In situ analysis of repair processes for oxidative DNA damage in mammalian cells

doi:10.1073/pnas.0406048101

In situ analysis of repair processes for oxidative DNA damage in mammalian cells

^*Department of Molecular Genetics, Institute of Development, Aging, and Cancer, Tohoku University, Seiryomachi 4-1, Sendai 980-8575, Japan; ^‡Research Laboratory for Molecular Genetics, Yamagata University, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan; ^§Biochemistry Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan; and ^¶Laboratory of Structural Biology, National Institute on Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709

Communicated by Philip C. Hanawalt, Stanford University, Stanford, CA, August 17, 2004 (received for review June 6, 2004)

Abstract

Oxidative DNA damage causes blocks and errors in transcription and replication, leading to cell death and genomic instability. Although repair mechanisms of the damage have been extensively analyzed in vitro, the actual in vivo repair processes remain largely unknown. Here, by irradiation with an UVA laser through a microscope lens, we have conditionally produced single-strand breaks and oxidative base damage at restricted nuclear regions of mammalian cells. We showed, in real time after irradiation by using antibodies and GFP-tagged proteins, rapid and ordered DNA repair processes of oxidative DNA damage in human cells. Furthermore, we characterized repair pathways by using repair-defective mammalian cells and found that DNA polymerase β accumulated at single-strand breaks and oxidative base damage by means of its 31- and 8-kDa domains, respectively, and that XRCC1 is essential for both polymerase β-dependent and proliferating cell nuclear antigen-dependent repair pathways of single-strand breaks. Thus, the repair of oxidative DNA damage is based on temporal and functional interactions among various proteins operating at the site of DNA damage in living cells.

Footnotes

↵ ∥ To whom correspondence should be addressed. E-mail: ayasui{at}idac.tohoku.ac.jp.
↵ † L.L. and S.N. contributed equally to this work.
Abbreviations: BER, base excision repair; CAF1-p150, chromatin assembly factor 1 p10 subunit; CHO, Chinese hamster ovary; DIQ, 1,5-dihydroxyisoquinoline; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; F, filter transparency; LIGIIIα, ligase IIIα; PARP, poly(ADP-ribose) polymerase; PCNA, proliferating cell nuclear antigen; POL β, DNA polymerase β; siRNA, short interference RNA; SSB, single-strand break.