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In Situ Visualization of Ultraviolet-Light-Induced DNA Damage Repair in Locally Irradiated Human Fibroblasts

2001; Elsevier BV; Volume: 117; Issue: 5 Linguagem: Inglês

10.1046/j.0022-202x.2001.01540.x

1523-1747

Sachiko Katsumi, Nobuhiko P. Kobayashi, Kyoko Imoto, Akemi Nakagawa, Yukio Yamashina, Tsutomu Muramatsu, Toshihiko Shirai, Sachiko Miyagawa, Shigeki Sugiura, Fumio Hanaoka, Tsukasa Matsunaga, Osamu Nikaido, Toshio Mori,

Light effects on plants

We have developed a novel method that uses a microfilter mask to produce ultraviolet-induced DNA lesions in localized areas of the cell nucleus. This technique allows us to visualize localized DNA repair in situ using immunologic probes. Two major types of DNA photoproducts [cyclobutane pyrimidine dimers and (6–4) photoproducts] were indeed detected in several foci per nucleus in normal human fibroblasts. They were repaired at those localized sites at different speeds, indicating that DNA photoproducts remain in relatively fixed nuclear positions during repair. A nucleotide excision repair protein, proliferating cell nuclear antigen, was recruited to the sites of DNA damage within 30 min after ultraviolet exposure. The level of proliferating cell nuclear antigen varied with DNA repair activity and diminished within 24 h. In contrast, almost no proliferating cell nuclear antigen fluorescence was observed within 3 h in xeroderma pigmentosum fibroblasts, which could not repair either type of photolesion. These results demonstrate that this technique is useful for visualizing the normal nucleotide excision repair process in vivo. Interestingly, however, in xeroderma pigmentosum cells, proliferating cell nuclear antigen appeared at ultraviolet damage sites after a delay and persisted as late as 72 h after ultraviolet exposure. This result suggests that this technique is also valuable for examining an incomplete or stalled nucleotide excision repair process caused by the lack of a single functional nucleotide excision repair protein. Thus, the technique provides a powerful approach to understanding the temporal and spatial interactions between DNA damage and damage-binding proteins in vivo. We have developed a novel method that uses a microfilter mask to produce ultraviolet-induced DNA lesions in localized areas of the cell nucleus. This technique allows us to visualize localized DNA repair in situ using immunologic probes. Two major types of DNA photoproducts [cyclobutane pyrimidine dimers and (6–4) photoproducts] were indeed detected in several foci per nucleus in normal human fibroblasts. They were repaired at those localized sites at different speeds, indicating that DNA photoproducts remain in relatively fixed nuclear positions during repair. A nucleotide excision repair protein, proliferating cell nuclear antigen, was recruited to the sites of DNA damage within 30 min after ultraviolet exposure. The level of proliferating cell nuclear antigen varied with DNA repair activity and diminished within 24 h. In contrast, almost no proliferating cell nuclear antigen fluorescence was observed within 3 h in xeroderma pigmentosum fibroblasts, which could not repair either type of photolesion. These results demonstrate that this technique is useful for visualizing the normal nucleotide excision repair process in vivo. Interestingly, however, in xeroderma pigmentosum cells, proliferating cell nuclear antigen appeared at ultraviolet damage sites after a delay and persisted as late as 72 h after ultraviolet exposure. This result suggests that this technique is also valuable for examining an incomplete or stalled nucleotide excision repair process caused by the lack of a single functional nucleotide excision repair protein. Thus, the technique provides a powerful approach to understanding the temporal and spatial interactions between DNA damage and damage-binding proteins in vivo. cyclobutane pyrimidine dimers nucleotide excision repair proliferating cell nuclear antigen (6–4) photoproducts xeroderma pigmentosum DNA damage in cells exposed to ultraviolet (UV) light plays a significant role in cell cycle arrest, activation of DNA repair, cell killing, mutation, and neoplastic transformation (Setlow, 1978Setlow R.B. Repair deficient human disorders and cancer.Nature. 