Replication Protein A Unfolds G-Quadruplex Structures with Varying Degrees of Efficiency

Abstract

Replication Protein A (RPA) is known to interact with G-rich sequences that adopt G-quadruplex (GQ) structures. Most studies in the literature have been performed on GQ formed by homogenous sequences, such as the human telomeric repeat, and RPA’s ability to unfold GQ structures of differing stability is not known. We compared the thermal stability of three potential GQ forming DNA sequences (PQS) to their stability against RPA mediated unfolding using single molecule FRET and bulk biophysical and biochemical experiments. One of these sequences is the human telomeric repeat and the other two located in the promoter region of tyrosine hydroxylase gene are highly heterogeneous sequences, which better represent PQS in the genome. The three GQ constructs have thermal stabilities that are significantly different from each other. Our measurements showed that the most thermally stable structure (Tm= 86 °C) was also the most stable against RPA mediated unfolding, although the least thermally stable structure (Tm= 69 °C) had at least an order of magnitude higher stability against RPA mediated unfolding compared to the structure with intermediate thermal stability (Tm= 78 °C). The significance of this observation becomes more evident when considered within the context of cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these, we conclude that thermal stability is not necessarily an adequate criterion for predicting physiological viability of GQ structures. Finally, we measured the time it takes for an RPA molecule to unfold a GQ from a fully folded to a fully unfolded conformation using a single molecule stopped-flow type method. All three GQ structures were unfolded within Δt≈0.30±0.10 sec, a surprising result as the unfolding time does not correlate with thermal stability or stability against RPA mediated unfolding. These results suggest that the limiting step in G-quadruplex unfolding by RPA is simply the accessibility of the structure to the RPA protein.

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

“…In 150 mM K + , RPA unfolds a majority of ssTEL4 GQs in the absence of POT1 at two orders of magnitude lower concentrations than its physiological levels ( Fig. 3 A and B), in agreement with an earlier study on a similar GQ construct (21). FRET value of the RPA-bound unfolded state (R, E F = 0.10 ± 0.03) is lower than 2P (E F = 0.16 ± 0.03), allowing us to distinguish between POT1-bound and RPA-bound unfolded states (Fig.…”

“…5). This construct has a CD spectrum similar to that of ssTEL4, and hence similar folding conformations are expected (21). RPA was less efficient in unfolding the GQ with a TT overhang, demonstrating that RPA also loads to the overhang for unfolding the GQ (Fig.…”

“…Biotinylated pdDNA molecules were immobilized on PEG-coated coverslips and imaged with a prism-type TIRF microscope. Steady-state smFRET and CD measurements were performed as described (21). The smFRET histograms were constructed such that each analyzed molecule contributes equally to the FRET histogram, and the total population of the histogram is normalized to 100%.…”

“…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)(18)(19) or ssDNA binding proteins (20,21). It remains unclear whether GQ formation of ssTEL plays any role in protection of telomeres.…”

5′ to 3′ Unfolding Directionality of DNA Secondary Structures by Replication Protein A

“…Since then, many studies have focused on the interaction between RPA and G4 to determine its unfolding mechanism. Electrophoretic mobility shift assays (EMSA) and fluorescence resonance energy transfer (FRET) experiments indicate that the initial binding of the human RPA (hRPA) requires a ssDNA region (27), probably generated by G4 structural breathing that transiently exposes ssDNA (28 -30), and that this initial step is rate-limiting (31,32). Second, an inverse relationship between telomeric G4 structural stability and hRPA binding has been established (27,33).…”