Valenzisi, P.; Parrillo, R.; Pichierri, P.; Franchitto, A.

G-quadruplexes (G4s) are non-canonical DNA structures that regulate transcription, replication, and DNA repair but, when unresolved, hinder replication fork progression and compromise genome stability. Here, we identify a WRNIP1-dependent mechanism that promotes the resolution of R-loop-associated G4 structures. Loss of WRNIP1 or disruption of its ubiquitin-binding zinc finger (UBZ) domain causes persistent G4/R-loop accumulation, leading to transcription-replication conflicts, DNA damage, and genome instability. We further show that WRNIP1 interacts with the G4 helicase FANCJ and that mutation of the UBZ domain disrupts this interaction, impairing FANCJ stability and recruitment to G4 sites. Mechanistically, WRNIP1 acts upstream of FANCJ by promoting ATM-dependent signaling required for FANCJ stabilization and chromatin association. ATM-mediated phosphorylation of FANCJ at Ser990 protects the helicase from ubiquitin-dependent proteasomal degradation. Together, these findings define a WRNIP1-ATM-FANCJ regulatory axis that promotes G4 resolution during DNA replication and preserves genome stability.