Materials and methods

Cells and culture conditions

The human primary cell strains Mori, Turu, and Sono were established as normal in this laboratory (^{Itoh et al. 1994,1995,1996a}). The human primary cell strain Ops1 was designated as a defect of DDB protein in this study. The human primary cell strain XP82TO (XP-E) was a gift from Dr. Seiji Kondo at Tokyo Medical and Dental University (^{Kondo et al. 1988}). The human primary cell strain, XP2SA (XP-V) was purchased from the Human Science Research Resources Bank (Osaka, Japan). All cells were cultured in Dulbecco's modified Eagle's minimum essential medium (ICN, Irvine, CA) supplemented with 10% (vol/vol) fetal bovine serum (ICN), penicillin G (100 units per ml), and streptomycin (100;g per ml) in a humidified 5% CO₂ incubator.

UV survival assay

Colony forming ability was performed as described elsewhere (^{Itoh et al. 1994,1995}). Cells were trypsinized and appropriate numbers of cells were plated on to 60;mm plates. After incubation for 10;h, they were washed with phosphate-buffered saline, irradiated with various doses of UVC (254;nm) at a fluence rate of 0.7;J per m² per s, and then fresh medium was applied. After 1–2;wk, the plates were washed with phosphate-buffered saline, fixed with 80% (vol/vol) methanol, stained with Giemsa, and colonies were counted. Survival curves were plotted on semilogarithmic paper. To determine the effect of caffeine on UV survival, cells were plated, washed, and irradiated as described above. Caffeine was added at a final concentration of 1;mM just after UV irradiation and the plates were incubated for 2;wk.

Measurement of UDS, recovery of RNA synthesis (RRS) and recovery of replicative DNA synthesis (RDS) after UV irradiation

UDS, RRS, and RDS were measured by a method described elsewhere (^{Itoh et al. 1994,1995,1996a}). To analyze effectively the results, 20;l aliquots of cell suspension were plated with a micropipette in two rows on a coverslip (18;mm;;18;mm) in all experiments. Briefly, UDS was measured as follows. Cells were irradiated with UVC (254;nm) at a dose of 30;J per m², immediately labeled with [³H]thymidine (40;Ci per ml) for 2.5;h, and fixed. Coverslips were mounted on glass slides, dipped in Kodak NTB-3 emulsion, and exposed for 24;h or 48;h at 4°C. Grains above nuclei were counted under a microscope. RRS was measured as follows. Briefly, cells were irradiated with UVC (254;nm) at a dose of 15;J per m². After UV irradiation, they were incubated for 23;h, and labeled with [³H]uridine (40;Ci per ml) for 1;h. Autoradiography was performed by the method described above. RDS was measured as follows. Briefly, cells were irradiated with UVC (254;nm) at a dose of 15;J per m². After UV irradiation, they were incubated for 6;h, and labeled with [³H]thymidine (15;Ci per ml) for 1;h. Autoradiography was performed as described above.

Measurement of DNA synthesis after UV irradiation

The rate of DNA synthesis after UV irradiation was measured as described (^{Itoh et al. 1996a}). Briefly, cells were irradiated or unirradiated with UVC (254;nm) at a dose of 5;J per m². After incubation for an appropriate time, they were pulse-labeled with [³H]thymidine (10;Ci per ml) for 30;min, and then harvested by adding 0.2;M NaOH. Each fraction was collected on Whatman Grade 17 paper strips, and the acid-insoluble radioactivities were counted in a liquid scintillation counter. Each point represents an average of four experiments.

Post-replication repair assay

Semiconservative DNA replication after UV irradiation was measured by a method described elsewhere (^{Itoh et al. 1995,1996a}). Cells were irradiated with UV light (254;nm) at a dose of 5;J per m², returned to fresh medium, and incubated for 60;min before being labeled for 30;min with 0.37;MBq per ml (10;Ci per ml) of [³H]thymidine. Cells were harvested, then irradiated with 20;Gy of X-rays on ice. A suspension of cells was layered on the top of 5;ml of 5%–20% (wt/vol) alkaline sucrose gradients with 0.1;M NaOH, and centrifuged at 237,600;;g for 90;min at 4°C with a RPS55T rotor (Hitachi, Tokyo, Japan). After centrifugation, drop fractions were collected on to Whatman Grade 17 paper strips, and the acid-insoluble radioactivities were counted in a liquid scintillation counter.

