Four-stranded G4 DNA Formation During Synthesis Phase of the Cell Cycle Protects Human Cells from Radiation Induced DNA Breaks.

The breaks induced during exposure to ionizing radiation are generally random. However, some studies show differences in radioprotection among the species within the same genus due to factors like DNA repair efficiency, genome complexity, chromatin organization, etc. A recent study linked the formation of G4 structures to differential radiosensitivity of the human genome. The present study investigates whether radiation-induced DNA damage varies across different phases of the cell cycle and explores the molecular basis for this. Neutral comet and immunofluorescence assays demonstrated that cells in S phase exhibit significantly fewer IR-induced DSBs compared to those in G1 or G2/M phases. In contrast, S phase cells remained fully susceptible to DSB induction by ROS-independent clastogens, indicating that S phase radioprotection is linked to ROS mitigation rather than intrinsic structural resistance alone. BG4 immunofluorescence revealed a higher abundance of transient G4 structures in replicating S phase cells. Depleting the BLM helicase via shRNA stabilized these G4 structures and further suppressed IR-induced DSB accumulation during S phase. Biochemical analyses using the Amplex Red assay confirmed that G4 DNA efficiently quenches IR-generated ROS, whereas non-G4 DNA shows negligible scavenging activity. Quantitative PCR assays corroborated that G4-enriched genomic loci sustain substantially less radiation damage than non-G4 control regions across all cell-cycle phases. Collectively, these findings demonstrate that elevated G-quadruplex formation during DNA replication acts as an intrinsic ROS sink that shields the genome against radiation injury. Alongside robust homologous recombination repair, G4-mediated radioprotection drives cell-cycle-dependent radiation tolerance, highlighting a structural mechanism governing human genome integrity.