The role of UV light-induced photoproducts in initiating base substitution mutation in human cells was examined by determining the frequency and spectrum of mutation in a supF tRNA gene in a shuttle vector plasmid transfected into DNA repair deficient cells (xeroderma pigmentosum complementation group A). To compare the role of two major UV-induced photoproducts, cis-syn cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone photoproducts(6–4PPs), each photoproduct was removed from UV-irradiated plasmid by photoreactivation before transfection. Removal of either CPDs or 6–4PPs by in vitro photoreactivation reduced the mutation frequency while keeping the mutation distribution and the predominance of G:C-A:T transitions as UV-irradiated plasmid without photoreactivation, indicating that both cytosine-containing CPDs and 6–4PPs were premutagenic lesions for G:C-A:T transitions. On the other hand, A:T-G:C transitions were not recovered from plasmids after the removal of 6–4PPs, whereas this type of mutation occurred at a significant level (11%) after the removal of CPDs. Thus, the premutagenic lesions for the A:T-G:C transition are 6–4PPs. Removal of both CPDs and 6–4PPs resulted in the disappearance of mutational hot spots and random distribution of mutation as observed in unirradiated control plasmids. However, the mutational spectrum of photoreactivated plasmids differed significantly from that of unirradiated plasmids. A characteristic feature is a high portion of A:T-T:A transversions (11%) in the photoreactivated plasmid. This mutation is due to nondipyrimidinic “minor” photoproducts, and the mutation spectrum suggests that TA*, the major photoproduct of thymidylyl-(3′-5′)-deoxyadenosine, is the premutagenic lesion for this mutation. This is the first report revealing the distinct mutagenic roles of the major UV photoproducts and “minor”photoproducts by the use of (6–4)photolyase.

It is now widely recognized that the transformation of normal cells into tumorigenic cells is a multistep process, and substantial evidence indicates that mutations play a fundamental role in cellular transformation and carcinogenesis. Thus, elucidation of mutagenic mechanisms is central to the understanding of carcinogenesis. UV light has been the most widely studied mutagen and is implicated as the major causative agent for skin cancer (1, 2, 3). UV light induces various classes of DNA damage that can lead to mutagenesis. These include two major UV-induced photoproducts, cis-syn CPDs3and 6–4PPs, as well as other minor photoproducts (1, 4, 5, 6). However, the relative roles of these forms of DNA damage on UV mutagenesis remain controversial, especially in mammalian cells.

Genetic analysis and direct DNA sequencing of mutated target genes suggest that both CPDs and 6–4PPs could be premutagenic lesions(7, 8, 9, 10). Several studies have focused on the specific question of which of these two major UV-induced DNA lesions is the mutagenic one. An approach to this question is to compare the frequency of each UV lesion with mutation frequency. Except for some hot spots,the observed spectrum of UV-induced mutations correlates well with the frequency of UV-induced 6–4PPs rather than CPDs (5, 6, 7, 11), suggesting that the 6–4PPs are more efficient premutagenic lesions than the CPDs. However, certain mutation hot spots do not correlate with the frequency of CPD/6–4PP damage (7, 11),raising the possibility that “minor” photoproducts also play a specific role as the premutagenic lesion at some hot spots. More direct evidence that 6–4PPs may play a dominant role in UV-induced mutagenesis has been obtained from Escherichia coli(12, 13, 14). Single-stand M13-based vectors containing a 6–4PP or CPD were introduced into E. coli cells, and the sequences of the recovered M13 phage DNA were determined. 6–4PPs at TT sites (TT 64PP) are highly mutagenic, causing 91% mutation, whereas the CPDs at TT sites (CPD TT) are not very mutagenic (7% mutation). UV irradiation caused primary base substitutions, and the G:C-A:T transition was the most dominant (1). Thus, photoproducts formed at dipyrimidine sites containing C are very mutagenic. However,M13 vectors containing only TT 64PP, TC 64PP, or TT CPD were used. As a result, the mutagenic properties of photoproducts induced at C-containing sites are unclear, and more studies are needed. Another approach was to take advantage of the photoreversibility of CPD to provide evidence of the premutagenic role of these lesions (8, 15, 16, 17, 18). In E. coli, photoreactivation of CPDs was more effective in the rescue of lethality rather than in the reduction of mutation (15, 16). On the other hand, in mammalian cells, the selective removal of CPDs from a UV-irradiated shuttle vector by CPD photolyase resulted in a marked reduction of mutation frequency. However, the mutational hot spot did not change drastically,indicating that mutagenic photoproducts were still present after photoreactivation (8, 17, 18). These results indicate that both CPDs and 6–4PPs are premutagenic lesions in both bacteria and mammalian cells, although 6–4PPs are more efficient premutagenic lesions in E. coli, whereas CPDs are more efficient premutagenic lesions in mammalian cells. Furthermore, the possibility that “minor” photoproducts play a significant role in UV-induced mutation cannot be ruled out because CPDs were the only substrate for CPD photolyase.

