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Molecular and Cellular Biology, June 2003, p. 3974-3981, Vol. 23, No. 11
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.11.3974-3981.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Spatial and Temporal Cellular Responses to Single-Strand Breaks in Human Cells

Satoshi Okano,1 Li Lan,1 Keith W. Caldecott,2 Toshio Mori,3 and Akira Yasui1*

Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, 980-8575 Sendai,1 Radioisotope Research Center, Nara Medical University, 634-8521 Nara, Japan,3 Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom2

Received 23 December 2002/ Returned for modification 10 February 2003/ Accepted 13 March 2003

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.

* Corresponding author. Mailing address: Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, 980-8575 Sendai, Japan. Phone: 81 22 717 8465. Fax: 81 22 717 8470. E-mail: ayasui{at}idac.tohoku.ac.jp.

Molecular and Cellular Biology, June 2003, p. 3974-3981, Vol. 23, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.11.3974-3981.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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