UV Light-induced DNA Damage and Tolerance for the Survival of Nucleotide Excision Repair-deficient Human Cells

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase ζ activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase ζ is involved in the tolerance of 6-4PPs in human cells. DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase ζ activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase ζ is involved in the tolerance of 6-4PPs in human cells. DNA lesions cause cell killing and mutations if they are not adequately repaired. Exposure of cells to UV light radiation results in formation of the two most common lesions, the cyclobutane pyrimidine dimer (CPD) 1The abbreviations used are: CPD, cyclobutane pyrimidine dimer; 6-4PP, 6-4 pyrimidine-pyrimidone; NER, nucleotide excision repair; pol, polymerase; siRNA, short interfering RNA; TLS, translesion synthesis; XP-A, xeroderma pigmentosum A. and the 6-4 pyrimidine-pyrimidone photoproduct (6-4PP) at adjacent pyrimidines (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995: 24-30Google Scholar). These lesions inhibit DNA replication and transcription and are normally removed from the genome by nucleotide excision repair (NER) in most organisms, including humans (2Hoeijmakers J.H. Nature. 2001; 411: 366-374Crossref PubMed Scopus (3164) Google Scholar). By contrast to a cis-syn CPD, which has only a modest effect on the DNA structure, a 6-4PP induces a large structural distortion and is repaired more rapidly than a CPD by NER (3Mitchell D.L. Photochem. Photobiol. 1988; 48: 51-57Crossref PubMed Scopus (311) Google Scholar). It is becoming widely appreciated that cells have developed a mechanism to tolerate damage in their genome without removing it. NER-deficient cells, such as XP12ROSV belonging to the xeroderma pigmentosum A (XP-A) group, for example, cannot remove UV light-induced DNA damage from their genome at all; but after irradiation with a dose of 0.3 J/m2 UV-C light, which creates >104 DNA lesions with a ratio of three CPDs to one 6-4PP in the genome, 50% of the XP-A cells continue to divide and form colonies (4Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1279) Google Scholar). This tolerance mechanism is known as postreplication repair. In human cells, a major mechanism for carrying out postreplication repair is translesion synthesis (TLS), which is performed by specialized DNA polymerases at the site of DNA damage (5Lehmann A.R. Mutat. Res. 2002; 509: 23-34Crossref PubMed Scopus (103) Google Scholar). The gene defective in XP variants (XP-V) encodes a DNA polymerase η (pol η), which can replicate undamaged and CPD-containing templates with equal efficiency in vitro (6Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Crossref PubMed Scopus (1153) Google Scholar, 7Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Crossref PubMed Scopus (673) Google Scholar). Although several DNA polymerases involved in TLS have been identified in vitro, it is still unclear which polymerase is responsible for the bypass of various types of DNA damage, including 6-4PPs in vivo. Many DNA lesions encounter replication before they are repaired in wild-type cells. Therefore, analysis of NER-deficient cells provides important information about cell death and tolerance after UV light irradiation in human cells. To understand the effects of DNA lesions and damage tolerance on cell killing, we established NER-defective human cell lines expressing photolyase(s). Photolyases recognize either CPDs or 6-4PPs and use visible light energy to repair the lesions. Although many organisms, including aplacental mammals (marsupials), possess photolyases for CPDs and/or 6-4PPs, no photolyase gene has been found in the human genome (8Yasui A. Eker A.P.M. Nickoloff J.A. Hoekstra M.F. DNA Damage and Repair: DNA Repair in Higher Eukaryotes. 2. Humana Press Inc., Totowa, NJ1998: 9-32Google Scholar, 9Wood R.D. Mitchell M. Sgouros J. Lindahl T. Science. 