In 1993, a light-dependent DNA repair activity specific for 6–4PPs was found in Drosophila melanogaster, and the enzyme was named 6–4(6–4)photolyase (19). Presently, the cDNAs of 6–4(6–4)photolyase have been cloned from Drosophila, Xenopus laevis, and Arabidopsis thaliana(20, 21, 22). It was believed that 6–4PPs could not be photorepaired to their original form by a simple photochemical splitting as in the case of CPD repair by CPD photolyase because of the structural differences between 6–4PPs and CPDs (23, 24). However, 6–4(6–4)photolyase repairs 6–4PPs to the original nondamaged configuration, and those repaired products do not induce mutation (25, 26, 27, 28). This permits the use of 6–4(6–4)photolyase to investigate the role of 6–4PPs as premutagenic lesions. Furthermore, the removal of both two major UV lesions by two types of photolyase may uncover the role of “minor” photoproducts as premutagenic lesions.

In this report, we removed CPDs and/or 6–4PPs from UV-irradiated shuttle vector plasmid pMY189 carrying the supF gene as the target for mutation by in vitro photoreactivation with CPD photolyase and/or 6–4(6–4)photolyase. Photoreactivated plasmid DNA was transfected into a repair-deficient XP-A cell line where the plasmids could be replicated by the human cell polymerase(s) without any effects of NER. The progeny plasmids were analyzed for the frequency of supF mutants,and the types and distributions of mutations were determined. We found an increase in the yield of replicated plasmids and a corresponding decrease in the frequency of supF mutants by photoreactivation of either UV lesion. Sequence analysis of the supF gene of mutant plasmids indicated that removal of either UV lesion does not alter the mutation spectrum drastically, and G:C-A:T transitions predominate. A notable difference between the two forms of photoreactivation was that A:T-G:C transitions were not recovered after the removal of 6–4PPs. Removal of both UV lesions resulted in the disappearance of mutational hot spots and a random distribution of mutations as was found in unirradiated controls. However, a high proportion of A:T-T:A transversions was recovered after the removal of both types of UV lesions.

Cells.

XP2OS(SV), an SV40-transformed cell line derived from a Japanese XP-A patient (29), was used in this study. Cells were cultured in Dulbecco’s modified MEM (Nikken, Kyoto, Japan) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT).

Plasmids and E. coli Strains.

The shuttle vector pMY189 (30), which was derived from pZ189 (31), was used in this study. pZ189/pMY189 contains the replication origin and the early region (coding for the large T antigen) of the SV40 virus, allowing its replication in human cells. It also contains sequences for its replication, maintenance, and selection(ampicillin resistance gene) in E. coli. The tyrosine amber suppressor tRNA supF gene was used as a target gene for mutations. The supF gene is able to abolish the effects of an amber mutation in the LacZ and gyrA genes of E. coli strain KS40/pKY241(32). pMY189 was constructed from pZ189 by inserting an M13 universal primer sequence at just upstream of the supF gene for the convenience of sequencing.

KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 [lacZ(am) CA7070 lacY1 HsdR HsdM D(araABC-leu)7679 galU galK rpsL thi; Ref.32]. Plasmid pKY241 contains a chloramphenicol-resistant marker and a gyrA (amber) gene. E. coli KS40/pKY241 cells carrying the active supF gene are sensitive to nalidixic acid, whereas cells carrying the mutated supF form colonies on plates containing nalidixic acid. Thus, when KS40/pKY241 cells are transformed with pMY189, mutations in supF can be selected on plates containing nalidixic acid, chloramphenicol, and ampicillin. To ensure the selection of the mutated supF gene,isopropyl-b-d-thiogalactoside and 5-bromo-4-chloro-3-indolyl-b-d-galactoside were added to the selection plates. E. coli cells containing the active supF gene produce blue colonies, whereas cells having a mutated supF gene produce white colonies.

Preparation of DNA Photolyase and Photoreactivation of UV-irradiated Plasmid.

