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. 2004 Sep 21;101(38):13738-43.
doi: 10.1073/pnas.0406048101. Epub 2004 Sep 13.

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

Li Lan  1 , Satoshi Nakajima, Yoshitsugu Oohata, Masashi Takao, Satoshi Okano, Mitsuko Masutani, Samuel H Wilson, Akira Yasui
Affiliations

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

Li Lan et al. Proc Natl Acad Sci U S A. .

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 beta 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 beta-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.

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Figures

Fig. 1.
Fig. 1.
Kinetics of repair intermediates measured by using antibodies. (A) Immunochemical detection of immediate products poly(ADP-ribose) (Left), γH2AX (Center), and 8-OHdG (Right) after laser-light irradiation of HeLa cells through F20 (1-pulse irradiation in array), F25 (1-pulse irradiation in array), and F30 (10 pulses) filters, respectively. (B) Immunohistochemistry of XRCC1 after irradiation through the F20 filter and PCNA and CAF1-p150 after irradiation through the F25 filter. (C) Time-dependent detection of repair protein with antibody against LIGIIIα after irradiation through the F20 filter. (D) Time course of the amount of accumulated LIGIIIα.(E) Time-dependent detection of 8-OHdG with its antibody after irradiation through the F30 filter with 10 pulses. (F) Time course of the amount of 8-OHdG after irradiation through the F30 filter with 10 pulses. Mean values with error bar obtained from more than three irradiated cells are shown.
Fig. 2.
Fig. 2.
Repair kinetics of SSBs and base damage in living cells. (A Left) Expression and accumulation of GFP-tagged XRCC1, POLβ, LIGIIIα, PCNA, and CAF1-p150 before and after irradiation through the F20 filter. (A Right) Expression and accumulation of GFP-tagged NTH1, OGG1, NEIL1, and NEIL2 before and after irradiation through the F25 filter. Before irradiation (left side of each image) and maximum accumulation after irradiation (right side of each image) are shown. (B) Accumulation patterns of GFP-tagged proteins in the proficient and deficient rodent cell lines. XRCC1 after irradiation through the F20 filter in wild-type (AA8, □) or deficient (EM9, ▪) cells; POL β after irradiation through the F20 filter in wild-type (MB36.3, ⋄) or deficient (MB38Δ4, ♦) cells; and NTH1 after irradiation through the F25 filter in wild-type (WTB, ○) or deficient (12–7B, •) cells. (C) Accumulation kinetics of GFP-tagged XRCC1 (□), POL β (⋄), LIGIIIα (○), CAF1-p150 (▵), and PCNA (▪) after irradiation through the F20 filter in HeLa cells. (D) Accumulation kinetics of GFP-tagged NTH1 (□), NEIL1 (○), NEIL2 (▵), and OGG1 (⋄) after irradiation through the F25 filter in HeLa cells. (E) Dissociation kinetics of GFP-tagged XRCC1 (□), POL β (⋄), LIGIIIα (○), CAF1-p150 (▵), and PCNA (▪) after irradiation through the F20 filter in HeLa cells. (F) Dissociation kinetics of GFP-tagged NTH1 (□), NEIL1 (○), NEIL2 (▵), and OGG1 (⋄) after irradiation through the F25 filter in HeLa cells.
Fig. 3.
Fig. 3.
POL β accumulation at SSBs and base damage. (A) Accumulation of GFP-tagged XRCC1 after irradiation without (▵) and with (▴) DIQ treatment, POL β after irradiation without (⋄) and with (♦) DIQ treatment, LIGIIIα after irradiation without (○) and with (•) DIQ treatment, and NTH1 after irradiation without (□) and with (▪) DIQ treatment in HeLa cells. XRCC1, POL β, and LIGIIIα are visualized after irradiation through the F20 filter, and NTH1 is visualized after irradiation through the F25 filter. (B) POL β accumulation in XRCC1-deficient and -proficient CHO cells. Accumulation of GFP-tagged POL β in XRCC1-deficient EM9 after irradiation through the F25 (•) or F20 (○) filter; wild-type AA8 after irradiation through the F25 (♦) or F20 (⋄) filter; and wild-type hXRCC1-expressing EM9, EFX, after irradiation through the F25 (▪) or F20 (□) filter. (C) Influence of photosensitizer RO-19-8022 on the accumulation of GFP-tagged OGG1 in EM9 cells after irradiation through the F25 filter. OGG1 accumulation without (□) or with (▪) photosensitization is shown. (D) Influence of RO-19-8022 on the accumulation of GFP-tagged POL β in EM9 cells after irradiation through the F25 filter. POL β accumulation without (□) or with (▪) photosensitization is shown. (E) Two domains of POL β, N-terminal 8-kDa domain with 5′-deoxyribose phosphate lyase activity and C-terminal 31-kDa polymerase domain, are shown. (F) Accumulation of the GFP-fused 8-kDa domain of POL β at irradiated sites in wild-type mouse (□), PARP1-mouse (⋄), and EM9 (XRCC1–)(○) CHO cells after irradiation through the F25 filter. (G) Accumulation of GFP-tagged 31-kDa domain of POL β in wild-type mouse (□), PARP1-mouse (⋄), and EM9 (XRCC1–)(○) CHO cells after irradiation through the F25 filter.
Fig. 4.
Fig. 4.
Accumulation of PCNA at SSBs and base damage. (A) Accumulation of GFP-tagged PCNA after irradiation in HeLa cells through the F20 filter without (○) or with (•) DIQ treatment and CAF1-p150 in HeLa cells after irradiation through F20 filter without (▵) or with (▴) DIQ treatment. (B) Accumulation of CAF1-p150 in CHO wild-type AA8 cells after irradiation through F25 (□) or F20 (▪) filters, EFX cells after irradiation through F25 (○) or F20 (•) filters, and XRCC1-defective EM9 cells after irradiation through F25 (▵) or F20 (▴) filters. (C) Accumulation of PCNA in CHO wild-type AA8 cells after irradiation through F25 (□) or F20 (▪) filters, EFX cells after irradiation through F25 (○) or F20 (•) filters, and XRCC1-defective EM9 cells after irradiation through F25 (▵) or F20 (▴) filters. (D) Influence of RNA interference by siRNA for XRCC1 on the suppression of hXRCC1 expression at mRNA (Left) and protein (Right) levels in HeLa cells. (E) Influence of RNA interference by siRNA for XRCC1 on the accumulation of PCNA after irradiation through the F20 filter in HeLa cells with mock (□) or siRNA (▪) treatment and POL β after irradiation through the F20 filter in HeLa cells with mock (○) or siRNA (•) treatment. (F) Accumulation of PCNA in POL β+ (□) and POL β– (▪) cells through the F20 filter. (G) Influence of RO-19-8022 on the accumulation of PCNA in CHO EM9 cells after irradiation through the F25 filter. Accumulation of GFP-tagged PCNA without (○) or with (•) photosensitization is shown.
Fig. 5.
Fig. 5.
Schematic description of repair pathways for oxidative DNA damage suggested by in vivo analysis.
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