Istituto di Genetica Molecolare del CNR, Pavia, Italy In mammalian cells DNA replication takes place in subnuclear sites called replication factories consisting of cluster of replicons and their associated replication machinery. Each factory undergoes an assembly- disassembly cycle according to a precise space- and time-dependent program, that parallels the replication of different portions of the genome. How this dynamic process is controlled is still elusive. We have recently observed that the dynamics of replication factories is perturbed in response to DNA damage. We have further investigated the fate of the factories in response to DNA damage and replicational stress in human normal cells and cell lines defective in DNA repair and/or checkpoint pathways. We have observed that in normal fibroblasts, etoposide but not UV irradiation or aphidicolin, induces the disassembly of replication factories and the dissociation of PCNA and DNA ligase I from the chromatin fraction. The effect of etoposide is prevented by agents that promote stalling of replication forks and by general inhibitors of protein kinases. Interestingly, disassembly of replication factories and dissociation of PCNA from chromatin do not occur in cells over-expressing a dominant negative version of ATR kinase indicating that the disassembly of the factories induced by etoposide requires a functional ATR. Moreover, UV-irradiation induces the dispersal of the factories in a subset of DNA repair and checkpoint deficient cells such as Bloom Syndrome cells. Our results demonstrate that the stability of replication factories after treatments that induce DNA damage and/or stalling of replication forks is controlled by the intra-S phase checkpoint.