2001; 291: 1284-1289Crossref PubMed Scopus (1112) Google Scholar). To differentiate between the effects of CPDs and 6-4PPs, we introduced into a XP-A cell line, XP12ROSV, a CPD photolyase gene (CPDphr) derived from a marsupial (Potorous tridactylis, rat kangaroo) and/or a 6-4PP photolyase gene (6-4PPphr) derived from a higher plant (Arabidopsis thaliana). We isolated cells showing a stable high expression of either one or both of the photolyases. Using these cell lines, we show the influence of each lesion on cell survival and the role of polymerase ζ (pol ζ) for bypass of 6-4PPs in vivo. Cell Lines and Culture Conditions—XP12ROSV, an SV40-transformed cell line derived from an XP-A patient, is mutated in the XPA gene and possesses no detectable NER activity (10Satokata I. Tanaka K. Miura N. Narita M. Mimaki T. Satoh Y. Kondo S. Okada Y. Mutat. Res. 1992; 273: 193-202Crossref PubMed Scopus (70) Google Scholar). Cells were cultured in Eagle's minimum essential medium containing 10% fetal calf serum. For the expression of photolyase genes in the XP-A cell line, the cDNA of a CPD photolyase gene (CPDphr) derived from the rat kangaroo P. tridactylis (11Yasui A. Eker A.P. Yasuhira S. Yajima H. Kobayashi T. Takao M. Oikawa A. EMBO J. 1994; 13: 6143-6151Crossref PubMed Scopus (178) Google Scholar) and the cDNA of a 6-4PP photolyase gene (6-4PPphr) derived from A. thaliana (12Nakajima S. Sugiyama M. Iwai S. Hitomi K. Otoshi E. Kim S.T. Jiang C.Z. Todo T. Britt A.B. Yamamoto K. Nucleic Acids Res. 1998; 26: 638-644Crossref PubMed Scopus (133) Google Scholar) were used. Because the A. thaliana gene contains putative signal sequences for transport into mitochondria and chloroplasts at the amino-terminal region, 2S. Nakajima, K. Yamamoto, and A. Yasui, unpublished data. 57 nucleotides encoding 19 amino acids from the putative start codon were deleted in the expression construct. cDNA for each gene was introduced into the vector pCY4B, which contains the cytomegalovirus enhancer, the chicken β-actin promoter, and a rabbit poly(A) signal (13Niwa H. Yamamura K. Miyazaki J. Gene. 1991; 108: 193-199Crossref PubMed Scopus (4617) Google Scholar). Two different markers for resistance to either neomycin or blasticidin S-hydrochloride were used in the plasmid constructs. XP12ROSV cells were transfected by a lipid-mediated method using FuGENE 6 (Roche). A cell line expressing both photolyases was established by introducing 6-4PPphr into the cell line expressing CPDphr. Stable transfectants were selected in a medium containing 600 μg/ml geneticin (Sigma) for neomycin or 10 μg/ml blasticidin S-hydrochloride (Funakoshi) and isolated by the cylinder technique. The limiting dilution method was used for secondary cloning. All transfectants were maintained in medium containing 200 μg/ml geneticin and/or 2 μg/ml blasticidin S-hydrochloride. Identification of Photolyases and UV Light-induced CPDs and 6-4PPs—Antibiotic-resistant cells were trypsinized, harvested by centrifugation, and suspended in extraction buffer A (50 mm Tris-HCl, pH 7.5, 0.3 m KCl, 2 mm dithiothreitol, protease inhibitor, and 0.1% Triton X-100). These cell suspensions were sonicated, an equal volume of extraction buffer B (50 mm Tris-HCl, pH 7.5, 2 mm dithiothreitol, protease inhibitors, and 0.1% Triton X-100) was added, and the suspension was then centrifuged. The supernatant was used for Western blotting. Antibodies against CPDs or 6-4PPs photolyases were raised against purified recombinant proteins of each photolyase produced in Escherichia coli. Measurement of CPDs and 6-4PPs—The rate of repair of CPDs and 6-4PPs was measured by an enzyme-linked immunosorbent assay using the monoclonal antibodies TDM-1 (specific for CPDs) and 64M2 (specific for 6-4PPs) (14Mori T. Nakane M. Hattori T. Matsunaga T. Ihara M. Nikaido O. Photochem. Photobiol. 