Suppression of replication fork progression in low dose specific p53 dependent S-phase DNA damage checkpoint.

The S-phase DNA damage checkpoint is activated by DNA damage to delay DNA synthesis allowing time to resolve the replication block. We previously discovered the p53-dependent S-phase DNA damage checkpoint in mouse zygotes fertilized with irradiated sperm. Here, we report that the same p53dependency holds in mouse embryonic fibroblasts (MEFs) at low doses of irradiation. DNA synthesis in p53wild- type (WT) MEFs was suppressed in a biphasic manner in which a sharp decrease below 2.5 Gy was followed by a more moderate decrease up to 10 Gy. In contrast, p53_/_ MEFs exhibited radioresistant DNA synthesis below 2.5 Gy whereas the cells retainedthe moderate suppression above 5 Gy. DNA fiber analysis revealed that 1Gy irradiation suppressed replication fork progression in p53WT MEFs, but not in p53_/_ MEFs. Proliferating cell nuclear antigen (PCNA), clamp loader of DNA polymerase, was phosphorylated in WT MEFs after 1Gy irradiation and redistributed to form foci in the nuclei. In contrast, PCNA was not phosphorylated and dissociated from chromatin in 1 Gy-irradiated p53_/_ MEFs. These results demonstrate that the novel low-dosespecific p53-dependent S-phase DNA damage checkpoint is likely to regulate the replication fork movement through phosphorylation of PCNA