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A Newly Identified Patient with Clinical Xeroderma Pigmentosum Phenotype has a Non-Sense in the DDB2 Gene and Incomplete Repair in (6-4) Photoproducts

Toshiki Itoh, Toshio Mori,† Hiroaki Ohkubo,* and Masaru Yamaizumi Department of Cell Genetics, *Gene Regulation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto, Japan; and †Radioisotope Center, Nara Medical University, Nara, Japan

We report here a patient (Ops1) with clinical photosen- pattern repair in cis-syn cyclobutane sitivity, including pigmented or depigmented macules pyrimidine dimers by repair kinetics using the enzyme- and patches, and multiple skin neoplasias (malignant linked immunosorbent assay. Moreover, Ops1 cells , basal cell carcinomas, and squamous cell were defective in a damage-specific DNA binding carcinomas in situ) in sun-exposed areas. These clinical protein and carried a non-sense mutation in the DDB2 features are reminiscent of xeroderma pigmentosum. gene. These results suggest that (i) the DDB2 gene is As cells from Ops1 showed normal levels in DNA somewhat related to skin carcinogenesis, photoaging repair synthesis in vivo (unscheduled DNA synthesis and skin, and the removal of (6-4) photoproducts; (ii) although it is believed that cyclobutane pyrimidine recovery of RNA synthesis after irradiation), dimers are the principal mutagenic lesion and (6-4) we performed a postreplication repair assay and recov- photoproducts are less likely to contribute to ultravi- ery of replicative DNA synthesis after ultraviolet irradi- olet-induced in mammals, Ops1 is one of ation to investigate if Ops1 cells belonged to a the ultraviolet-induced mutagenic models induced by xeroderma pigmentosum variant pattern. Ops1 cells (6-4) photoproducts. Key words: damage-specific DNA were normal, but there was an incomplete pattern binding protein/nucleotide excision repair/postreplication repair in (6-4) photoproducts in contrast to a normal repair. J Invest Dermatol 113:251–257, 1999

eroderma pigmentosum (XP) is a rare autosomal levels of caffeine enhanced the lethal effect of UV light in XP-V recessive disease characterized by a clinical and cells (Arlett et al, 1975; Cleaver and Kraemer, 1995). As the cellular hypersensitivity to ultraviolet (UV) light defective gene in XP-V has not yet been cloned, it is necessary (Cleaver and Kraemer, 1995). Patients exhibit to detect abnormal semiconservative DNA replication after UV dermatologic abnormalities, including thickening irradiation, that is, postreplication repair (PRR), for the definite andX hyperpigmentation of sun-exposed skin, and develop sunlight- diagnosis of XP-V (Lehmann et al, 1975, 1977; Rude and Friedberg, induced malignancies at an earlier age than usual. Cells from XP 1977; Cleaver and Kraemer, 1995). In addition, we recently patients show hypersensitivity to killing by UV irradiation and reported a simple alternative method for the diagnosis of XP-V severely reduced levels of unscheduled DNA synthesis (UDS). This (Itoh et al, 1996a). This technique provided a systematic procedure cellular phenotype correlates with a defect in nucleotide-excision- for diagnosis in photosensitive patients with NER defective XP or repair (NER) of UV-induced DNA lesions. By cell fusion analyses, PRR defective XP (Itoh et al, 1996a). XP was defined as having seven genetic complementation groups A damage-specific DNA binding (DDB) protein was first (XP-A through XP-G). A separate group XP variant (XP-V), had reported by Feldberg and Grossman (1976). It has a broad proficient NER. XP-V showed an exaggerated delay in recovery specificity, binding to DNA damaged by UV light, ionizing of replicative DNA synthesis (RDS) after UV irradiation (Lehmann radiation, NaHSO3, OsO4, and an enzymatic superoxide-generating et al, 1975, 1977; Rude and Friedberg, 1977; Cleaver and Kraemer, system (Feldberg and Grossman, 1976; Feldberg, 1980; Feldberg et al, 1982; Carew and Feldberg, 1985). In particular, a DDB 1995), and continuous postirradiation treatment with nontoxic protein bound with a high affinity to UV photoproducts: trans,syn-I cyclobutane pyrimidine dimers (CPD) (6-4) photoproducts [(6-4) PP], and Dewar isomer of the (6-4) PP, but with only a marginal Manuscript received November 12, 1998; revised April 12, 1999; affinity to cis-syn CPD. A binding with the psoralen-thymine accepted for publication April 21, 1999. monoadduct was not measurably detected (Reardon et al, 1993). Reprint requests to: Toshiki Itoh, Department of Cell Genetics, NER was originally defined in terms of the removal of cis-syn Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kuhonji 4-24-1, Kumamoto, 862-0976, Japan. CPD from DNA in both and (Friedberg E-mail: [email protected] et al, 1995). Moreover, NER defective XP (XP-A through XP- Abbreviations: (6-4) PP, (6-4) photoproducts; CPD, cyclobutane pyrimi- G) is defective in the removal of cis-syn CPD and the psoralen- dine dimers; DDB, damage-specific DNA binding; NER, nucleotide- thymine monoadduct, and is not usually sensitive to most of these excision-repair; PRR, postreplication repair; RRS, recovery of RNA agents, except for UV photoproducts (Friedberg et al, 1995). Thus, synthesis; RDS, recovery of replicative DNA synthesis; UDS, unscheduled it seemed that DDB protein was somewhat related with DNA DNA synthesis; XP, xeroderma pigmentosum. repair, but was not directly related with NER or NER defective XP.