Salmonella typhimurium CPD photolyase (33) and Xenopus laevis 6–4(6–4)photolyase (21) were used in this study. S. typhimurium photolyase and Xenopus photolyase were overexpressed in E. coliand purified as described by Li et al.(33) and Todo et al.(21), respectively.

pMY189 plasmid DNA was irradiated with 1 kJ/m2 of 254 nm of UV light using a germicidal lamp as described previously (21). Concentrated buffer was added to give a solution of 0.03 mg of DNA/ml in 10 mm Tris-HCl (pH 7.4), 5 mm DTT. One hundred thirty mg of purified CPD photolyase or 130 mg of purified 6–4(6–4)photolyase were added, and samples were illuminated with photoreactivating light for 3 h at room temperature as described previously (21). After photoreactivation, the samples were treated with proteinase K (final concentration of 0.1 mg/ml) in the presence of SDS (final concentration of 0.4%) at 500°C for 90 min. The samples were extracted with phenol and chloroform, and plasmid DNA was recovered by ethanol precipitation.

Measurement of CPDs and 6–4(6–4)Photoproducts by ELISA.

Direct binding of monoclonal antibodies to CPDs and 6–4(6–4)photoproducts was measured by ELISA as described (34, 35).

Transfection of Human Cells.

Human cells were trypsinized, washed, and suspended in Dulbecco’s PBS solution (pH 7.5). Cells (2 × 107) plus 20 mg of pMY189 in PBS solution (0.2 ml) were placed in an electroporation chamber (electrodes 0.3-cm apart;PDS Inc., Madison, WI), and the cells were transfected with the plasmids by electric pulses (600 V, five times). The cells were plated in five 10-cm dishes and incubated at 370°C for 72 h in a CO2 incubator.

Plasmid Recovery, Selection of Mutated supF, and DNA Sequencing.

The extrachromosomal plasmid DNA was recovered using a small-scale alkaline lysis procedure and digested by the restriction endonuclease Dpn I (TAKARA, Kyoto, Japan) to eliminate nonreplicated input plasmids retaining the bacterial methylation pattern.

E. coli KS40/pKY241 was transformed by the plasmid. For the selection of mutated supF, the transformed bacterial cells were plated on Luria-Bertani agar containing nalidixic acid,chloramphenicol, ampicillin,isopropyl-b-d-thiogalactoside, and 5-bromo-4-chloro-3-indolyl-b-d-galactoside. A fraction of the cells was plated on Luria-Bertani agar containing chloramphenicol and ampicillin to measure the total number of transformants.

Mutated plasmids were purified from overnight cultures, and the nucleotide sequences of the supF gene of the plasmid were determined with the -21 M13 primer and Big Dye Terminator cycle Sequencing Kit using a 310 automatic DNA sequencer (Applied Biosystems). To obtain independent mutant clones, fewer than five mutants were isolated from each independent transfection.

Statistics.

Statistical comparisons were performed with Fisher’s exact test for difference in proportions (36). Ps for a one-tailed test are presented.

Removal of CPDs and 6–4PPs from UV-irradiated Plasmid DNA.

The shuttle vector plasmid pMY189 was irradiated in vitrowith 254-nm UV. CPDs and 6–4PPs were removed from UV-irradiated DNA using two types of DNA photolyase, CPD photolyase, and 6–4(6–4)photolyase. To confirm the photoenzymatic reversal of photolesions by DNA photolyase, we measured the amount of CPDs and 6–4PPs on UV-irradiated plasmids by ELISA using monoclonal antibody specific for CPDs or 6–4PPs. ELISA showed that photoreactivation of UV-irradiated plasmid DNA with the two types of DNA photolyase eliminates each type of UV photoproduct without affecting the other types of damage (Fig. 1).

6–4PPs can be converted to their Dewar isomers by irradiation with wavelengths of light between 280 and 360 nm (1, 6). To test the possibility that 6–4PPs were converted to Dewar isomers by photoreactivating light, the amount of Dewar isomers in photoreactivated DNA was determined by ELISA using a monoclonal antibody specific for Dewar isomers. We did not detect Dewar isomers within the limits of detection (data not shown). Thus, we can exclude the possibility of contamination by Dewar isomers of the photoreactivated plasmid.

Plasmid Survival.

After UV irradiation and photoreactivation, plasmids were transfected into XP-A cells and incubated for 3 days to permit replication of the plasmid DNA and fixation of mutations. The progeny plasmids were purified from the transfected human cells and used to transform indicator bacteria. The survival of the plasmid was determined as the relative yield of bacterial colonies obtained after transformation with the purified progeny plasmids. The recovery of UV-irradiated plasmid from the XP-A cell line was reduced to 0.29% compared to unirradiated plasmid at a dose of 1 kJ/m2 (Table 1). Removal of either CPDs or 6–4PPs by DNA photolyase resulted in a slight increase in plasmid recovery, 11.6% and 2.3%, respectively. On the other hand, the removal of both types of UV damage drastically enhanced plasmid recovery to 94.3% of that of unirradiated plasmid.

Plasmid Mutagenesis.

