Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding

Abstract

G-quadruplexes (G4s) are unusual DNA structures and can stall DNA replication, causing genomic instability for the cell. Although the solved crystal structure of the DHX36 helicase demonstrated that G4 was specifically targeted by a DHX36-specific motif (DSM), lack of complete structural details for general G4-resolving helicases without specific target motifs remains a barrier to the complete understanding of the molecular basis underlying the recognition and unfolding of G4s. Herein, we present the first X-ray crystal structure of the Thermus oshimai Pif1 (ToPif) complexed with a G4, thereby mimicking the physiological G4 formed during DNA replication. Strictly different from the previous determined G4-helicase structure of DHX36, our structure revealed that ToPif1 recognizes the entire native G4 via a cluster of amino acids at domains 1B/2B constituting a G4-Recognizing Surface (GRS). The overall topology of the G4 structure solved in this work maintains its three-layered propeller-type G4 topology, with no significant reorganization of G-tetrads upon protein binding. The three G-tetrads in G4 were differentially recognized by GRS residues mainly through electrostatic, ionic interactions and hydrogen bonds formed between the GRS residues and the ribose-phosphate backbone. Our structure explains how helicases from distinct superfamilies adopt different strategies for recognizing and unfolding G4s.