DHX36-mediated G-quadruplex unfolding is ATP-independent?

TL;DR

This study challenges the ATP-independent G-quadruplex unfolding mechanism of DHX36, providing experimental evidence that supports a different, ATP-dependent process involving G4 binding and translocation.

Contribution

The paper offers new experimental insights that refute previous ATP-independent G4 unfolding models of DHX36, emphasizing the importance of ATP in its mechanism.

Findings

DHX36 does not unfold G4 DNA without ATP.

FRET oscillations reflect G4 binding, not unfolding.

Experimental data support an ATP-dependent translocation model.

Abstract

Chen et al. solved the crystal structure of bovine DHX36 bound to a DNA with a G-quadruplex (G4) and a single-stranded DNA segment. They believed that the mechanism they proposed may represent a general model for describing how a G4-unfolding helicase recognizes and unfolds G4 DNA. Their conclusion is interesting, however, we noticed that their linear DNA substrate (DNAMyc) that harbors a Myc-promoter-derived G4-forming sequence was directly used without pre-folding. This raises the question whether the structure they obtained really reflects DHX36-mediated G4 recognition and unfolding, or just only represents a DHX36-binding-induced quasi-folded G4 structure. By a combination of polymerase extension, DMS footprinting, stopped-flow, and smFRET assays, we obtained clear evidences that do not support their ATP-independent one-base translocation structural model. We further revealed that the oscillation of FRET signal they observed should correspond to a repetitive G4 binding, but not unfolding, by DHX36.