The molecular basis for the arising replication stress and DNA damage following p53 loss

p53 has critical functions in regulating cell cycle in response to cellular stresses, preventing the accumulation of damaged cells. Recent studies demonstrated that p53 acts to avoid replication-induced DNA-damage by preventing replication-stress. Here we studied the molecular basis for the replication-stress and DNA-damage induced by p53 loss. We focused on: 1.p53-downregulated cells. 2.Cells from Li-Fraumeni syndrome (LFS) patients that spontaneously lose the wild type p53(WTp53) in culture. To determine the outcome of WTp53-loss, we performed high resolution DNA replication dynamics analyses, accompanied with DNA-damage analyses. We found in both cellular systems following WTp53-loss, replication-stress and high DNA-damage levels. Exogenous nucleoside supply rescued both the replication-stress and DNA-damage. We further focused on the LFS cells, that enter an early proliferating crisis followed by a spontaneous escape of cells from the proliferation crisis and immortalization. To determine if replication-stress contributes to both the early crisis and the late immortalization, we performed replication dynamics analyses on early and late LFS cells. We found that LFS cells show a loss of the WTp53 and induction of replication-stress with high DNA-damage levels before the proliferation crisis. However, cells that escaped from the crisis exhibit normal replication dynamics and low levels of DNA-damage. Strikingly, exogenous nucleoside supply to early LFS cells enhanced cell proliferation and further rescued the replication-stress and DNA-damage. Analyses of the escaped immortalized cells revealed a significant up-regulation of nucleoside biosynthesis genes, suggesting that the cellular adaptation mechanism to the p53-loss involves rescue of the replication-stress through regulation of the nucleotide pool.