G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K+, POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA’s access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na+, in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.

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