Identification of HELLS as a Novel DNA G-quadruplex Helicase

Guanine quadruplexes (G4s) are unique secondary structures of nucleic acids with functions in many biological processes. Understanding the biological functions of DNA G4s requires the knowledge about their recognitions by cellular proteins. Affinity pull-down coupled with LC-MS/MS analysis was previously employed to identify G4-binding proteins (G4BPs); the approach, however, has limitations in capturing weak and transient interactions, potentially leading to false-positives. Here, we developed a photoclick chemistry-based method, in combination with LC-MS/MS, for uncovering G4BPs. By incorporating a photoactivatable ortho-nitrobenzylamine (o-NBA) moiety into G4 DNA probes and employing UVA irradiation along with stringent washing, we identified 99 proteins enriched with G4 structures derived from the human telomere. We further validated the abilities of one of these proteins, HELLS, in binding and resolving G4 structures both in vitro and in chromatin. Together, we developed a photoclick chemistry-based approach for identifying novel G4BPs. Our work led to the discovery of new G4BPs, and uncovered novel functions of HELLS in recognizing and unwinding G4 structures in vitro and in cells.