Abstract
Tumor suppressor p53 Binding Protein 1(53BP1) is recruited rapidly to sites of DNA double-strand breaks (DSBs) and participates in the cellular response to DNA damage. Formation of 53BP1 foci is important for both DNA damage repair and signal transduction. We exploited property of 53BP1 that accumulate in DSB sites and developed biosensor detecting DNA damage using the specical form of 53BP1-GFP fusion protein that could express stably in the cell. Futhermore, we built a system that can identify the difference of cell cycle stage by intranuclear localization patterns of PCNA-DsRed. U2OS cell lines (U2RDP-LE53-21) expressing both these fluorescent fusion proteins make it possible to observe DSBs occurred in different cell cycle stage, these cells remain living. We will reveal that DNA damage and behavior of 53BP1 in the different cell cycle stage, and attempt screening chemotherapy drugs and radiosensitizers that induce DSBs in cancer cells.
In this report, we will show how 53BP1 foci are observed in normal culturing conditions, and also how DSBs 53BP1 foci are induced by chemical agents such as Neocarcinostatine (NCS) or DNA Topoisomerase I inhibitor Camptothesin (CPT) that are well-known DSBs inducers. Also, 53BP1 foci induced by radiation as mono-DSBs were detected by confocal laser scanning microscope and its kinetics were revealed. We performed three-dimensional restructuring of DSBs 53BP1 foci and quantifying their steric volumes, and examined kinetics in specific cell stage using newly developed micro-imaging software.