Mutations in the supF target gene were detected in the indicator E. coli strain containing suppressible (amber) mutations in the lacZ gene and gyrA genes (E. coli cells having a wild allele of the gyrA gene are sensitive to nalidixic acid). The mutation frequency was defined as the ratio of the number of white and nalidixic acid-resistant colonies:the total number of bacterial colonies obtained without nalidixic acid selection. UV irradiation of pMY189 increased the proportion of plasmids having a mutated supF gene (Table 2). The background plasmid mutation frequency was 1.6 × 10−4 and increased 260-fold (to 4.2 × 10−2) following the treatment of plasmids with 1 kJ/m2 UV. Removal of either or both types of UV damage by DNA photolyase reduced the mutation frequency. The mutation frequency, when either CPDs or 64PPs or both were removed, was 4.2 × 10−3,1.8 × 10−2, and 1.2 × 10−3, respectively (Table 2).

Sequence Analysis.

Sequence analysis of 261 supF mutant plasmids recovered after passage of the UV-treated pMY189 through the XP-A cells was carried out. As shown in Table 3, base sequence changes in plasmids were classified as single base substitutions, tandem base substitutions, multiple base substitutions(base substitutions more than three bases apart), frameshifts (single base insertions or deletions), and large deletions.

Large deletions (average size, 83.1 bp; range, 7–210 bp) were found in 33.3% of the mutants derived from unirradiated plasmids. However, when plasmids were irradiated with UV, none of 53 mutants sequenced showed deletions. The frequency of plasmids with deletions was still low(2%) after treatment with either CPD photolyase (2%) or 6–4(6–4)photolyase (0%), but it increased to 18.2% when plasmids were treated with both types of photolyase.

The mutant plasmids without deletions contained base substitutions and frameshifts. Whereas base substitutions were most frequent(66.7–100%), frameshifts were scarce (0-4%). Consistent with previous studies (1, 8, 9, 10, 11), a high frequency of tandem base substitutions was observed with UV-irradiated plasmid (37.7%),whereas in unirradiated plasmid (4.4%), they were rare.

Types of single and tandem base substitution mutations in supF mutant plasmids are shown in Table 4. A significantly smaller portion of mutant plasmids had transitions(40.7%), with a greater portion having transversions (59.3%) in the unirradiated plasmids. In contrast, 85.6% of single or tandem base substitutions observed in the UV-irradiated plasmids were G:C-A:T transitions, and the portion of mutant plasmids with transversions was small (14.4%). Such a high proportion of transitions was also observed after removal of either CPDs or 6–4PPs (73.7% and 80.9%,respectively), but it was reduced to 44.5% when both forms of UV damage were removed.

Mutational Spectrum.

The distribution of single and tandem base substitutions in the supF gene after replication in the XP-A cells is shown in Fig. 2. All mutations were found between bp 56 and 198. The point mutations observed in UV-irradiated plasmids were not distributed randomly in the supF gene but appeared preferentially at certain sites. About half the G:C-A:T transitions were found in two strong hotspots at positions 123 and 168. Lesser hot spots were seen at positions 108, 156, and 169.

As reported previously (1, 8, 9, 10, 11), most of the sequence alterations determined were single or tandem base substitutions at positions of potential pyrimidine dimers. Thus, the assumption was made that the mutations originated from lesions at dipyrimidine sites, and the sites of premutagenic photoproducts for the mutants listed in Fig. 2 were therefore expected. In Tables 5 and 6, each type of base substitution mutation was classified with the expected sites of UV-induced mutations.

We have investigated the mutational properties of the two photolesions, CPDs and 6–4PPs, in repair-deficient human cells. We used an approach for the selective removal of CPDs or 6–4PPs from a UV-irradiated shuttle vector by photoreactivation with CPD photolyase or 6–4(6–4)photolyase before transfection. Removal of photolesions by photoreactivation was confirmed by ELISA (Fig. 1). ELISA showed that photoreactivation eliminates each type of photolesion completely within the limit of detection. Consistent with the results of ELISA,photoreactivation of both types of photolesion eliminated the effects of UV irradiation on the recovery of transfected plasmid (Table 1) and reduced the frequency of mutations (Table 2). Furthermore, the types and spectra of mutations in the plasmids after photoreactivation of both types of photolesion (-CPD/-64PP plasmid) became similar to those from unirradiated control plasmids; i. e., (a) a high frequency of deletion mutations compared to CPD and 6–4PP-containing plasmids, (18.2% in -CPD/-64 plasmid and 31.8%in control plasmid, whereas CPD and 6–4PP-containing plasmids had 0–2%; Table 3); (b) the portion of mutant plasmids having transversions (55.5%) was larger than that with transitions (44.5%;Table 4); (c) mutational hot spots disappeared from the-CPD/-64PP plasmids (Fig. 2). These results indicate that photoreactivation with each type of DNA photolyase selectively eliminated either type of photolesion almost completely and that the photoreactivated plasmids specifically manifested the effects of the remaining photolesion. In the case of the removal of CPDs or 6–4PPs, the mutagenic effects of 6–4PPs or CPDs plus other minor photoproducts were manifested selectively, and in the case of the removal of both types of lesion, the mutagenic effects of the remaining minor photoproducts alone became apparent.