0022-202X/99/$14.00 · Copyright © 1999 by The Society for Investigative Dermatology, Inc. 251 252 ITOH ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

In the course of surveying photosensitive patients, we had a as a defect of DDB protein in this study. The human primary cell strain chance to diagnose an interesting case. The patient, a 62 y old XP82TO (XP-E) was a gift from Dr. Seiji Kondo at Tokyo Medical and Japanese woman (Ops1), showed a clinical XP phenotype. In this Dental University (Kondo et al, 1988). The human primary cell strain, study, we extensively examined biochemical characteristics of Ops1 XP2SA (XP-V) was purchased from the Human Science Research Resources Bank (Osaka, Japan). All cells were cultured in Dulbecco’s cells including NER or PRR assays established previously (Itoh modified Eagle’s minimum essential medium (ICN, Irvine, CA) supple- et al, 1994, 1995, 1996a). Interestingly, while the cells showed mented with 10% (vol/vol) fetal bovine serum (ICN), penicillin G (100 NER activity within a normal range, the repair kinetics of (6-4) units per ml), and streptomycin (100 µg per ml) in a humidified 5% PP was selectively impaired. Moreover, Ops1 cells showed a CO2 incubator. defective DDB protein. These results suggest the possibility that Ops1 had a phenotype other than the already known XP, as Ops1 UV survival assay Colony forming ability was performed as described showed normal NER and PRR, and was involved in the repair of elsewhere (Itoh et al, 1994, 1995). Cells were trypsinized and appropriate specific types of DNA damage. numbers of cells were plated on to 60 mm plates. After incubation for 10 h, they were washed with phosphate-buffered saline, irradiated with MATERIALS AND METHODS 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 Cells and culture conditions The human primary cell strains Mori, with phosphate-buffered saline, fixed with 80% (vol/vol) methanol, stained Turu, and Sono were established as normal in this laboratory (Itoh et al, with Giemsa, and colonies were counted. Survival curves were plotted on 1994, 1995, 1996a). The human primary cell strain Ops1 was designated 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.

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, Figure 2. Rate of DNA synthesis after UV irradiation in Ops1 cells. and stained with Giemsa. Caffeine was added at a final concentration of Cells were irradiated with UV light at a dose of 5 J per m2. After 0, 2, 4, 1 mM just after UV irradiation. Each point represents an average of three 6, or 8 h incubation, cells were pulse-labeled with [3H]thymidine (10 µCi or four experiments; error bar: SEM. Mori cells (u) were used as a normal per ml) for 30 min. Mori cells (u) were used as a normal control. Each control. Ops1 cells (s); Ops1 cells with 1 mM caffeine (d); XP2SA (XP- value is the mean for three or four experiments; error bar: SEM. Ops1 cells V) cells (n); XP2SA cells with 1 mM caffeine (m). (s); XP2SA cells (XP-V) (n).