Detection of CPD and (6-4) PP by enzyme-linked immunosorbent assay

Direct binding of monoclonal antibodies to CPD or (6-4) PP was measured by enzyme-linked immunosorbent assay as described (^{Mori et al. 1991}). Cells were prelabeled with 1.85;kBq per ml of [2-¹⁴C]thymidine for 3;d. Cells were then cultured for 2;d in a radioisotope-free media. After washing with phosphate-buffered saline, cells were UV irradiated and incubated for various times for repair. Immediately after irradiation or after post-UV incubation, cells were harvested and genomic DNA was purified using the phenol/isoamyl alcohol/chloroform procedure or QIAamp Bood Kit (Qiagen, Hilden, Germany). DNA concentration was calculated from the absorbance at 260;nm, and ¹⁴C-radioactivity of DNA was measured by liquid scintillation counter (LSC-5100, Aloka, Tokyo, Japan). The DNA was extracted from UV-irradiated (10;J per m²) or unirradiated cells at various time intervals after irradiation. The extracted DNA was sonicated, denatured, and applied to 1% protamine sulfate-pretreated polyvinyl chloride microtiter plates. TDM2 for CPD or 64M2 for (6-4) PP was used as the first antibody and biotinylated F(ab)₂ fragment of goat anti-mouse IgG (H;+;L) (Zymed Laboratories, San Francisco, CA) was used as the second antibody. After incubation with streptavidin-peroxidase conjugate (Zymed Laboratories), samples were incubated in a substrate solution containing 0.04% o-phenylene diamine and 0.007% H₂O₂. After a 30;min incubation at 37°C, 2;M H₂SO₄ was added to stop the reaction, and absorbance at 492;nm was measured with Titertek Multiskan Plus MKII (Labsystems, Helsinki, Finland). The mean values of three wells were calculated, and values obtained from unirradiated samples were subtracted as background.

Gel mobility shift assay

A gel shift assay was performed as previously described (^{Chu & Chang 1988;Keeney et al. 1992}) with some modifications. Briefly, the 40;bp oligonucleotide described above was labeled with [³²P]dCTP and irradiated with UVC (254;nm) at a dose of 9000;J per m². Binding reactions (10;l) contained approximately 0.2;ng [³²P]DNA with 10% (vol/vol) glycerol, 0.35;mM poly(dI–dC), 0.1;mg per ml bovine serum albumin, 5;mM MgCl₂, 60;mM KCl, and 50;mM Tris–HCl, pH;8.0. The reactions were incubated for 15;min at 30°C, and electrophoresed on 5% polyacrylamide gels in 1;;Tris–borate/EDTA electrophoresis (TBE) buffer. Then the gels were dried, and analyzed using a Fujix Bio-Analyzer BAS-2000 (Fuji Photo Film) for an appropriate time.

Identification of genomic DDB2 clones

A human phage library (YG-LI020) was purchased from the Human Science Research Resources Bank (Osaka, Japan) and screened with three DDB2 cDNA probes. The cDNA probes were made by polymerase chain reaction (PCR) with three sets of primers (–30: 5'-AGCACAGTACCCCTTCACAC and 425: 5'-GGTGGGTTTGTCCTTGATGC; 265: 5'-GGGCTCCAGCAGTCCTTTT and 634: 5'-GTGACCACCATTCGGCTACT; 632: 5'-CTAGTAGCCGAATGGTGGTC and 1347: 5'-AAATCACCACCTCTGCTTGC). Positive clones were plaque-purified and identified by restriction digestion (EcoRI, BglII, HindIII, and PstI; Boehringer, Mannheim, Germany). Sequencing analysis was performed using a Dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA) with a Model 373S DNA sequencer (PE Applied Biosystems).

