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Abstract
DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB.
ACKNOWLEDGMENTS
We thank B. Stillman (Cold Spring Harbor Laboratory) for providing us with the anti-CAF-1 p150 antibody. We also thank Y. Watanabe (Olympus) and D. Suzuki (Hamamatsu) for technical support in microscopy. We thank S. McCready for editing the text.
This work was supported by Grants-in-Aid for Scientific Research (no. 12143201 and no. 13480162) from the Ministry of Education, Science, Sports and Culture of Japan and by the Mitsubishi Foundation to A.Y.