Distinct and overlapping roles of human RecQ helicases in the recognition and unwinding of different G-quadruplexes

G-quadruplexes (G4s) are non-canonical DNA structures widely distributed across the genome, enriched at oncogenic promoters and telomeres. They play crucial roles in transcription, replication and telomere maintenance; G4 formation and regulated unwinding are important for genome stability and their dysregulation has been linked to cancer development. The RecQ and Iron-Sulfur (FeS) helicase families were shown to regulate G4 structures. RecQ helicases are conserved in evolution, with 5 paralogs in humans (RecQ1, BLM, WRN, RecQ4, RecQ5), all involved in cancer development. Although there are evidences for RecQ resolving G4s, a systematic analysis is needed to clarify their specificity toward different G4 topologies and understand their distinct or overlapping roles in G4 metabolism. We produced the catalytic domains of each helicase and analyzed their activity towards DNA Forks, D-loops, R-loops, and eleven physiologically relevant unimolecular G4s, with different genomic origins and topologies. This analysis revealed distinct patterns. BLM and WRN showed high affinity and robust unwinding, with significant discrimination between different G4s. As the helicase activity can be influenced by G4 thermal stability in various ionic conditions, CD spectroscopy assessed the stability of each G4 in multiple buffers, confirming that helicase preferences were not simply due to relative stabilities. BLM ability to unwind G4s remained unaffected by increasing K+ concentrations, whereas WRN activity decreased. In contrast, RecQ1, RecQ4, and RecQ5 exhibited little to no G4 binding or unwinding activity. The crystal structure of a bacterial RecQ bound to a resolved G4 suggested the presence of a putative guanine-binding pocket within RecQ helicases. Site-directed mutagenesis experiments on BLM and WRN to disrupt this pocket confirmed this hypothesis. Crystallization experiments to elucidate the structural determinants of RecQ-G4s interaction were unsuccessful. In parallel, we expressed and purified the E. coli DinG FeS helicase and, in collaboration with the University of Naples, systematically analyzed its interactions with different G4s, revealing a preference for parallel conformations. G4 ligands reduced or abolished DinG helicase activity without affecting its binding. Overall, our findings highlight the specificity of helicase interactions with G4s and the importance of investigating multiple G4 structures to fully understand the role of helicases in G4 metabolism.