Direct sequencing of DNA and restriction analysis

Genomic DNA was extracted from fibroblasts using Easy DNA kit (Invitrogen, Carlsbad, CA). The amplification of genomic DNA for the analysis of the change at nucleotide 937 (Ops1) was performed by PCR using the DDB2 gene-specific oligonucleotides followed by their position in relation to the ATG translational start codon: 833: 5'-AAGCCAGCTTCCTCTACTCG and 1000: 5'-ATGGGTGTGAGGTGCTGGA. PCR reaction was performed using KOD' polymerase (Toyobo, Osaka, Japan) in 30 cycles at 94°C, 30;s; 60°C, 2;s; 74°C, 30;s. The PCR fragments were purified (QIAquick gel extraction kit, Qiagen), and were directly sequenced with primers 833, 1000, I-239; 5'-TCTTCCTTTCCGCTCCTGTC as described above. A genomic DNA fragment including a non-sense mutation (nucleotide 937) was amplified by PCR using primers 833 and I-177: 5'-GGCAGAAGTGGGATAAAAGG. PCR reaction and purification of PCR fragments were performed as described above. The amplified fragments were digested with BglII (Boehringer Mannheim) at 37°C for 6;h. The digested DNA was separated by electrophoresis in 1.5% 1;;TBE agarose gel and was stained with ethidium bromide.

Results

Case report

Ops1, a 62;y old Japanese woman, was first recognized to be photosensitive at the age of about 20. There were no complications during pregnancy, labor, or delivery. The parents;were consanguineous. She showed average development. At present, she has clinical sensitivity to UV light including pigmented or depigmented macules and patches on her face, neck, chest, and extremities, especially the dorsa of the hands. The sun-exposed skin showed slight dryness and xerosis. Furthermore, she had multiple skin neoplasias (five malignant melanoma and 14 basal cell carcinoma on her face, and two of squamous cell carcinoma in situ on her forearm and leg). Therefore, after examination at several hospitals, Ops1 was diagnosed as having XP.

Normal post-UV survival with/without caffeine

Cellular hypersensitivity to UV light was not shown by colony forming assay with or without caffeine in Ops1 cells (Figure 1).

Figure 1.

UV survival of Ops1 cells. Appropriate numbers of cells were inoculated on to 60;mm dishes. After UV irradiation at a fluence rate of 0.7;J per m² per s, cells were incubated for 14;d, fixed with 80% methanol, and stained with Giemsa. Caffeine was added at a final concentration of 1;mM just after UV irradiation. Each point represents an average of three or four experiments; error bar:;SEM. Mori cells () were used as a normal control. Ops1 cells (); Ops1 cells with 1;mM caffeine (); XP2SA (XP-V) cells (); XP2SA cells with 1;mM caffeine ().

Full figure and legend (11K)

Normal UDS level in;vivo

The level of UDS in Ops1 cells was compared with that of cells from three normal subjects (Mori, Turu, and Sono). The level of UDS in Ops1 cells was normal (Table 1) and post-UV survival in Ops1 cells was also normal (Figure 1), suggesting that Ops1 cells belonged to PRR defective XP, that is, XP variant (XP-V), not to NER defective XP.

Table 1 - DNA repair levels in vivo in Ops1 cells.

Full table

Normal RRS and RDS levels in;vivo

XP-V cells specifically showed a reduced RDS level and this reduction was enhanced in the presence of caffeine in spite of having normal levels in UDS and RRS (^{Itoh et al. 1996a}). The levels of RRS and RDS with or without caffeine were within normal ranges (Table 1). These results indicated that Ops1 did not belong to XP-V.

