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(PP246) The autophosphorylation of DNA-PKcs in irradiated human cells.
Okabe, Atsushi1, 2, Takakura, Kaoru2, Okayasu, Ryuichi1, 1 International Space Radiation Laboratory, Chiba, Chiba, Japan2 Division of Natural Sciences, Mitaka, Tokyo, Japan
ABSTRACT- We used an antibody to DNA-PKcs phosphorylated at threonine 2609(T2609) to study the status of autophosphorylation of DNA-PKcs, which is one of the key proteins for Non Homologous End Joining(NHEJ). The number of foci of T2609 induced by X rays in normal human fibroblast cells (HFLIII) and Ligase IV deficient cells (180BR) were counted under fluorescent microscope. DNA-PKcs was autophosphorylated 10 minutes after irradiation and the number of foci reached the peak at 1hour after irradiation in both cell lines. Although the number of foci were gradually reduced and returned to almost the background levels after 24 hours, the remaining number of foci in 180BR cells was much higher than that in normal cells. To investigate the interaction with other proteins, the colocalization of T2609 was studied with phosphorylated ATM at serine 1981(S1981) and gamma H2AX. The focus of T2609 was formed over nucleus and did not correspond with the sites of S1981 and gamma H2AX foci. However the foci of T2609 were accumulated and almost all of them were colocalized with S1981 and gamma H2AX 4 hours after irradiation. In unirradiated cells, gamma H2AX and S1981 foci were not formed at the sites of T2609 foci, although there were some foci of phosphorylated DNA-PKcs. After high LET carbon ion(70keV/um) irradiation, the peak for the number of T2609 foci appearance was significantly delayed in HFLIII and 180BR when compared with X-irradiated cells. This delay was more distinct in irradiated 180BR cells. These results indicate both DNA-PKcs and ATM may function at DSB sites simultaneously. Besides, DNA-PKcs should be phosphorylated not at the sites of DSB. Therefore, the phosphorylation of DNA-PKcs should have not only repair function but also other functions. The results from experiments using high LET heavy ion irradiation are also presented.
Key words: autophosphorylation of DNA-PKcs, DNA damage, high LET irradiation
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