Table I. DNA repair levels in vivo in Ops1 cells

Cell straina Grains/nucleus Range of relative rate (%)b

UDS Ops1/Moric 14.4 Ϯ 0.3/13.9 Ϯ 0.3 99–108 Ops1/Turuc 12.8 Ϯ 0.3/ 9.8 Ϯ 0.3 124–138 Ops1/Sonoc 8.9 Ϯ 0.2/ 8.3 Ϯ 0.2 102–112 RRS Ops1/Morid 48.6 Ϯ 3.9/40.2 Ϯ 3.9 101–145 With caffeine Without caffeine With caffeine Without caffeine

RDS 59.9 Ϯ 6.4/66.1 Ϯ 5.9 72.2 Ϯ 4.2/62.0 Ϯ 5.9 74–110 100–136 Ops1/Morid

aMori, Turo, and Sono are normal control cells. bRange of relative rate are the values (grain-numbers) of Ops1 cells compared with those of normal cells. cThe values represent the means Ϯ SEM of 300 nuclei per cell strain in each coverslip. These were examined under the same conditions. dThe values represent the means Ϯ SEM of 50 nuclei per cell strain. VOL. 113, NO. 2 AUGUST 1999 DEFECTIVE DDB2 GENE AND (6-4) PP REPAIR 253

Measurement of UDS, recovery of RNA synthesis (RRS) and counted in a liquid scintillation counter. Each point represents an average recovery of replicative DNA synthesis (RDS) after UV of four experiments. irradiation UDS, RRS, and RDS were measured by a method described elsewhere (Itoh et al, 1994, 1995, 1996a). To analyze effectively the results, µ Post-replication repair assay Semiconservative DNA replication after 20 l aliquots of cell suspension were plated with a micropipette in two UV irradiation was measured by a method described elsewhere (Itoh et al, rows on a coverslip (18 mm ϫ 18 mm) in all experiments. Briefly, UDS 1995, 1996a). Cells were irradiated with UV light (254 nm) at a dose of was measured as follows. Cells were irradiated with UVC (254 nm) at a 2 2 3 µ 5 J per m , returned to fresh medium, and incubated for 60 min before dose of 30 J per m , immediately labeled with [ H]thymidine (40 Ci per being labeled for 30 min with 0.37 MBq per ml (10 µCi per ml) of ml) for 2.5 h, and fixed. Coverslips were mounted on glass slides, dipped [3H]thymidine. Cells were harvested, then irradiated with 20 Gy of X-rays in Kodak NTB-3 emulsion, and exposed for 24 h or 48 h at 4°C. Grains on ice. A suspension of cells was layered on the top of 5 ml of 5%–20% above nuclei were counted under a microscope. RRS was measured as (wt/vol) alkaline sucrose gradients with 0.1 M NaOH, and centrifuged at follows. Briefly, cells were irradiated with UVC (254 nm) at a dose of 15 J 237,600 ϫ g for 90 min at 4°C with a RPS55T rotor (Hitachi, Tokyo, per m2. After UV irradiation, they were incubated for 23 h, and labeled 3 µ Japan). After centrifugation, drop fractions were collected on to Whatman with [ H]uridine (40 Ci per ml) for 1 h. Autoradiography was performed Grade 17 paper strips, and the acid-insoluble radioactivities were counted by the method described above. RDS was measured as follows. Briefly, in a liquid scintillation counter. 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 µCi per ml) for 1 h. Autoradiography was performed as described above. Detection of CPD and (6-4) PP by enzyme-linked immunosorbent assay Direct binding of monoclonal antibodies to CPD or (6-4) PP was Measurement of DNA synthesis after UV irradiation The rate of measured by enzyme-linked immunosorbent assay as described (Mori et al, DNA synthesis after UV irradiation was measured as described (Itoh et al, 1991). Cells were prelabeled with 1.85 kBq per ml of [2-14C]thymidine 1996a). Briefly, cells were irradiated or unirradiated with UVC (254 nm) for 3 d. Cells were then cultured for 2 d in a radioisotope-free media. at a dose of 5 J per m2. After incubation for an appropriate time, they After washing with phosphate-buffered saline, cells were UV irradiated were pulse-labeled with [3H]thymidine (10 µCi per ml) for 30 min, and and incubated for various times for repair. Immediately after irradiation or then harvested by adding 0.2 M NaOH. Each fraction was collected on after post-UV incubation, cells were harvested and genomic DNA was Whatman Grade 17 paper strips, and the acid-insoluble radioactivities were 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 ϩ 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,2MH2SO4 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.