A characteristic feature of UV mutagenesis is the high frequency of G:C-A:T transitions at dipyrimidine sites including CC-TT double base mutations (8, 9, 10, 11, 37, 38). This feature unique to UV was also observed in the UV-induced mutation of plasmid DNA (82.7% of base substitutions are G:C-A:T transitions and 24% of base substitutions are CC-TT tandem substitutions). Although the mutagenicity was substantially reduced by the removal of each UV lesion, the tendency toward having a larger portion of G:C-A:T transitions was preserved(63.2% in -CPD plasmid and 80.9% in -64PP plasmid), and the position of mutational hot spots did not change dramatically (Table 4 and Fig. 2). These findings indicate that both CPDs and 6–4PPs are premutagenic lesions for G:C-A:T transitions. On the other hand, the frequency of CC-TT tandem substitution was significantly reduced by the removal of either photoproduct. In particular, removal of CPDs dramatically reduced the frequency of CC-TT tandem substitution (7% in -CPD plasmid, P = 0.007; 13% in -64PP plasmid),indicating that although both CPDs and 6–4PPs are premutagenic lesions for CC-TT tandem substitutions, CPDs induce this type of mutation more effectively than 6–4PPs.

Although A:T-G:C transitions occurred less frequently than G:C-A:T transitions, they represent a significant component of UV-induced mutation. In contrast to G:C-A:T transitions, the frequency of A:T-G:C transitions showed an apparent difference between the contribution of CPDs and 6–4PPs. In the -CPD plasmids, 14.3% of transitions (8 of 56)were the A:T-G:C type, whereas in the -64PP plasmids, all transitions obtained (38 sites) were the G:C-A:T type, and no mutant having the A:T-G:C transition was recovered (P = 0.02). Thus, A:T-G:C transitions are induced at 6–4PPs specifically. As shown in Table 5, almost all of 6–4PP-specific A:T-G:C transitions were formed at TT sites. In fact, in COS cells, TT 6–4PP is more mutagenic than TT CPD and primarily elicits 3′T-C substitutions(39). 6–4PP-specific A:T-G:C transitions might be due to the following two reasons. First, duplexes with G opposite the 3′T of the 6–4PPs are thermodynamically more stable than that with A(40). The second reason is the properties of mammalian DNA polymerases. Recently, a new type of DNA polymerase, DNA polymeraseη, which is defective in XP-variant cells, was reported(41, 42, 43). DNA polymerase h replicates CPD-containing, but not 6–4PP-containing DNA templates. Furthermore, DNA polymerase ηincorporates dATP at the site opposite to TT CPDs, indicating that DNA polymerase h can bypass TT-CPDs without inducing mutation. This might be the reason why A:T-G:C transitions were not recovered from the plasmid containing CPDs. 6–4PP-specific A:T-G:C transitions might reflect the absence of a DNA polymerase that bypasses TT-6–4PPs in human cells.

When both types of photolesions were removed from UV-irradiated plasmids, the hot spots disappeared and the mutations became randomly distributed. However, the distribution of mutants in the -CPD/-64PP plasmids differ significantly from that in the unirradiated plasmids. In fact, A:T-G:C transitions and A:T-T:A transversions occurred in the-CPD/-64PP plasmids at significant levels (6.2% and 11.1%,respectively) but were rare in unirradiated samples (Table 4 and 5). The premutagenic lesions for these mutations were not CPDs and 6–4PPs, but they may have been other minor UV-induced photoproducts.

As minor UV-induced photoproducts, thymine glycol (44),pyrimidine hydrate (45), 8,8-adenine dehydrate (46, 47), and thymidylyl-(3′-5′)-deoxyadenosine (TA*; Ref.48) have been described. Of these, TA* is the most probable candidate for causing A:T-T:A transversions. Although TA* was produced by 254-nm irradiation of DNA with a quantum yield of 10–100 less than dipyrimidine products, it is highly mutable in SOS-induced E. coli (82% of the recovered phages were mutants; Refs. 48 and 49). The most abundant mutation was a 3′A-T substitution (49). Five of eight A:T-T:A transversions observed in -CPD/-64PP plasmids were induced at TA sequences and constituted a weak hot spot (positions 134–135, the predicted A:T-T:A transversions were indicated in white letters in Fig. 2; In Table 5, they are classified as TT and CT sites because Table 5 was based on the assumption that mutations are originated from the dipyrimidine sites). Thus, it is reasonable to conclude that TA* is the premutagenic lesion for A:T-T:A transversions. Of course, we cannot exclude the possibility that A:T-T:A transversions were made by other unknown photoproducts. Furthermore, it is not proven clearly whether NER defects in XP-A cells are defective for the excisions of all of the minor photoproducts. Further studies of the minor UV photoproducts are needed.

