| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
In this study, we checked the localization of human REV proteins in cells, and we also analyzed the effect of UV-induced DNA damage, to the REV protein localization. The expression vectors for GFP-fusion REV proteins were produced and introduced into COS7 cells. The intracellular localization of the GFP-fusion REV proteins were analyzed using a confocal laser microscope. It was revealed that REV1 protein localizes in nucleus and forms a lot of tiny foci in about 3% of cells. REV7 was found to localize mainly in nucleus and partially in cytoplasm without focus formation, and REV3 could not be analyzed in detail because of its low expression in cells. Thereafter, we focused on the analysis of REV1 localization and its relation with UV-induced DNA damage. Cells with GFP-REV1 expression were UV-irradiated and REV1 focus formation was analyzed. It was revealed that REV1 focus formation was observed in about 3% of cells under normal condition and the percentage of the focus forming cells was increased up to about 25% 8 hours after 8J/m^2 irradiation. The percentage was found to increase in a UV dose-dependent and a time-dependent manner. Most of the REV1 foci were co-localized with PCNA, a marker of the DNA replication fork. And the REV1 foci were also co-localized with Pol κ and Pol η, the other proteins involved in translesion DNA synthesis. The domain of REV1 required for the foci formation was also analyzed by using truncation mutants of REV1 fused with GFP. It was found that C-terminal region of REV1,where the binding domain for REV7,Pol κ and Pol η exists, was required for the focus formation. These results indicate that REV1 focus formation is induced by UV irradiation and may be associated with UV-induced DNA damage. The foci may be also involved the PCNA-associated DNA replication. REV1 functions as a terminal deoxycytidyl transferase at abasic lesions on template DNA in vitro. Our data support the REV1 function in vivo.
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