1991; 54: 225-232Crossref PubMed Scopus (391) Google Scholar). Cells were grown until confluent, washed twice with Hanks' buffer, and irradiated with 20 J/m2 UV light (0.87 J/m2/s at 254 nm). Irradiation was followed immediately by illumination with white fluorescent lamps at room temperature for the time indicated. The light used for illumination was a Toshiba FL20SS fluorescent light, and cells were exposed from the bottom of the dishes through a box made of transparent poly(methyl methacrylate) plate and filled with water to absorb the heat generated by the light. Other illumination conditions have been described previously (11Yasui A. Eker A.P. Yasuhira S. Yajima H. Kobayashi T. Takao M. Oikawa A. EMBO J. 1994; 13: 6143-6151Crossref PubMed Scopus (178) Google Scholar). After illumination, cells were harvested and genomic DNA was extracted using an Easy-DNA kit (Invitrogen). Genomic DNA was then treated with RNase A, extracted with phenol and phenol-chloroform, precipitated and washed with ethanol, and resuspended in TE (10 mm Tris-HCl and 1 mm EDTA, pH 7.5) buffer. Genomic DNA at 30 or 300 ng/ml was used to detect CPDs and 6-4PPs, respectively. UV Light Survival—Exponentially growing fibroblasts were plated at 1.5–3 × 103 cells per 100-mm Petri dish and cultured for 8–12 h. Cells were then washed twice with Hanks' buffer and irradiated with UV-C light (254 nm) at either 0.05 or 0.25 J/m2/s. Immediately after irradiation, plates were filled with Hanks' buffer and illuminated with visible light for 90 min or kept in the dark at room temperature. After illumination, the Hanks' buffer was removed and fresh medium was added. Cells were cultured for 12–14 days. Colonies were fixed and stained with 0.3% crystal violet in methanol, and the number of colonies was counted. Preparation of Short Interference RNA (siRNA) for pol ζ and Transfection—Double-stranded siRNA of 19-mer with a protruding 3′-TT sequence (total 21-mer) was synthesized and purified by a Silencer siRNA construction kit (Ambion). 5′-AAATGTCGGAGCCAACCTCAGCCTGTCTC-3′ and 5′-AACTGAGGTTGGCTCCGACATCCTGTCTC-3′ oligonucleotides were synthesized (Japan BioService) and then used for pol ζ siRNA synthesis. The position of siRNA corresponded to the coding region (nucleotides 9167–9185) of the pol ζ catalytic subunit REV3 gene (GenBank™ accession number AF071798) (15Lin W. Wu X. Wang Z. Mutat. Res. 1999; 433: 89-98Crossref PubMed Scopus (94) Google Scholar). Cells were plated at 2 × 105 cells per 60-mm Petri dish and cultured overnight. Cells were transfected with 2 nm siRNA for pol ζ by using OligofectAMINE (Invitrogen). After 40 h these cells were subjected to the UV light survival assay. Reverse Transcription PCR—Total RNA was extracted from cells by the High Pure RNA isolation kit (Roche Applied Science). cDNAs were synthesized using the First Strand cDNA synthesis kit (Roche Applied Science). The reverse transcription PCR primers were designed as 5′-CTTTCCTTACCTCTATGTGCC-3′ and 5′-TGTAGGAGGTAGGGAATATGC-3′, which amplify the coding region of polymerase ζ (nucleotides 71 to 485). Quantitative reverse transcription PCR was performed by using Light Cycler (Roche). Light-dependent Repair of UV Light-induced Lesions in Human Cells—We established human XP-A cell lines expressing either a marsupial CPD photolyase or a plant 6-4PP photolyase. The cell line expressing CPD photolyase was further transfected to produce a cell line expressing both CPD and 6-4PP photolyases. By immunofluorescence microscopy we found that >95% of the cells express either or both of the photolyases (not shown). Fig. 1 presents a Western blotting analysis showing the expression of each photolyase in the established cell lines. Using these cell lines, we analyzed the light-dependent decrease of CPDs and 6-4PPs by antibodies against each lesion (Fig. 2). Cells were irradiated with 20 J/m2 UV-C light and exposed to visible light. In cells expressing the CPD photolyase alone, the amount of CPDs decreased to ∼10% after 2 h of illumination. In contrast to CPDs, in cells expressing 6-4PP photolyase almost all 6-4PPs disappeared after 2 h of illumination. In cells expressing both photolyases, both CPDs and 6-4PPs disappeared more rapidly than in cells expressing either photolyase. 