Normal levels in the rate of post-UV DNA synthesis and PRR

To confirm these results, we took the following two approaches. One approach is the rate of post-UV DNA synthesis. XP-V cells showed a defect in the rate of post-UV DNA synthesis (Figure 2) (^{Lehmann et al. 1975;Rude & Friedberg 1977;Cleaver & Kraemer 1995;Itoh et al. 1996a}). The rate of post-UV DNA synthesis showed a normal pattern (Figure 2).

Figure 2.

Rate of DNA synthesis after UV irradiation in Ops1 cells. Cells were irradiated with UV light at a dose of 5;J per m². After 0, 2, 4, 6, or 8;h incubation, cells were pulse-labeled with [³H]thymidine (10;Ci per ml) for 30;min. Mori cells () were used as a normal control. Each value is the mean for three or four experiments; error bar:;SEM. Ops1 cells (); XP2SA cells (XP-V) ().

Full figure and legend (13K)

Another approach is a PRR assay. By PRR assay, DNA synthesized after UV irradiation was of the same size as that of unirradiated Ops1 cells (Figure 3a). Under this condition, the DNA synthesized after UV irradiation in XP-V cells was found to be smaller than that in unirradiated cells (Figure 3b) (^{Itoh et al. 1995,1996a}). Furthermore, the effect of caffeine on UV survival was not found as shown in Figure 1. Therefore, it was confirmed that Ops1 cells did not belong to XP-V.

Figure 3.

Comparison of the size of DNA synthesized after UV irradiation in Ops1, and XP2SA cells with that in unirradiated cells. Cells were irradiated with UV light at a dose of 5;J per m², grown for 1;h, and incubated for 30;min with [³H]thymidine (10;Ci per ml). Centrifugation was performed for 1.5;h at 237,600 g. The top of the gradient is to the right. , unirradiated cells (control); , UV irradiated cells. (a) Ops1; (b) XP2SA (XP-V).

Full figure and legend (18K)

Defective repair kinetics of (6-4) PP in contrast to normal repair kinetics of CPD in Ops1 cells

Although we could not detect the abnormality in DNA repair markers, to investigate the repair phenotype of UV-induced DNA lesions in Ops1 cells, we measured the repair kinetics of CPD and (6-4) PP using the enzyme-linked immunosorbent assay. Ops1 cells surprisingly showed an incomplete pattern repair in (6-4) PP despite showing a normal pattern repair in cis-syn CPD (Figure 4). NER defective XP showed defective repair kinetics in both CPD and (6-4) PP (^{Friedberg et al. 1995;Itoh et al. 1996b}). Furthermore, (6-4) PP were rapidly removed within 1;h after UV irradiation, similar to what occurred in normal cells, but were more slowly removed from that in Ops1 cells (Figure 4). This removal pattern of (6-4) PP was similar to that in normal cells at a dose of more than 30;J per m² (^{Nakagawa et al. 1998}).

Figure 4.

The repair kinetics of CPD and (6-4) PP from genomic DNA in Ops1 and normal (Mori) cells. Mori ( three experiments) and Ops1 ( two experiments) cells were UV irradiated (10;J per m²) and incubated for repair. The percentage of the initial number of photolesions was determined at various times after UV irradiation using the standard enzyme-linked immunosorbent assay technique with monoclonal antibodies specific for each type of lesion. Each point shows the mean;;SEM.

Full figure and legend (15K)

Defect of DDB protein in Ops1 cells

A DDB protein bound with a high affinity to (6-4) PP, but with only a marginal affinity to cis-syn CPD (^{Reardon et al. 1993}). As Ops1 cells did not show defective repair in cis-syn CPD, but was apparent in (6-4) PP (Figure 4), we performed a gel mobility shift assay to detect for DDB protein in relation with binding to (6-4) PP in Ops1 cells. Ops1 cells showed a defect of DDB activity as shown in Figure 5.

Figure 5.