Figure 4. The repair kinetics of CPD and (6-4) PP from genomic Figure 3. Comparison of the size of DNA synthesized after UV DNA in Ops1 and normal (Mori) cells. Mori (u; three experiments) irradiation in Ops1, and XP2SA cells with that in unirradiated cells. and Ops1 (d; two experiments) cells were UV irradiated (10 J per m2) Cells were irradiated with UV light at a dose of 5 J per m2, grown for and incubated for repair. The percentage of the initial number of 1 h, and incubated for 30 min with [3H]thymidine (10 µCi per ml). photolesions was determined at various times after UV irradiation using the Centrifugation was performed for 1.5 h at 237,600 ϫ g. The top of the standard enzyme-linked immunosorbent assay technique with monoclonal gradient is to the right. u, unirradiated cells (control); j, UV irradiated antibodies specific for each type of lesion. Each point shows the cells. (a) Ops1; (b) XP2SA (XP-V). mean Ϯ SEM. 254 ITOH ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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 µg 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.

Gel mobility shift assay A gel shift assay was performed as previously DNA was separated by electrophoresis in 1.5% 1 ϫ TBE agarose gel and described (Chu and Chang, 1988; Keeney et al, 1992) with some modifica- was stained with ethidium bromide. tions. 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. RESULTS Binding reactions (10 µl) contained approximately 0.2 ng [32P]DNA with 10% (vol/vol) glycerol, 0.35 mM poly(dI–dC), 0.1 mg per ml bovine Case report Ops1, a 62 y old Japanese woman, was first serum albumin, 5 mM MgCl2, 60 mM KCl, and 50 mM Tris–HCl, pH 8.0. recognized to be photosensitive at the age of about 20. There The reactions were incubated for 15 min at 30°C, and electrophoresed on were no complications during pregnancy, labor, or delivery. The 5% polyacrylamide gels in 1 ϫ Tris–borate/EDTA electrophoresis (TBE) parents were consanguineous. She showed average development. buffer. Then the gels were dried, and analyzed using a Fujix Bio-Analyzer At present, she has clinical sensitivity to UV light including BAS-2000 (Fuji Photo Film) for an appropriate time. pigmented or depigmented macules and patches on her face, neck, chest, and extremities, especially the dorsa of the hands. The sun- Identification of genomic DDB2 clones A human λ phage library exposed skin showed slight dryness and xerosis. Furthermore, she (YG-LI020) was purchased from the Human Science Research Resources had multiple skin neoplasias (five malignant and 14 basal Bank (Osaka, Japan) and screened with three DDB2 cDNA probes. The cell carcinoma on her face, and two of squamous cell carcinoma cDNA probes were made by polymerase chain reaction (PCR) with three sets of primers (–30: 5Ј-AGCACAGTACCCCTTCACAC and 425: 5Ј- in situ on her forearm and leg). Therefore, after examination at GGTGGGTTTGTCCTTGATGC; 265: 5Ј-GGGCTCCAGCAGTCC- several hospitals, Ops1 was diagnosed as having XP. TTTT and 634: 5Ј-GTGACCACCATTCGGCTACT; 632: 5Ј-CTA- GTAGCCGAATGGTGGTC and 1347: 5Ј-AAATCACCACCTCT- Normal post-UV survival with/without caffeine Cellular GCTTGC). Positive clones were plaque-purified and identified by restric- hypersensitivity to UV light was not shown by colony forming tion digestion (EcoRI, BglII, HindIII, and PstI; Boehringer, Mannheim, assay with or without caffeine in Ops1 cells (Fig 1). Germany). Sequencing analysis was performed using a Dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA) with a Normal UDS level in vivo The level of UDS in Ops1 cells was Model 373S DNA sequencer (PE Applied Biosystems). compared with that of cells from three normal subjects (Mori, Turu, and Sono). The level of UDS in Ops1 cells was normal Direct sequencing of DNA and restriction analysis Genomic DNA (Table I) and post-UV survival in Ops1 cells was also normal was extracted from fibroblasts using Easy DNA kit (Invitrogen, Carlsbad, (Fig 1), suggesting that Ops1 cells belonged to PRR defective XP, CA). The amplification of genomic DNA for the analysis of the change that is, XP variant (XP-V), not to NER defective XP. at nucleotide 937 (Ops1) was performed by PCR using the DDB2 gene- specific oligonucleotides followed by their position in relation to the ATG Normal RRS and RDS levels in vivo XP-V cells specifically translational start codon: 833: 5Ј-AAGCCAGCTTCCTCTACTCG and showed a reduced RDS level and this reduction was enhanced in 1000: 5Ј-ATGGGTGTGAGGTGCTGGA. PCR reaction was performed the presence of caffeine in spite of having normal levels in UDS using KODЈ polymerase (Toyobo, Osaka, Japan) in 30 cycles at 94°C, and RRS (Itoh et al, 1996a). The levels of RRS and RDS with 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, or without caffeine were within normal ranges (Table I). These 1000, I-239; 5Ј-TCTTCCTTTCCGCTCCTGTC as described above. A results indicated that Ops1 did not belong to XP-V. genomic DNA fragment including a non-sense mutation (nucleotide 937) was amplified by PCR using primers 833 and I-177: 5Ј-GGCA- Normal levels in the rate of post-UV DNA synthesis and GAAGTGGGATAAAAGG. PCR reaction and purification of PCR frag- PRR To confirm these results, we took the following two ments were performed as described above. The amplified fragments were approaches. One approach is the rate of post-UV DNA synthesis. digested with BglII (Boehringer Mannheim) at 37°C for 6 h. The digested XP-V cells showed a defect in the rate of post-UV DNA synthesis VOL. 113, NO. 2 AUGUST 1999 DEFECTIVE DDB2 GENE AND (6-4) PP REPAIR 255

