Single-molecule imaging reveals a common mechanism shared by G-quadruplex-resolving helicases.

G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.