Carcinogenesis is believed to be etiologically related to somatic cell mutations arising from unrepaired lesions. XP patients have a deficiency in NER of UV lesions, and unrepaired UV lesions result in a high level of skin tumors. On the other hand, trichothiodystrophy and Cockayne syndrome patients, other disorders having a defect in NER, do not develop skin tumors in body exposed to UV light(1, 2, 3). The high mutation frequency due to a defect in NER in these cells was primarily caused by CPDs (50, 51). These results suggest that carcinogenic effects of 6–4PPs are different from those of CPDs and that unrepaired 6–4PPs resulting in a high mutagenic potential may play an important role in the molecular events leading to skin cancer development in XP patients(50). As shown in this paper, our system is useful for determining the distinctive roles of CPDs, 6–4PPs, and minor photoproducts. Our system, when applied to the trichothiodystrophy,Cockayne syndrome, and XP-variant cells, would be able to clarify the role of each type of UV lesion on carcinogenesis.

Fig. 1.

Removal of CPDs (A) and 6–4PPs(B) from UV-irradiated plasmid DNA by photoreactivation. Plasmid pMY189 was UV-irradiated (1 kJ/m2) and then illuminated with visible light with CPD photolyase (UV-CPD),(6–4)photolyase (UV-64), or both photolyases (UV-64/CPD). The percentage of the initial number of photolesions was determined using a standard ELISA technique with a monoclonal antibody for each photolesion. Each point shows the means of three to four determinations.

Fig. 1.

Removal of CPDs (A) and 6–4PPs(B) from UV-irradiated plasmid DNA by photoreactivation. Plasmid pMY189 was UV-irradiated (1 kJ/m2) and then illuminated with visible light with CPD photolyase (UV-CPD),(6–4)photolyase (UV-64), or both photolyases (UV-64/CPD). The percentage of the initial number of photolesions was determined using a standard ELISA technique with a monoclonal antibody for each photolesion. Each point shows the means of three to four determinations.

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Fig. 2.

Localization of base substitution mutation found in pMY189 that is unirradiated (NO UV), UV-treated(UV), or UV-treated followed by photoreactivation(UV-CPDs, UV-64PPs, and UV-CPDs-64PPs) and propagated in the XP-A cell line. The sequence shown contains the marker supFgene. Base substitutions are indicated below the altered bp as a change in the sequence presented. Tandem or closely spaced base substitutions are underlined. The substituted bases predicted to derive from the TA* photoproduct are shown as white letters in the UV-CPDs-64PPs sequence(see text).

Fig. 2.

Localization of base substitution mutation found in pMY189 that is unirradiated (NO UV), UV-treated(UV), or UV-treated followed by photoreactivation(UV-CPDs, UV-64PPs, and UV-CPDs-64PPs) and propagated in the XP-A cell line. The sequence shown contains the marker supFgene. Base substitutions are indicated below the altered bp as a change in the sequence presented. Tandem or closely spaced base substitutions are underlined. The substituted bases predicted to derive from the TA* photoproduct are shown as white letters in the UV-CPDs-64PPs sequence(see text).

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1

Supported by a grant-in-aid for Scientific Research (09308020, 11480140, and 11878093) and on Priority Area(08280101) from Ministry of Education, Science, Sports and Culture of Japan.

3

The abbreviations used are: CPD, cyclobutane pyrimidine dimer; XP, xeroderma pigmentosum; XP-A, XP complementation group A; 6–4PP, pyrimidine (6–4) pyrimidone photoproduct; NER, nucleotide excision repair; kJ, kilojoule.