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 ϫ TBE agarose gel. Lane M shows size markers (100 bp DNA ladder). Mori was used as a normal control.

(Fig 2) (Lehmann et al, 1975; Rude and Friedberg, 1977; Cleaver removed within 1 h after UV irradiation, similar to what occurred and Kraemer, 1995; Itoh et al, 1996a). The rate of post-UV DNA in normal cells, but were more slowly removed from that in Ops1 synthesis showed a normal pattern (Fig 2). cells (Fig 4). This removal pattern of (6-4) PP was similar to that Another approach is a PRR assay. By PRR assay, DNA in normal cells at a dose of more than 30 J per m2 (Nakagawa synthesized after UV irradiation was of the same size as that of et al, 1998). unirradiated Ops1 cells (Fig 3a). Under this condition, the DNA synthesized after UV irradiation in XP-V cells was found to be Defect of DDB protein in Ops1 cells A DDB protein bound smaller than that in unirradiated cells (Fig 3b) (Itoh et al, 1995, with a high affinity to (6-4) PP, but with only a marginal affinity 1996a). Furthermore, the effect of caffeine on UV survival was not to cis-syn CPD (Reardon et al, 1993). As Ops1 cells did not show found as shown in Fig 1. Therefore, it was confirmed that Ops1 defective repair in cis-syn CPD, but was apparent in (6-4) PP cells did not belong to XP-V. (Fig 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 Defective repair kinetics of (6-4) PP in contrast to normal cells showed a defect of DDB activity as shown in Fig 5. repair kinetics of CPD in Ops1 cells Although we could not detect the abnormality in DNA repair markers, to investigate the Non-sense mutation in DDB2 gene in Ops1 cells Recent repair phenotype of UV-induced DNA lesions in Ops1 cells, we studies showed the DDB protein to be a heterodimer of 127 kDa measured the repair kinetics of CPD and (6-4) PP using the enzyme- (p127; DDB1) and 48 kDa (p48; DDB2) subunits (Keeney et al, linked immunosorbent assay. Ops1 cells surprisingly showed an 1993; Dualan et al, 1995). Although we could not detect mutations incomplete pattern repair in (6-4) PP despite showing a normal in DDB1 cDNA of Ops1 cells (data not shown), we found a non- pattern repair in cis-syn CPD (Fig 4). NER defective XP showed sense mutation in DDB2 cDNA of Ops1 cells (data not shown). defective repair kinetics in both CPD and (6-4) PP (Friedberg et al, Furthermore, to evaluate the functional integrity of alleles in Ops1 1995; Itoh et al, 1996b). Furthermore, (6-4) PP were rapidly cells, we characterized the genomic organization of the DDB2 256 ITOH ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY gene and found it to be comprised of 10 exons (Fig 6a). To normal UDS levels, as the patient had the possibility to belong to confirm that Ops1 is homozygous for the non-sense mutation, we PRR defective XP (XP-V), the patient was diagnosed using PRR performed direct sequencing using PCR products of genomic assay (alkaline density sucrose gradient) or RDS levels with/without DNA. Ops1 carries a homozygous C→T transition at nucleotide caffeine (Cleaver and Kraemer, 1995; Itoh et al, 1995, 1996a). On position 937 in exon 7 in genomic DNA, generating a non-sense the basis of the evidence, we performed the diagnostic procedures mutation from CGA (Arg) to TGA (Stop) at codon 313 (Fig 6b). and determined that Ops1 is distinct from the already known XP This would be expected to produce a protein truncated by (NER defective XP or PRR defective XP). As XP-E is still 115 amino acids. Moreover, as this mutation carried BglII site inconclusive as described above, however, we cannot completely (AGATCC→AGATCT) (Fig 6b), we performed restriction analysis exclude the possibility that Ops1 is a disease related to XP-E of the genomic DNA fragment of DDB2 gene. The DNA fragment at present. of Ops1 was completely digested with BglII whereas that of normal Finally, both cis-syn CPD and (6-4) PP are mutagenic, but it is cells (Mori) was not digested (Fig 6c). These results demonstrated believed that cis-syn CPD is the principal mutagenic lesion in that Ops1 is homozygous for the non-sense mutation of DDB2 gene. mammalian cells (Brash, 1988; Tornaletti and Pfeifer, 1996). (6-4) PP, although clearly mutagenic in Escherichia coli (Horsfall and DISCUSSION Lawrence, 1994; Tornaletti and Pfeifer, 1996), are repaired much faster than cis-syn CPD in mammalian cells (Mitchell and Nairn, In this study, we reported a photosensitive patient (Ops1) with 1989; Tornaletti and Pfeifer, 1996) and are thus somewhat less multiple skin neoplasias and a new type of the non-sense mutation likely to contribute to UV-induced mutations in mammals. In this in DDB2 gene. Interestingly, Ops1 cells showed an incomplete study, however, we have indicated the possibility that Ops1 presents repair of pattern in (6-4) PP in spite of having a normal repair of itself as one of the mutagenic models induced by (6-4) PP in pattern in cis-syn CPD (Fig 4). In contrast, Ops1 cells had a mammals. normal DNA repair capacity in vivo (Table I, Figs 1–3). 