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

Abstract

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 lessstable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.telomere protection | DNA damage response | single molecule imaging H uman telomeres consist of 2,000-30,000 bp of doublestranded TTAGGG repeats and terminate with a 50-to 200-nt-long, single-stranded 3′ G overhang (1). Telomeric termini need to be protected against DNA damage signals to ensure genome integrity. Replication protein A (RPA) nonspecifically binds ssDNA, is highly abundant in eukaryotes, and plays a role in DNA replication and repair (2). RPA binding to ssDNA, including telomeric overhangs, activates the ataxia telangiectasia and Rad3-related checkpoint (2, 3). The protection of telomeres (POT1)/telomere protection protein (TPP1) subunit of the shelterin complex contributes to telomere protection by specifically binding to the G overhang (4, 5). RPA is 1,000-fold more abundant than POT1/TPP1 and has a similar affinity for singlestranded telomeric DNA (ssTEL) (6, 7). Therefore, POT1/TPP1 alone may not be able to effectively compete against RPA binding (8, 9). Efficient protection of ssTEL against RPA binding may require association of POT1/TPP1 to the rest of the shelterin complex along double-stranded telomeric tracts or other telomere-associated proteins (6, 9).Another potentially significant factor that could influence the competition between RPA and POT1/TPP1 is the ability of ssTEL to form G-quadruplex (GQ) structures (10, 11). Recent studies have shown that human telomeres form GQ structures in vivo (12, 13) and in cell extracts (14). At physiologically relevant ionic conditions (∼150 mM K + ), GQs are thermodynamically more stable than competing Watson-Crick pairing (15). These structures were often regarded as obstacles for recruitment of telomerase (16) and translocation of the DNA replication machinery (17), and their unfolding requires helicase activity (17-19) or ssDNA binding proteins (20,21). It remains unclear whether GQ formation of ssTEL plays any role in protection of telomeres. ssTEL sequences fold into parallel...

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

“…We propose that one of the six RPA DBDs, likely DBD-F, interferes with Exo1-ssDNA contacts to displace Exo1 from DNA. Our results, in concert with other biochemical studies indicating that RPA also inhibits Fen1 at Okazaki fragment flaps and Pot1 at telomeric DNA (59)(60)(61)(62), suggest a general mechanism where RPA physically strips other proteins from DNA. Indeed, we demonstrate that the tetrameric E. coli SSB can strip hExo1 from DNA (Fig.…”

Understanding the stability of DNA G-quadruplex units in long human telomeric strands

“…The telomeric 3 0 -overhang participates in capping and regulation of telomeres. Folding of a transiently exposed telomeric 3 0 -overhang into tandem G4s might provide protection from binding of non-shelterin proteins or modulate their binding, as suggested by recent in vitro studies [14,15]. G4s at human telomeric 3 0 -overhang might have a capping function, when normal capping is impaired, as it has been suggested in a budding yeast [6].…”

“…In mammalian cells, indirect evidence suggests that G4s may occur at telomeres during replication and at the 3 0 -overhangs, challenging telomere maintenance, at least in the absence of proteins able to unfold G4s or in the presence of ligands able to stabilize these structures [7e10]. Telomeric G4s modulate in vitro telomerase activity [11e13] and binding of other proteins involved in telomere biology, as recently demonstrated by single-molecule approaches [14,15].…”

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase

“…Some G4-specific helicases show topological specificity. For example, the telomere protein TPP1 more efficiently unwinds antiparallel G4 structures like Q6 than it does parallel G4 (34). In contrast, RNA helicase RHAU preferentially binds to parallel G4 structures like that of ILPR (35).…”