Damaged DNA binding activity in Ops1 cells. Gel mobility shift assay was performed as described in Materials and Methods. Binding assay samples including 0.2;ng of labeled probes were incubated with 3–9;g of whole cell extracts (WCE) from the Mori (normal), XP82TO (XP-E), and Ops1 cells. The damaged DNA binding protein is indicated by an arrow. "UV damaged" or "undamaged" indicate the addition of the unlabeled, UV irradiated competitor DNA (20;ng per assay) or the unlabeled, undamaged competitor DNA (20;ng per assay) in the binding assay sample, respectively. F: unbound free DNA probes.

Full figure and legend (172K)

Non-sense mutation in DDB2 gene in Ops1 cells

Recent studies showed the DDB protein to be a heterodimer of 127;kDa (p127; DDB1) and 48;kDa (p48; DDB2) subunits (^{Keeney et al. 1993;Dualan et al. 1995}). Although we could not detect mutations in DDB1 cDNA of Ops1 cells (data not shown), we found a non-sense mutation in DDB2 cDNA of Ops1 cells (data not shown). Furthermore, to evaluate the functional integrity of alleles in Ops1 cells, we characterized the genomic organization of the DDB2 gene and found it to be comprised of 10 exons (Figure 6a). To confirm that Ops1 is homozygous for the non-sense mutation, we performed direct sequencing using PCR products of genomic DNA. Ops1 carries a homozygous CT transition at nucleotide position 937 in exon 7 in genomic DNA, generating a non-sense mutation from CGA (Arg) to TGA (Stop) at codon 313 (Figure 6b). This would be expected to produce a protein truncated by 115 amino acids. Moreover, as this mutation carried BglII site (AGATCCAGATCT) (Figure 6b), we performed restriction analysis of the genomic DNA fragment of DDB2 gene. The DNA fragment of Ops1 was completely digested with BglII whereas that of normal cells (Mori) was not digested (Figure 6c). These results demonstrated that Ops1 is homozygous for the non-sense mutation of DDB2 gene.

Figure 6.

Mutation analysis of DDB2 gene. The procedure for mutation analysis is described in Materials and Methods. (a) Map of the human DDB2 genomic structure. Exon lengths deduced from direct comparison of the sequence of the DDB2 cDNA with the genomic copy are indicated as boxes. The numbers within the boxes are the exon number and size, respectively. Arrowheads indicate the positions of the mutations of Ops1 cells. (b) Direct sequencing around the termination mutation at nucleotide 937 (Exon 7) in Ops1. The positions of the mutations are indicated by arrows. (c) BglII digestion of the amplified DNA of the DDB2 gene. PCR products were digested with BglII at 37°C for 6;h and were analyzed by 1.5% 1; TBE agarose gel. Lane M shows size markers (100;bp DNA ladder). Mori was used as a normal control.

Full figure and legend (26K)

Discussion

In this study, we reported a photosensitive patient (Ops1) with multiple skin neoplasias and a new type of the non-sense mutation in DDB2 gene. Interestingly, Ops1 cells showed an incomplete repair of pattern in (6-4) PP in spite of having a normal repair of pattern in cis-syn CPD (Figure 4). In contrast, Ops1 cells had a normal DNA repair capacity in;vivo (Table 1, Figure 1, Figure 2 and Figure 3). We addressed this discrepancy as follows. Using radioimmunoassays to;detect (6-4) PP, it was found that (6-4) PP were formed with a 6-fold greater frequency in linker DNA than in nucleosomal DNA (^{Mitchell et al. 1990}) indicating that the formation of (6-4) PP is generally suppressed in nucleosome cores. Recent studies showed that the p48 subunit (DDB2 gene) activated the p127 subunit of the DDB protein complex (^{Hwang et al. 1998}), and the p127 subunit was tightly associated with chromatin after UV irradiation (^{Otrin et al. 1997}). Furthermore, although approximately 60% of (6-4) PP was rapidly removed within 1;h after UV irradiation, 40% remained in Ops1 cells in contrast to 20% in normal cells (Figure 4). Therefore, it is possible that a major part of (6-4) PP in linker DNA was rapidly removed using normal NER, but the unsuppressed formation of (6-4) PP in nucleosomal DNA remained in Ops1 cells. The DDB protein in Ops1 cells did not bind to nucleosomal DNA owing to the non-sense mutation in the DDB2 gene (Figure 5 and Figure 6b). This evidence suggested that the DDB2 gene acted on the suppression of DNA lesions against various DNA damage, including (6-4) PP, except for CPD in nucleosome core DNA.