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 We are grateful to Dr. Seiji Kondo, Dr. Mariko Iki and Dr. Hidenari Kusakabe (Mitchell et al, 1990) indicating that the formation of (6-4) PP is for XP82TO and Ops1 cells. We also thank Dr. Hironori Niki and Mrs. Yuka generally suppressed in nucleosome cores. Recent studies showed Itoh for preparation of the manuscript. This work was supported partly by grants that the p48 subunit (DDB2 gene) activated the p127 subunit of from the Ministry of Education, Science, Sports and Culture of Japan (to T.I. and the DDB protein complex (Hwang et al, 1998), and the p127 M.Y.), by the Naito Foundation (to T.I.), and by the Kao Foundation for Arts subunit was tightly associated with chromatin after UV irradiation and Sciences (to T.I.). (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 REFERENCES (Fig 4). 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It was reported that XP-E had biochemical heterogeneity localization and cDNA cloning of the genes (DDB1 and DDB2) for the p127 regarding DDB protein (Kataoka and Fujiwara, 1991; Keeney et al, and p48 subunits of a human damage-specific DNA binding protein. Genomics 1992; Friedberg et al, 1995). As the literature on XP-E is very 29:62–69, 1995 Feldberg RS: On the substrate specificity of a damage-specific DNA binding protein complex and confusing, and XP-E gene has not been cloned, from human cells. Nucleic Acids Res 8:1133–1143, 1980 XP-E is still inconclusive. Two of 13 of XP-E patients showed a Feldberg RS, Grossman L: A DNA binding protein from human placenta specific defect in the DDB protein (Keeney et al, 1992). Recently, three for ultraviolet damaged DNA. Biochemistry 15:2402–2408, 1976 XP-E patients with a defect in the DDB protein were reported Feldberg RS, Lucas JL, Dannenberg A: A damage-specific DNA binding protein. Large scale purification from human placenta and characterization. J Biol Chem (Otrin et al, 1998). All of these patients showed the mildest form 257:6394–6401, 1982 of XP, and the cells showed 40%–50% of normal UDS levels Friedberg EC, Walker GC, Siede W: Nucleotide excision repair in mammalian (Cleaver and Kraemer, 1995; Otrin et al, 1998), that is, the defect cells. In: DNA Repair and Mutagenesis. Washington: American Society of of NER. The level of UDS in Ops1 cells, however, was carefully Microbiology, 1995, pp 283–365 Horsfall MJ, Lawrence CW: Accuracy of replication past the T-C (6-4) adduct. J determined by counting 300 nuclei per cell strain in each coverslip Mol Biol 235:465–471, 1994 and the results were confirmed under different coverslips (Table I). Hwang BJ, Toering S, Francke U, Chu G: p48 activates a UV-damaged-DNA Each pair of Ops1 and reference cells (Mori, Turu, and Sono) were binding factor and is defective in xeroderma pigmentosum group E cells that compared by Student’s t test. We could not detect the differences lack binding activity. Mol Cell Biol 18:4391–4399, 1998 Itoh T, Ono T, Yamaizumi M: A new UV–sensitive syndrome not belonging between Ops1 and normal (Mori and Sono) cells statistically to any complementation groups of xeroderma pigmentosum or Cockayne (p Ͼ 0.10). Grains of Ops1 cells were significantly higher than syndrome: siblings showing biochemical characteristics of Cockayne syndrome those of Turu cells (p Ͻ 0.001). We determined that Ops1 cells without typical manifestations. Mutat Res 314:233–248, 1994 s showed normal UDS levels from these results. To diagnose whether Itoh T, Fujiwara Y, Ono T, Yamaizumi M: UV syndrome, a new general category of photosensitive disorder with defective DNA repair, is distinct from xeroderma a patient is XP, it is first necessary to measure UDS levels (Cleaver pigmentosum variant and rodent complementation group 1. Am J Hum Genet and Kraemer, 1995; Itoh et al, 1996a). In the case of reduced UDS 56:1267–1276, 1995 levels, as the patient had the possibility to belong to one of the Itoh T, Ono T, Yamaizumi M: A simple method for diagnosing xeroderma NER defective XP groups (XP-A through XP-G), the patient was pigmentosum variant. J Invest Dermatol 107:349–353, 1996a Itoh T, Shiomi T, Shiomi N, et al: Rodent complementation group 8 (ERCC8) diagnosed using the complementation assay (Itoh et al, 1994, 1996a; corresponds to Cockayne syndrome complementation group A. Mutat Res Cleaver and Kraemer, 1995). On the other hand, in the case of 362:167–174, 1996b VOL. 113, NO. 2 AUGUST 1999 DEFECTIVE DDB2 GENE AND (6-4) PP REPAIR 257

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