1978; 271: 713-717Crossref PubMed Scopus (388) Google Scholar;Suzuki et al., 1981Suzuki F. Han A. Lankas G.R. Utsumi H. Elkind M.M. Spectral dependencies of killing, mutation, and transformation in mammalian cells and their relevance to hazards caused by solar ultraviolet radiation.Cancer Res. 1981; 41: 4916-4924PubMed Google Scholar;Maher et al., 1982Maher V.M. Rowan L.A. Silinskas K.C. Kateley S.A. McCormick J.J. Frequency of UV-induced neoplastic transformation of diploid human fibroblasts is higher in xeroderma pigmentosum cells than in normal cells.Proc Natl Acad Sci USA. 1982; 79: 2613-2617Crossref PubMed Scopus (84) Google Scholar;Otoshi et al., 2000Otoshi E. Yagi T. Mori T. et al.Respective roles of cyclobutane pyrimidine dimers, (6–4) photoproducts, and minor photoproducts in ultraviolet mutagenesis of repair-deficient xeroderma pigmentosum A cells.Cancer Res. 2000; 60: 1729-1735PubMed Google Scholar;Zhou and Elledge, 2000Zhou B.-B.S. Elledge S.J. The DNA damage response: putting checkpoints in perspective.Nature. 2000; 408: 433-439Crossref PubMed Scopus (2637) Google Scholar). Two major types of DNA lesions produced by UVB (280–315 nm) and UVC (200–280 nm) are cyclobutane pyrimidine dimers (CPD) and (6–4) photoproducts (6–4PP) (Clingen et al., 1995Clingen P.H. Arlett C.F. Cole J. et al.Correlation of UVC and UVB cytotoxicity with the induction of specific photoproducts in T-lymphocytes and fibroblasts from normal human donors.Photochem Photobiol. 1995; 61: 163-170Crossref PubMed Scopus (59) Google Scholar). 6–4PP are formed at a rate that is 15%-33% that of CPD (Mitchell, 1988Mitchell D.L. The relative cytotoxicity of (6–4) photoproducts and cyclobutane dimers in mammalian cells.Photochem Photobiol. 1988; 48: 51-57Crossref PubMed Scopus (310) Google Scholar;Clingen et al., 1995Clingen P.H. Arlett C.F. Cole J. et al.Correlation of UVC and UVB cytotoxicity with the induction of specific photoproducts in T-lymphocytes and fibroblasts from normal human donors.Photochem Photobiol. 1995; 61: 163-170Crossref PubMed Scopus (59) Google Scholar;Eveno et al., 1995Eveno E. Bourre F. Quilliet X. et al.Different removal of ultraviolet photoproducts in genetically related xeroderma pigmentosum and trichothiodystrophy diseases.Cancer Res. 1995; 55: 4325-4332PubMed Google Scholar). Although 6–4PP are removed from the global genome at a much higher speed than CPD, both types of lesions are repaired by nucleotide excision repair (NER) in normal human cells (Mitchell et al., 1985Mitchell D.L. Haipek C.A. Clarkson J.M. (6–4) Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers.Mutat Res. 1985; 143: 109-112Crossref PubMed Scopus (241) Google Scholar). Defects in NER are associated with a rare autosomal recessive human disease termed xeroderma pigmentosum (XP), which is primarily characterized by extreme UV sensitivity and an increased incidence of sunlight-induced skin cancers (Kraemer et al., 1987Kraemer K.H. Lee M.M. Scotto J. Xeroderma pigmentosum.Arch Dermatol. 1987; 123: 241-250Crossref PubMed Scopus (964) Google Scholar). There are seven different genetic complementation groups of XP (XP-A to XP-G) and a variant form. XP-A cells, which lack a functional XPA protein, have the most severe NER defects of all XP groups. NER is initiated by recognition of DNA damage, which can occur either by the binding of XPC/hHR23B to damaged DNA (Sugasawa et al., 1998Sugasawa K. Ng J.M.Y. Masutani C. et al.Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair.Mol Cell. 1998; 2: 223-232Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar) or by the stalling of RNA polymerase at a DNA lesion (Hanawalt et al., 1994Hanawalt P.C. Donahue B.A. Sweder K.S. Collision or collusion?.Curr Biol. 1994; 4: 518-521Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The duplex DNA around the lesion is subsequently opened by the concerted action of RPA, XPA, and TFIIH (Evans et al., 1997Evans E. Fellows J. Coffer A. Wood R.D. Open complex formation around a lesion during nucleotide excision repair provides a structure for cleavage by human XPG protein.EMBO J. 1997; 16: 625-638https://doi.org/10.1093/emboj/16.3.625Crossref PubMed Scopus (204) Google Scholar;Mu et al., 1997Mu D. Wakasugi M. Hsu D.S. Sancar A. Characterization of reaction intermediates of human excision repair nuclease.J Biol Chem. 1997; 272: 28971-28979Crossref PubMed Scopus (142) Google Scholar). This allows incisions of the damaged strand on both sides of the lesion by the structure-specific endonucleases XPG (O'Donovan et al., 1994O'Donovan A. Davies A.A. Moggs J.G. West S.C. Wood R.D. XPG endonuclease makes the 3′ incision in human DNA nucleotide excision repair.Nature. 