Quantifying the impact of small molecule ligands on G-quadruplex stability against Bloom helicase

Abstract

G-quadruplex (GQ) stabilizing small molecule (SM) ligands have been used to stabilize human telomeric GQ (hGQ) to inhibit telomerase activity, or non-telomeric GQs to manipulate gene expression at transcription or translation level. GQs are known to inhibit DNA replication unless destabilized by helicases, such as Bloom helicase (BLM). Even though the impact of SM ligands on thermal stability of GQs is commonly used to characterize their efficacy, how these ligands influence helicase-mediated GQ unfolding is not well understood. Three prominent SM ligands (an oxazole telomestatin derivative, pyridostatin, and PhenDC3), which thermally stabilize hGQ at different levels, were utilized in this study. How these ligands influence BLM-mediated hGQ unfolding was investigated using two independent single-molecule approaches. While the frequency of dynamic hGQ unfolding events was used as the metric in the first approach, the second approach was based on quantifying the cumulative unfolding activity as a function of time. All three SM ligands inhibited BLM activity at similar levels, 2–3 fold, in both approaches. Our observations suggest that the impact of SM ligands on GQ thermal stability is not an ideal predictor for their inhibition of helicase-mediated unfolding, which is physiologically more relevant.

E. coli Rep helicase and RecA recombinase unwind G4 DNA and are important for resistance to G4-stabilizing ligands

“…The binding rate calculated from over 150 traces is similar for all three proteins (Figure 3C , I ). Helicase binding is followed by a long period of FRET fluctuations, which is similar to the repetitive G4 resolving activity seen in other G4 helicases such as RHAU, BLM, and WRN (Figure 3B , II) ( 19 , 20 , 25 ). We interpret this pattern as emerging from repetitive cycles of unwinding and refolding of G4 which acts as a physical barrier.…”

“…G4-interacting small molecules enhance G4 stability, thereby inhibiting cellular pathways including telomerase activity ( 20 , 65 ). G4 ligand binding to G4 structure can also downregulate gene expression ( 65 ).…”

“…A recent study reported that a bacterial RecQ helicase, which possesses a guanine-specific binding pocket that is essential for G4 unwinding, resolves G4 through repetitive cycles of unwinding and refolding ( 23 ). In addition, several reports have shown that Pif1, BLM and DHX36 exhibit similar repetitive unfolding of G4 and can successfully dislodge G4-stabilizing ligands ( 20 , 24 , 25 ). In some cases, helicase activity was limited by G4 ligand binding ( 18 , 26 ).…”

Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction

“…The characteristic dwell times ( t 2 ) were 2.1-fold and 2.5-fold longer in LiCl compared to KCl for 12G-Tract and 16G-Tract constructs, respectively ( Supplementary Figure S3 ). In addition, we compared the effect of a GQ stabilizing small molecule ( 36 ) on binding frequencies for 12G-Tract and 16G-Tract constructs in KCl ( Supplementary Figure S2B , C). For both 12G-Tract and 16G-Tract constructs, the binding frequencies were 2.9 fold lower with the small molecule compared to its absence.…”

Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling

“…The specific durations of PDS treatment are indicated accordingly in the text and/or in the figure legends. We emphasize that the addition of PDS only increases the melting temperature (thermostability) of already folded native G4s 66 – 68 , resulting in a slight enrichment in the frequency of stable native G4s at forks, and provided improved in situ probing of their presence, effects, and regulations on replication forks.…”