1. ¹Department of Late Effect Studies, Radiation Biology Center, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto, Japan
2. ²Department of Radiation System Biology, Radiation Biology Center, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto, Japan
3. ³Graduate School of Biosphere Science, Hiroshima University, Hiroshima, Japan
4. ⁴Medical Research Institute, Kanazawa Medical University, Uchinada-machi, Kahoku-gun, Ishikawa, Japan

Correspondence: Dr T Shimura, Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, NIH Bldg 37, Rm 5056 37 Convent Dr Bethesda, MD 20892-4255, USA. E-mail: shimurat@mail.nih.gov

Received 27 June 2005; Revised 8 March 2006; Accepted 13 March 2006; Published online 8 May 2006.

Abstract

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 p53 dependency holds in mouse embryonic fibroblasts (MEFs) at low doses of irradiation. DNA synthesis in p53 wild-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 retained the moderate suppression above 5 Gy. DNA fiber analysis revealed that 1 Gy irradiation suppressed replication fork progression in p53 WT MEFs, but not in p53-/- MEFs. Proliferating cell nuclear antigen (PCNA), clamp loader of DNA polymerase, was phosphorylated in WT MEFs after 1 Gy 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-dose-specific p53-dependent S-phase DNA damage checkpoint is likely to regulate the replication fork movement through phosphorylation of PCNA.