1994; 371: 432-435Crossref PubMed Scopus (395) Google Scholar) and ERCC1/XPF (Sijbers et al., 1996Sijbers A.M. de Laat W.L. Ariza R.R. et al.Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease.Cell. 1996; 86: 811-822Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar), which are followed by excision of the lesion-containing oligonucleotide (Huang et al., 1992Huang J.-C. Svoboda D.L. Reardon J.T. Sancar A. Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5′ and the 6th phosphodiester bond 3′ to the photodimer.Proc Natl Acad Sci USA. 1992; 89: 3664-3668Crossref PubMed Scopus (377) Google Scholar). The gap in the duplex is filled in by a proliferating cell nuclear antigen (PCNA) dependent DNA polymerase (Nichols and Sancar, 1992Nichols A.F. Sancar A. Purification of PCNA as a nucleotide excision repair protein.Nucl Acids Res. 1992; 20: 2441-2446Crossref PubMed Scopus (181) Google Scholar;Shivji et al., 1992Shivji M.K.K. Kenny M.K. Wood R.D. Proliferating cell nuclear antigen is required for DNA excision repair.Cell. 1992; 69: 367-374Abstract Full Text PDF PubMed Scopus (732) Google Scholar) and is then sealed by a DNA ligase to regenerate the intact DNA structure (de Boer and Hoeijmakers, 2000de Boer J. Hoeijmakers J.H.J. Nucleotide excision repair and human syndromes.Carcinogenesis. 2000; 21: 453-460Crossref PubMed Scopus (553) Google Scholar). The NER reaction mechanism proposed above has been elucidated mainly by in vitro repair experiments with purified proteins in a reconstituted system. It has not been verified yet, however, whether this NER reaction indeed occurs in human cells in vivo, as cells consist of a much more complex environment including nuclear compartments (Misteli, 2001Misteli T. Protein dynamics. Implications for nuclear architecture and gene expression.Science. 2001; 291: 843-847https://doi.org/10.1126/science.291.5505.843Crossref PubMed Scopus (528) Google Scholar) and chromatin organization than does an in vitro system. In addition, in vivo analysis has a potential for allowing us to visualize the distribution and dynamics of NER proteins in relation to the sites of DNA damage within an individual cell. For this purpose, it is essential to detect UV-induced DNA damage in situ, particularly the two major types of damage as those are repaired at different speeds. We have raised monoclonal antibodies specific for particular photoproducts (TDM-2 for CPD, 64M-2 for 6–4PP) (Mori et al., 1991Mori T. Nakane M. Hattori T. Matsunaga T. Ihara M. Nikaido O. Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6–4) photoproduct from the same mouse immunized with ultraviolet-irradiated DNA.Photochem Photobiol. 1991; 54: 225-232Crossref PubMed Scopus (389) Google Scholar) and have established a method that allows us to visualize the induction and repair of photoproducts in individual human cells (Nakagawa et al., 1998Nakagawa A. Kobayashi N. Muramatsu T. et al.Three-dimensional visualization of ultraviolet-induced DNA damage and its repair in human cell nuclei.J Invest Dermatol. 