Table 1

Effects of photoreactivation after UV irradiation on the yield of plasmids after replication in XP-A cells

UV irradiationPhotoproducts removed by photoreactivationRelative no. of bacterial colonies (%)a
0 J/m2  100 
1 kJ/m2 None 0.29 
1 kJ/m2 -CPDs 11.6 
1 kJ/m2 -6-4PPs 2.3 
1 kJ/m2 -CPDs/6-4PPs 94.3 
UV irradiationPhotoproducts removed by photoreactivationRelative no. of bacterial colonies (%)a
0 J/m2  100 
1 kJ/m2 None 0.29 
1 kJ/m2 -CPDs 11.6 
1 kJ/m2 -6-4PPs 2.3 
1 kJ/m2 -CPDs/6-4PPs 94.3 
a

Relative number of bacterial colonies observed after transformation by plasmids harvested from a XP-A cell line transfected with pMY189 treated as indicated. The data show mean values from three independent transfection experiments.

Table 2

Analysis of mutants obtained by transformation of E. coli with progeny of pMY189 generated during replication in XP-A cells

UV irradiationPhotoproducts removed by photoreactivationFrequency of supF mutants (SD)a
0 J/m2  1.6 (±1.0) × 10 
1 kJ/m2 None 4.2 (±2.3) × 10 
1 kJ/m2 -CPDs 4.2 (±2.6) × 10 
1 kJ/m2 -6-4PPs 1.8 (±0.5) × 10 
1 kJ/m2 -CPDs/6-4PPs 1.2 (±0.6) × 10 
UV irradiationPhotoproducts removed by photoreactivationFrequency of supF mutants (SD)a
0 J/m2  1.6 (±1.0) × 10 
1 kJ/m2 None 4.2 (±2.3) × 10 
1 kJ/m2 -CPDs 4.2 (±2.6) × 10 
1 kJ/m2 -6-4PPs 1.8 (±0.5) × 10 
1 kJ/m2 -CPDs/6-4PPs 1.2 (±0.6) × 10 
a

The data show mean values from three independent transfection experiments and the standard deviation is indicated in parenthesis.

Table 3

Analysis of sequence alterations generated in the supF gene by replication of UV-irradiated plasmids in XP-A cellsa

No UV+UV+UV-CPDs+UV-6-4PPs+UV-CPDs/6-4PPs
Independent plasmids sequenced 69 (100) 53 (100) 50 (100) 34 (100) 55 (100) 
Point mutations 46 (66.7) 52 (98.1) 47e (94.0) 34e (100) 43 (78.2) 
Single base substitution 22 (31.9) 21 (39.6) 31 (62) 23 (67.6) 22 (40.0) 
Tandem base substitutionb 3 (4.4) 20 (37.7) 12f (24.0)  6g (17.7)  8h (14.6) 
Multiple base substitutionc 21 (30.4) 11 (20.8)  4 (8.0)  5 (14.7) 13 (23.6) 
Frame shift 0 (0%) 1 (1.9)  2 (4)  0 (0)  2 (3.6) 
Single base insertion 0 (0) 0 (0)  2d (4.0)  0 (0)  0 (0) 
Single base deletion 0 (0) 1 (1.9)  0 (0)  0 (0) 21d (3.6) 
Deletion 22 (31.8 0 (0)  1 (2.0)  0 (0) 10 (18.2) 
Insertion 1 (1.5) 0 (0)  0 (0)  0 (0)  0 (0) 
No UV+UV+UV-CPDs+UV-6-4PPs+UV-CPDs/6-4PPs
Independent plasmids sequenced 69 (100) 53 (100) 50 (100) 34 (100) 55 (100) 
Point mutations 46 (66.7) 52 (98.1) 47e (94.0) 34e (100) 43 (78.2) 
Single base substitution 22 (31.9) 21 (39.6) 31 (62) 23 (67.6) 22 (40.0) 
Tandem base substitutionb 3 (4.4) 20 (37.7) 12f (24.0)  6g (17.7)  8h (14.6) 
Multiple base substitutionc 21 (30.4) 11 (20.8)  4 (8.0)  5 (14.7) 13 (23.6) 
Frame shift 0 (0%) 1 (1.9)  2 (4)  0 (0)  2 (3.6) 
Single base insertion 0 (0) 0 (0)  2d (4.0)  0 (0)  0 (0) 
Single base deletion 0 (0) 1 (1.9)  0 (0)  0 (0) 21d (3.6) 
Deletion 22 (31.8 0 (0)  1 (2.0)  0 (0) 10 (18.2) 
Insertion 1 (1.5) 0 (0)  0 (0)  0 (0)  0 (0) 
a

Data in parentheses are percent of total mutations per treatment group.

b

Two base substitutions 0–1 bases apart or three adjacent base substitutions.

c

More than two base substitutions more than three bases apart.

d

These mutants also showed single base substitutions.

e

P < 0.01 versus +UV.

f

P < 0.1 versus +UV.

g

P < 0.05 versus +UV.

h

P < 0.05 versus No UV.

