Zaatra, W.; Philippos, G.; Smirnov, P.; Cochavi, S. M.; Otonicar, J.; Chan, M.; Harel, M.; Devens, F.; Eliassaf, A.; Grimes, K.; Irony-Tur Sinai, M.; Sigismondo, G.; Gieiger, T.; Korbel, J.; Krijgsveld, J.; Shalev, O.; Kerem, B.; Ernst, A.

P53 plays a critical role in preventing DNA damage accumulation by activating repair processes, inducing cell cycle arrest, or triggering apoptosis. Loss of p53 leads to replication stress and genomic instability, yet the mechanisms underlying these effects and their contribution to catastrophic events like chromothripsis remain poorly understood. Using patient-derived fibroblasts with germline p53 variants that spontaneously undergo chromothripsis, as well as p53-downregulated fibroblasts, we discovered that p53 loss disrupts the balance between nucleotide consumption and production through increasing transcription and decreasing nucleotide biosynthesis, respectively. This imbalance results in insufficient nucleotide pools, leading to replication stress. We further showed that this replication stress triggers telomere dysfunction and micronuclei formation, ultimately causing chromothripsis. Supplementing nucleosides or inhibiting transcription rescued replication stress and genomic instability. We also observed the emergence of dominant chromothriptic clones, which exhibited normal DNA replication, telomere stabilization, and extrachromosomal circular DNA structures, highlighting critical features for clonal selection and expansion.