1998; 110: 143-148https://doi.org/10.1046/j.1523-1747.1998.00100.xCrossref PubMed Scopus (72) Google Scholar). When cells are UV irradiated on a dish, however, DNA damage is induced throughout the cell nucleus where most NER proteins are diffusely located. Thus, it is difficult to detect the dynamic interactions between damage and NER proteins in situ. Because of the lack of suitable methods, some important questions remain unanswered, as follows. It is not known whether CPD and 6–4PP remain in a fixed nuclear position during DNA repair. It has not been directly shown whether NER proteins are recruited to the sites of CPD and 6–4PP for repair. It has not been shown how levels of NER proteins vary when recruited to the sites of DNA damage during repair. Moreover, it remains unclear how the lack of one functional NER protein influences the association of other NER proteins with DNA damage sites during repair. A method that produces UV-induced DNA damage at defined areas in the cell nucleus and subsequently detects them in situ is the best possible approach to resolve these issues. In this study, we developed a method to induce DNA photoproducts within localized areas of the cell nucleus using a microfilter. We then visualized the induction and repair of two major types of photoproducts (CPD and 6–4PP) at these localized nuclear sites using an immunologic method in normal and XP-A fibroblasts. Finally, we demonstrated that an NER protein, PCNA, was quickly recruited to the sites of DNA damage in normal cells. In contrast, in XP-A cells, PCNA appeared at the UV damage sites after a delay and persisted as late as 72 h after UV exposure. Normal human fibroblasts (MSU-2) from a newborn foreskin were kindly provided by Dr. James E. Trosko (Michigan State University, East Lansing, MI) (Mori et al., 1989Mori T. Wani A.A. D'Ambrosio S.M. Chang C.-C. Trosko J.E. In situ pyrimidine dimer determination by laser cytometry.Photochem Photobiol. 1989; 49: 523-526Crossref PubMed Scopus (22) Google Scholar). XP-A fibroblasts (XP12BE or GM05509) were obtained from the Coriell Cell Repositories (Camden, NJ). Fibroblasts between passages 10 and 18 were used for these experiments. Cells were cultured in Dulbecco's modified Eagle's medium (Nissui Seiyaku, Tokyo) supplemented with 10% fetal bovine serum (FBS; Dainippon Pharmaceutical, Osaka, Japan). Cells (106) were cultured in 35 mm glass bottom dishes (MatTek, Ashland, MA) for 48 h. Cells were masked with a polycarbonate isopore membrane filter (pore size, 3 µm; Millipore, Bedford, MA) and were then UV irradiated with five low-pressure mercury lamps (GL-10, Toshiba, Tokyo; predominantly 254 nm UV) at a dose rate of 1.67 J per m2 per s, which was monitored with a UV radiometer (UVR-1, Topcon, Tokyo). Immediately after micropore UV irradiation (100 J per m2) or at various times after UV irradiation, cells were treated with ice-cold detergent solution (0.5% Triton-X 100, 0.2 mg per ml ethylenediamine tetraacetic acid, and 1% bovine serum albumin in phosphate-buffered saline) for 15 min and were then fixed with methanol:acetone (1:1) for 10 min at -20°C. CPD or 6–4PP were then visualized by the immunologic method using TDM-2 or 64M-2 monoclonal antibody as described before (Nakagawa et al., 1998Nakagawa A. Kobayashi N. Muramatsu T. et al.Three-dimensional visualization of ultraviolet-induced DNA damage and its repair in human cell nuclei.J Invest Dermatol. 1998; 110: 143-148https://doi.org/10.1046/j.1523-1747.1998.00100.xCrossref PubMed Scopus (72) Google Scholar). Detergent-insoluble PCNA was visualized with the same method except we used a PCNA monoclonal antibody (Clone 24, Transduction Laboratory, Lexington, KY) at 1:50 dilution and omitted the denaturation of DNA. Nuclear DNA was counterstained with propidium iodide (PI; Sigma). The dishes were washed with phosphate-buffered saline, mounted in drops of ProLong Antifade (Molecular Probes, Eugene, OR), and coverslipped. Fluorescent images were obtained using an InSIGHTplus-IQ laser-scanning confocal microscope (Meridian Instruments, Okemos, MI). For double staining of CPD and PCNA, cells were allowed to repair for 15 min or 24 h after micropore UV irradiation, and were treated with detergent and fixed as described above. Cells were then sequentially labeled with a PCNA rabbit antibody (FL-261, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:25 dilution and Alexa Fluor 488 goat antirabbit IgG conjugate (Molecular Probes) at 1:50 dilution. After denaturing the DNA, cells were sequentially labeled with TDM-2 at 1:1500 dilution and Alexa Fluor 594 goat antimouse IgG conjugate at 1:100 dilution. Fluorescence microscopy was performed on a Leica DMIRB. The fluorescent images of CPD and PCNA were superimposed using Adobe Photoshop software. We developed a method to induce DNA photoproducts