6-4PPs disappeared almost completely after 60 min, and CPDs disappeared after 2 h of light illumination as well. These data indicate that almost all CPDs and 6-4PPs are accessible to the foreign photolyases. The presence of either lesion may influence the repair of the other lesion.Fig. 2Removal of UV light-induced lesions from genomic DNA of the cells by illumination with visible light. A, XP-A/vector cells. B, XP-A/CPDphr cells. C, XP-A/6-4PPphr cells. D, XP-A/CPD-phr+6-4PPphr cells. The amounts of CPD (squares) or 6-4PP (circles) lesions relative to those of non-illuminated cells are shown. The UV light dose was 20 J/m2.View Large Image Figure ViewerDownload (PPT) Fig. 3A shows survival of UV light-irradiated cells with and without visible light illumination. The repair of each UV lesion by either photolyase contributes to survival increase. After UV light irradiation and light illumination, cells expressing both photolyases have a survival level slightly higher than that of XP-A cells expressing wild-type XPA cDNA (Fig. 3B). To compare the effects of photorepair between CPD and 6-4PP photolyases, the fluence decrement was used, which is defined as the UV light dose whose effect is annulled by light exposure and corresponds to the effect of repair on survival increase (16Harm H. Biological Effects of Ultraviolet Radiation. Cambridge University Press, Scholar). The fluence of the cells irradiated with and J/m2 are similar after the repair of CPDs and 6-4PPs (Fig. immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as that of CPDs, indicating that a single 6-4PP is severalfold more toxic than a CPD lesion in NER-deficient human cells. of ζ expressing each photolyase repair one of damage by the other photoproduct must be for of the most for damage tolerance is for CPD, pol η can bypass CPDs with high efficiency and In the of 6-4PP, a in vitro of translesion synthesis that pol η or pol the of 6-4PP and that pol ζ as an at S. Prakash L. 2002; PubMed Scopus Google Scholar). Because CPDs and 6-4PPs are by UV-C light and repaired by NER in wild-type cells, it is not to which polymerase is responsible for the bypass of each lesion in vivo. To the of pol ζ in we which lesion of photorepair the effect of pol ζ by RNA Therefore, siRNA designed for the of pol was introduced into the cell lines. siRNA for pol ζ decreased the level of pol ζ to of the (Fig. and decrease the XP-A cells with vector to UV-C light, indicating that pol ζ contributes to the TLS of UV light damage (Fig. Although a similar level of was for pol it not influence the UV light resistance of the cells (not that the amount of pol ζ may be important and that it the UV light In cells expressing CPD photolyase (Fig. pol ζ the effect of CPD that the pol ζ activity is not for the bypass of was no in the repair rate of CPD by photolyase between and cells (not shown). In contrast to CPD the repair of 6-4PPs the survival of the cells with pol ζ (Fig. the effect of pol ζ can be by 6-4PPs but not by CPD These data show for the time that pol ζ contributes to bypass 6-4PPs in cells. We have established human cell lines in which either UV light-induced CPD 6-4PP can be repaired by visible light illumination after UV light Because cell lines express high of either or both photolyases, we were to the effects of almost repair of each or both lesions at time after UV light has not been in H. Z. M. T. H. Todo T. T. H. M. Mutat. Res. 1999; PubMed Scopus Google Scholar, A.R. J. A. Res. Google Scholar). First of we found that most CPDs and 6-4PPs in human cells are accessible and are repaired by the foreign photolyases illumination with visible light. Because 6-4PPs are produced in to the higher of and CPDs are produced in DNA as Res. PubMed Scopus Google Scholar, D.L. J. PubMed Google the of repair to CPDs in