Pyridine-bis(benzimidazole) induces DNA damage at G-quadruplex loci and promotes synthetic lethality with DNA repair inhibition.

G-quadruplexes (G4s) are noncanonical DNA structures that play key roles in regulating replication, transcription, and genome stability. Here, we investigate the effects of pyridine-bis(benzimidazole) (PyBI), a selective parallel and hybrid G4 stabilizer, on genome stability in cells. Biophysical and biochemical assays confirm PyBI's strong affinity for parallel G4s, leading to replication fork stalling and transcriptional repression of G4-associated oncogenes. Cleavage under targets and tagmentation (CUT&Tag) sequencing reveals a PyBI-induced genome-wide increase in G4 peaks, particularly at promoter and transcription start site regions. This G4 induction and stabilization triggers replication stress, G2/M arrest, and apoptosis. DNA repair pathway profiling shows that PyBI-induced G4 stabilization activates both homologous recombination and nonhomologous end joining (NHEJ), with a predominant role for NHEJ, as indicated by higher 53BP1 colocalization at G4 sites. Genome-wide mapping of PyBI-induced DNA breaks further supports a direct link between G4 stabilization and DNA damage. Moreover, PyBI shows synthetic lethality in DNA repair-deficient contexts, highlighting its therapeutic potential. These findings provide mechanistic insights into the genotoxic effects of PyBI and highlight its potential as a novel anticancer agent targeting G4-mediated genome instability.