within localized areas of the cell nucleus Figure 1a. A polycarbonate isopore membrane filter (pore size, 3 µm) was applied as an irradiation mask to produce a UV microbeam, as the membrane did not transmit 254 nm UV light (data not shown) except through its pores. When microfilter-masked UV is used to irradiate cells on a culture dish, small defined areas of the cells that correspond to the location and the size of filter pores are UV irradiated. The two major types of DNA damage resulting from the micropore UV irradiation were then visualized by the immunologic method utilizing monoclonal antibodies specific for particular photolesions. The nuclear localization of immunofluorescent CPD foci is shown in Figure 1(b). The size and shape of CPD foci roughly resembled those of the filter pores. The focus number per nucleus was not constant, because pores were not uniformly located in the filter. The immunofluorescent focus size could be varied by using filters with different pore sizes Figure 1c. Moreover, the immuno fluorescent intensities of CPD foci increased with increasing UV doses Figure 1d. Thus, we have succeeded for the first time in producing different numbers of UV DNA lesions at defined nuclear sites with certain sizes and subsequently detecting them in situ. Utilizing this microfilter DNA lesion assay, we studied the induction and repair of CPD Figure 2a, b, c, d, e and f and 6–4PP Figure 2g, h, i, j, k and l in normal human fibroblasts. No DNA damage was observed in the unirradiated control Figure 2a, g, but UV irradiation immediately produced several fluorescent (CPD or 6–4PP) foci per nucleus Figure 2b, h. The CPD fluorescence gradually weakened over time after UV exposure, but the focus pattern was maintained even at 24 h after UV irradiation Figure 2c, d, e and f. The 6–4PP fluorescence weakened more quickly and had completely disappeared within 3 h Figure 2i, j, k and l. Similar experiments were performed using NER-defective XP-A fibroblasts Figure 3a, b, c, d, e, f, g, h, i, j, k and l. Again, no DNA damage was seen in unirradiated cells Figure 3a, g, but damage-specific fluorescent foci (CPD or 6–4PP) immediately appeared in UV-irradiated cells Figure 3b, h. As predicted from the defective repair capacity of these cells, the bright fluorescence and the focal pattern of DNA damage persisted over the entire repair period of 24 h Figure 3c, b, c, d, e and f, i, j, k and l, indicating that the XP-A cells cannot repair CPD or 6–4PP produced by the micropore UV irradiation. The sum of these results indicates that the repair kinetics of both types of DNA photoproducts induced by partial-cell UV irradiation is similar to that found after whole-cell irradiation, both in normal and in XP-A cells (Nakagawa et al., 1998Nakagawa A. Kobayashi N. Muramatsu T. et al.Three-dimensional visualization of ultraviolet-induced DNA damage and its repair in human cell nuclei.J Invest Dermatol. 1998; 110: 143-148https://doi.org/10.1046/j.1523-1747.1998.00100.xCrossref PubMed Scopus (72) Google Scholar). Moreover, the results demonstrate that DNA photoproducts remain in relatively fixed nuclear positions during repair and that the NER of DNA photoproducts does not require the dynamic movement of DNA as is observed during DNA replication (Nakamura et al., 1986Nakamura H. Morita T. Sato C. Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus.Exp Cell Res. 1986; 165: 291-297Crossref PubMed Scopus (348) Google Scholar). This in turn suggests that NER proteins must be recruited to the sites of DNA damage for repair. To test this hypothesis, we examined whether a detergent-insoluble form of PCNA is recruited to the sites of DNA damage. PCNA is a processivity factor for DNA polymerase and is essential for both NER and DNA replication (Celis and Madsen, 1986Celis J.E. Madsen P. Increased nuclear cyclin/PCNA antigen staining of non S-phase transformed human amnion cells engaged in nucleotide excision DNA repair.FEBS Lett. 1986; 209: 277-283Abstract Full Text PDF PubMed Scopus (151) Google Scholar). It is accepted that PCNA participates in a repair synthesis step of NER by forming a trimeric ring around duplex DNA near a gap site in UV-irradiated human cells (Aboussekhra and Wood, 1995Aboussekhra A. Wood R.D. Detection of nucleotide excision repair incisions in human fibroblasts by immunostaining for PCNA.Exp Cell Res. 1995; 221: 326-332https://doi.org/10.1006/excr.1995.1382Crossref PubMed Scopus (73) Google Scholar). This form of PCNA is thus resistant to extraction with nonionic detergents, although PCNA is largely in a soluble form in non-S-phase cells in the absence of UV (Toschi and Bravo, 1988Toschi L. Bravo R. Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis.J Cell Biol. 1988; 107: 1623-1628Crossref PubMed Scopus (226) Google Scholar). Normal human fibroblasts were doubly stained for CPD and for detergent-insoluble PCNA 15 min after micropore UV irradiation Figure 2s, t and u. It is clear that both fluorescent signals essentially overlapped with each other, indicating that PCNA is quickly recruited to the sites of DNA damage and then undergoes NER in situ. Based on these results, we were interested to know how levels of detergent-insoluble PCNA vary in damage sites during DNA repair. PCNA was visualized in normal human fibroblasts Figure 2m, n, o, p, q and r under the same conditions as in the DNA damage repair experiments reported above. PCNA fluorescence was not observed without UV exposure Figure 2m or immediately after UV irradiation Figure 2n, when NER had not begun. We found several bright fluorescent foci per nucleus at 0.5 h and 3 h after micropore UV irradiation, however Figure 2o, p. The fluorescence of the PCNA foci gradually weakened at 9 h and became minimal at 24 h after UV exposure Figure 2q, r, indicating that more DNA damage was being repaired at 0.5 h and 3 h than at 9 h and 24 h. These results are consistent with these studies and our previous studies (Nakagawa et al., 1998Nakagawa A. Kobayashi N. Muramatsu T. et al.Three-dimensional visualization of ultraviolet-induced DNA damage and its repair in human cell nuclei.J Invest Dermatol. 1998; 110: 143-148https://doi.org/10.1046/j.1523-1747.1998.00100.xCrossref PubMed Scopus (72) Google Scholar), which showed that 6–4PP were completely repaired within 3 h of UV irradiation and that CPD were repaired more efficiently at early repair times than at 9 and 24 h. Thus, detergent-insoluble PCNA directly relates to active NER and varies in amount at DNA damage sites according to NER activity. To investigate how the lack of one functional NER protein influences the association of other NER proteins with damage sites during DNA repair, detergent-insoluble PCNA was visualized in XP-A fibroblasts as described above Figure 3m, n, o, p, q and r. In contrast to normal cells, no and little PCNA fluorescence was observed at 0.5 h and 3 h, respectively, after micropore UV irradiation Figure 3o, p. This strengthens the concept that PCNA foci observed in normal cells reflect NER of CPD and 6–4PP, and that functional XPA protein is essential for the association of other NER proteins with DNA damage sites. Interestingly, however, we detected several fluorescent PCNA foci per nucleus at 9 and 24 h after UV irradiation Figure 3q, r. We confirmed that PCNA foci began to appear in XP-A cells at 3–4 h after UV exposure (data not shown) and persisted as late as 72 h after UV irradiation, by which time some XP-A cells started to detach from the culture dish Figure 4e, f, g and h. The fluorescent intensities of PCNA foci gradually increased, reaching a maximal level around 9 h after UV irradiation, and PCNA levels did not decrease thereafter. Based on these results, it is clear that the PCNA foci observed in XP-A cells are quite different from those observed in normal cells, and that they are not involved in NER of CPD or 6–4PP (Miura et al., 1992Miura M. Domon M. Sasaki T. Kondo S. Takasaki Y. Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation.Exp Cell Res. 1992; 201: 541-544Crossref PubMed Scopus (33) Google Scholar). The PCNA foci in XP-A cells are definitely related to UV-induced DNA damage, however, as they overlapped with the sites of DNA damage Figure 3s, t and u. Thus, the lack of functional XPA protein results in the delayed and prolonged expression of detergent-insoluble PCNA foci at the sites of DNA damage. Because of the lack of suitable experimental methods, it has been difficult to study how NER of photolesions is processed in human cells in vivo, and particularly to visualize the temporal and spatial interactions between the specific DNA damage and NER proteins. Although a new fluorescence redistribution after photobleaching (FRAP) technology has recently been developed and has contributed to understanding the dynamics of NER proteins (Houtsmuller et al., 1999Houtsmuller A.B. Rademakers S. Nigg A.L. Hoogstraten D. Hoeijmakers J.H.J. Vermeulen W. Action of DNA repair endonuclease ERCC1/XPF in living cells.Science. 1999; 284: 958-961https://doi.org/10.1126/science.284.5416.958Crossref PubMed Scopus (296) Google Scholar), it does not offer any information about DNA damage. Thus, to overcome those difficulties, we developed a novel method that produces UV-induced DNA damage within localized areas of the cell nucleus and allows us to subsequently visualize them in situ. Indeed, both CPD and 6–4PP were produced and visualized as several foci per nucleus. It is worth noting that those fluorescent images are very specific for the particular type of DNA damage because of the absence of any nonspecific fluorescence in unirradiated control samples. Normal human cells repaired 6–4PP more quickly than CPD at the localized sites, whereas XP-A cells were unable to repair either type of photoproduct. These repair patterns are quite similar to those obtained after whole-cell irradiation with a physiologic UV dose (Nakagawa et al., 1998Nakagawa A. Kobayashi N. Muramatsu T. et al.Three-dimensional visualization of ultraviolet-induced DNA damage and its repair in human cell nuclei.J Invest Dermatol. 1998; 110: 143-148https://doi.org/10.1046/j.1523-1747.1998.00100.xCrossref PubMed Scopus (72) Google Scholar). The results are consistent with the evidence that micropore UV irradiation at 100 J per m2 produced a sublethal effect on normal human cells, although the dose completely killed cells after whole-cell irradiation (data not shown). Thus, the method for visualizing repair of photoproducts within localized nuclear areas was successfully established. Two major types of DNA damage were repaired within the focused pattern in the nucleus, indicating that DNA photoproducts remain in relatively fixed nuclear positions during repair, which is what is observed for DNA double strand break repair (Nelms et al., 1998Nelms B.E. Maser R.S. MacKay J.F. Lagally M.G. Petrini J.H.J. In situ visualization of DNA double-strand break repair in human fibroblasts.Science. 1998; 280: 590-592https://doi.org/10.1126/science.280.5363.590Crossref PubMed Scopus (420) Google Scholar). This in turn suggests that NER proteins must be recruited to the sites of DNA damage for repair. The most exciting result is that an NER protein, PCNA, was recruited to the sites of DNA damage within 30 min after UV exposure and levels of PCNA then diminished within 24 h depending on DNA repair activity in normal cells. In contrast, almost no PCNA foci were observed at least until 3–4 h after irradiation in XP-A cells. These results demonstrate that we succeeded in temporally and spatially visualizing the normal NER process in human cells, which consists of interactions between the particular DNA damage and PCNA. Interestingly, however, we detected PCNA foci at the UV damage sites at 9 and 24 h after UV irradiation in XP-A cells. One possible explanation of this finding is that the PCNA foci are due to DNA damage repair that occurs normally in XP-A cells. XP-A cells can repair oxidative DNA damage and single strand breaks, which are minor types of DNA lesions induced by UV (Cadet et al., 1992Cadet J. Anselmino C. Douki T. Voituriez L. Photochemistry of nucleic acids in cells.J Photochem Photobiol B Biol. 1992; 15: 277-298Crossref PubMed Scopus (187) Google Scholar), in a PCNA-dependent fashion (Okano et al., 2000Okano S. Kanno S. Nakajima S. Yasui A. Cellular responses and repair of single-strand breaks introduced by UV damage endonuclease in mammalian cells.J Biol Chem. 2000; 275: 32635-32641Crossref PubMed Scopus (37) Google Scholar). Indeed, nuclear PCNA staining appears immediately after oxidative damage induction (Balajee et al., 1999Balajee A.S. Dianova I. Bohr V.A. Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells.Nucl Acids Res. 1999; 27: 4476-4482Crossref PubMed Scopus (42) Google Scholar) or X-ray irradiation (Miura et al., 1996Miura M. Sasaki T. Takasaki Y. Characterization of X-ray-induced immunostaining of proliferating cell nuclear antigen in human diploid fibroblasts.Radiat Res. 1996; 145: 75-80Crossref PubMed Scopus (20) Google Scholar), and then disappears or diminishes by 12 h. The PCNA foci in XP-A cells appeared after a delay, however, and persisted with bright signals for almost 3 d, indicating that these foci were very unlikely to be involved in such DNA repair. Another possible explanation is that the formation of PCNA foci is attributable to an incomplete or stalled NER of CPD or 6–4PP. Indeed, the bright CPD foci were detected in parallel with the PCNA foci from 9 h to 72 h after UV exposure Figure 4a, b, c and d. Thus, the latter possibility seems more likely. In a repair synthesis step of NER, replication factor C (RFC) binds to the primer-template DNA junction at a gap site and then functions to load PCNA onto the DNA in an adenosine-triphosphate-dependent manner, which creates a sliding clamp for DNA polymerase (Tsurimoto and Stillman, 1991Tsurimoto T. Stillman B. Replication factors required for SV40 DNA replication in vitro.J Biol Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar). Repair-defective XP-A cells are unable to create gaps in UV-damaged DNA because of their inability to make dual incisions, but they may form open structures of DNA around CPD or 6–4PP sites by using normal TFIIH. Such open structures contain duplex-single-stranded junctions that are similar to the primer-template junction at a gap site. Thus, it is conceivable that the sequential binding of RFC, PCNA, and DNA polymerase occurs at the duplex-single-stranded junction of the open DNA structure, but cannot proceed with repair replication because of the absence of a single-stranded gap with a 3′-OH terminus. Therefore, XP-A cells may form and keep PCNA foci at DNA damage sites for a long time after UV irradiation. XP-G cells, which are also unable to perform dual incisions, formed a similar pattern of PCNA foci to that observed in XP-A cells (data not shown), further supporting this hypothesis. Although more experiments are clearly needed to explain the appearance of PCNA foci in XP-A cell nuclei, this may give us a valuable opportunity to examine the interactions between DNA damage and NER protein complex. If the lack of XPA protein interrupts the normal NER process and keeps it stalled for a long time, many NER proteins except XPA might be slowly recruited to the sites of DNA damage and form repairosome-like structures (Svejstrup et al., 1995Svejstrup J.Q. Wang Z. Feaver W.J. et al.Different forms of TFIIH for transcription and DNA repair: holo-THIIH and a nucleotide excision repairosome.Cell. 1995; 80: 21-28Abstract Full Text PDF PubMed Scopus (239) Google Scholar). Thus, it may be possible to visualize such structures in situ. These data prove that development of the UV microfilter DNA lesion assay is an important first step toward understanding the molecular mechanisms of UV-induced DNA damage repair in situ. Indeed, this technology can be applicable for visualizing the interactions between DNA damage and individual NER proteins or the NER protein complex. Moreover, this assay is not limited to visualizing DNA repair. After the induction of UV-induced DNA damage, several cellular response processes (termed the DNA damage response pathway) are initiated, such as activation of DNA repair, accumulation of p53 protein, and overexpression of certain p53-regulated genes that lead either to cell cycle arrest or to apoptosis (Zhou and Elledge, 2000Zhou B.-B.S. Elledge S.J. The DNA damage response: putting checkpoints in perspective.Nature. 2000; 408: 433-439Crossref PubMed Scopus (2637) Google Scholar). The damage response pathway is a signal transduction pathway consisting of sensors, transducers, and effectors. Thus, this assay can also be applied to new approaches aimed at characterizing interactions between DNA damage and damage-response proteins. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.