XP-E was designated a fifth complementation group based on findings using a cell fusion technique, which showed the highest UDS level to be in NER defective XP groups (^{Kraemer et al. 1975}). It was reported that XP-E had biochemical heterogeneity regarding DDB protein (^{Kataoka & Fujiwara 1991;Keeney et al. 1992;Friedberg et al. 1995}). As the literature on XP-E is very complex and confusing, and XP-E gene has not been cloned, XP-E is still inconclusive. Two of 13 of XP-E patients showed a defect in the DDB protein (^{Keeney et al. 1992}). Recently, three XP-E patients with a defect in the DDB protein were reported (^{Otrin et al. 1998}). All of these patients showed the mildest form of XP, and the cells showed 40%–50% of normal UDS levels (^{Cleaver & Kraemer 1995;Otrin et al. 1998}), that is, the defect of NER. The level of UDS in Ops1 cells, however, was carefully determined by counting 300 nuclei per cell strain in each coverslip and the results were confirmed under different coverslips (Table 1). Each pair of Ops1 and reference cells (Mori, Turu, and Sono) were compared by Student's t;test. We could not detect the differences between Ops1 and normal (Mori and Sono) cells statistically (p >;0.10). Grains of Ops1 cells were significantly higher than those of Turu cells (p <;0.001). We determined that Ops1 cells showed normal UDS levels from these results. To diagnose whether a patient is XP, it is first necessary to measure UDS levels (^{Cleaver & Kraemer 1995;Itoh et al. 1996a}). In the case of reduced UDS levels, as the patient had the possibility to belong to one of the NER defective XP groups (XP-A through XP-G), the patient was diagnosed using the complementation assay (^{Itoh et al. 1994,1996a;Cleaver & Kraemer 1995}). On the other hand, in the case of normal UDS levels, as the patient had the possibility to belong to PRR defective XP (XP-V), the patient was diagnosed using PRR assay (alkaline density sucrose gradient) or RDS levels with/without caffeine (^{Cleaver & Kraemer 1995;Itoh et al. 1995,1996a}). On the basis of the evidence, we performed the diagnostic procedures and determined that Ops1 is distinct from the already known XP (NER defective XP or PRR defective XP). As XP-E is still inconclusive as described above, however, we cannot completely exclude the possibility that Ops1 is a disease related to XP-E at present.

Finally, both cis-syn CPD and (6-4) PP are mutagenic, but it is believed that cis-syn CPD is the principal mutagenic lesion in mammalian cells (^{Brash 1988;Tornaletti & Pfeifer 1996}). (6-4) PP, although clearly mutagenic in Escherichia coli (^{Horsfall & Lawrence 1994;Tornaletti & Pfeifer 1996}), are repaired much faster than cis-syn CPD in mammalian cells (^{Mitchell & Nairn 1989;Tornaletti & Pfeifer 1996}) and are thus somewhat less likely to contribute to UV-induced mutations in mammals. In this study, however, we have indicated the possibility that Ops1 presents itself as one of the mutagenic models induced by (6-4) PP in mammals.

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Acknowledgments

We are grateful to Dr. Seiji Kondo, Dr. Mariko Iki and Dr. Hidenari Kusakabe for XP82TO and Ops1 cells. We also thank Dr. Hironori Niki and Mrs. Yuka Itoh for preparation of the manuscript. This work was supported partly by grants from the Ministry of Education, Science, Sports and Culture of Japan (to T.I. and M.Y.), by the Naito Foundation (to T.I.), and by the Kao Foundation for Arts and Sciences (to T.I.).