the may be by the presence of proteins D.L. Nucleic Acids Res. PubMed Scopus Google Scholar). we found that of CPDs are repaired by CPD photolyase only h after UV light irradiation of 20 J/m2 (Fig. 2). results have been in of and are by the of M. F. EMBO J. PubMed Scopus Google Scholar). A of DNA proteins may DNA lesions and accessible to a repair F. EMBO J. 1999; PubMed Google Scholar, M. 1999; PubMed Scopus Google Scholar). The repair of CPDs with that of 6-4PPs in Fig. may be by the severalfold more CPDs produced by UV light. Although the of the NER to CPDs may be less than that of we that a major for the repair of CPDs by NER is a number of NER for CPD repair in cells S. A. 1999; PubMed Scopus Google Scholar). show that an almost repair of either CPDs or 6-4PPs almost to the increase in cell survival (Fig. A and that the number of 6-4PPs produced by UV-C light is only about of the number of CPDs A. J. R. EMBO J. PubMed Scopus Google then 6-4PPs may be about three more toxic than CPDs in human cells without This with NER-defective E. in which repair by CPD photolyase survival by more than that by 6-4PP photolyase (12Nakajima S. Sugiyama M. Iwai S. Hitomi K. Otoshi E. Kim S.T. Jiang C.Z. Todo T. Britt A.B. Yamamoto K. Nucleic Acids Res. 1998; 26: 638-644Crossref PubMed Scopus (133) Google Scholar). This between human and E. cells that the UV light resistance of E. cells more on the repair of CPDs than is the for human cells. The photorepair of CPD the cell survival of NER-deficient cells as A. W. J. PubMed Scopus Google that the tolerance for UV light-induced damage in the S. is similar to that in E. coli. CPD photolyases in E. and an important role for UV light It be to the repair of CPDs or 6-4PPs by photolyase cell survival in NER-deficient of other and to the of TLS for UV light-induced damage of the This may the of photolyase genes in various In vitro data have that several DNA polymerases to bypass CPD in human cell (5Lehmann A.R. Mutat. Res. 2002; 509: 23-34Crossref PubMed Scopus (103) Google Scholar). pol η can bypass CPD with the efficiency and as it but pol η cannot bypass 6-4PP and only one the of 6-4PP R.E. L. Prakash S. Prakash L. 2001; PubMed Scopus Google Scholar). In contrast to pol pol ζ can bypass CPDs with efficiency it cannot the bypass of 6-4PP, it can from the the of 6-4PP R.E. L. Prakash S. Prakash L. Nature. PubMed Scopus Google Scholar). pol cannot the nucleotides CPD but can lesion bypass by from the nucleotides by other polymerases pol pol cannot from the nucleotides 6-4PP R.E. Prakash L. Prakash S. S. A. 2002; PubMed Scopus Google Scholar). Although pol can bypass CPD with a efficiency with pol η and can bypass 6-4PP as conditions A. Iwai S. Hanaoka F. R. EMBO J. PubMed Scopus Google is no that pol cells UV light in the of NER the killing effect of 6-4PPs is similar to that of CPDs, that a 6-4PP is more to than a Therefore, it is important to tolerance to 6-4PPs is in human cells. data that the of pol by siRNA the UV light sensitivity of NER-deficient cells. This that in NER-defective cells many of the UV light lesions are by pol The repair by 6-4PP but not by CPD the of pol indicating that pol ζ is involved in the tolerance of This is the showing that 6-4PP is by the pol translesion synthesis in cells, including and human cells. analysis using RNA interference to various genes involved in TLS their in vivo. In we established and analyzed NER-deficient human cells expressing photolyase for either CPDs 6-4PPs, and the major are as A 6-4PP is severalfold more toxic than a CPD unless it is repaired by The killing effect by the of pol ζ after UV light irradiation is by repair with 6-4PP photolyase but not with a CPD photolyase. These data the of NER as as TLS for the survival of UV light-irradiated human cells. to in vitro data to the analysis of cells. We Miyazaki and Tanaka for with the expression vector and the XP-A cell line expressing wild-type XPA respectively. We for of the