Table 4

Types of single or tandem base substitutions generated in the supF gene by replication of UV-irradiated pMY189 with or without photoreactivation in XP-A cella

Base pair substitutionNo. of mutations observed
No UV+UV+UV-CPDs+UV-6-4PPs+UV-CPDs/6-4PPs
Transitions 83 (40.7) 89 (85.6) 56b (73.7) 38 (80.9) 36 (44.5) 
G:C A:T 33 (40.7) 86 (82.7) 48c (63.2) 38 (80.9) 31 (38.3) 
A:T G:C 0 (0) 3 (2.9)  8b (10.5)  0d (0)  5e (6.2) 
Transversions 48 (59.3) 15 (14.4) 20b (26.3)  9 (19.1) 45 (55.5) 
G:C T:A 21 (25.9) 4 (3.8)  9b (11.8)  2 (4.3) 20 (24.7) 
G:C C:G 25 (30.9) 3 (2.9)  5 (6.6)  4 (8.5) 13e (16.0) 
A:T T:A 1 (1.3) 8 (7.7)  4 (5.3)  2 (4.3)  9e (11.1) 
A:T C:G 1 (1.3) 0 (0)  2 (2.6)  1 (2.1)  3 (3.7) 
Total 81 (100) 104 (100) 76 (100) 47 (100) 81 (100) 
Base pair substitutionNo. of mutations observed
No UV+UV+UV-CPDs+UV-6-4PPs+UV-CPDs/6-4PPs
Transitions 83 (40.7) 89 (85.6) 56b (73.7) 38 (80.9) 36 (44.5) 
G:C A:T 33 (40.7) 86 (82.7) 48c (63.2) 38 (80.9) 31 (38.3) 
A:T G:C 0 (0) 3 (2.9)  8b (10.5)  0d (0)  5e (6.2) 
Transversions 48 (59.3) 15 (14.4) 20b (26.3)  9 (19.1) 45 (55.5) 
G:C T:A 21 (25.9) 4 (3.8)  9b (11.8)  2 (4.3) 20 (24.7) 
G:C C:G 25 (30.9) 3 (2.9)  5 (6.6)  4 (8.5) 13e (16.0) 
A:T T:A 1 (1.3) 8 (7.7)  4 (5.3)  2 (4.3)  9e (11.1) 
A:T C:G 1 (1.3) 0 (0)  2 (2.6)  1 (2.1)  3 (3.7) 
Total 81 (100) 104 (100) 76 (100) 47 (100) 81 (100) 
a

Data in parentheses are percent of total base substitution mutations per treatment group.

b

P < 0.1 versus +UV.

c

P < 0.01 versus +UV.

d

P < 0.05 versus +UV-CPDs.

e

P < 0.03 versus No UV.

Table 5

Sites of single base pair substitutions generated in the supF gene by replication of UV-irradiated plasmids pMY189 in XP-A cell

Mutation siteNo. mutations observed
No UV+UV+UV-CPDs+UV-6-4PPs+UV-CPDs/6-4PPs
Transitions 28 47 32 26 27 
G:C A:T      
TC 15 20 10 13 
C
CC 
TC18 11 11 
CC
TC
CC
A:T G:C      
TT 
CT 
TT
TT
CT
Transversions 47 12 35 
G:C T:A      
TC 
C
CC 
C
TC
CC
TC
CC
G:C C:G      
TC 
C
CC 
a 
TC
CC
TC11 
CC
A:T T:A      
T
TT 4b 
CT 1b 
CT
A:T C:G      
T
a 
Mutation siteNo. mutations observed
No UV+UV+UV-CPDs+UV-6-4PPs+UV-CPDs/6-4PPs
Transitions 28 47 32 26 27 
G:C A:T      
TC 15 20 10 13 
C
CC 
TC18 11 11 
CC
TC
CC
A:T G:C      
TT 
CT 
TT
TT
CT
Transversions 47 12 35 
G:C T:A      
TC 
C
CC 
C
TC
CC
TC
CC
G:C C:G      
TC 
C
CC 
a 
TC
CC
TC11 
CC
A:T T:A      
T
TT 4b 
CT 1b 
CT
A:T C:G      
T
a 
a

No dipyrimidine was found.

b

Mutants were predicted to be derived from TA

*

photoproducts(see text).

Table 6

Sites of tandem base pair substitutions generated in the supF gene by replication of UV-irradiated plasmids pMY189 in XP-A cell

SiteMutationNo UV+UV+UV-CPDs+UV-6-4PPs+UV-CPDs/6-4PPs
CC TT 17 
 TA 
 GG 
 AT 
 Ta 
TT AA 
CT TA 
 TG 
 AC 
 AA 
CCC TTT 
TCC GTT 
 GGT 
TCCC GTTa 
CTTC AA(T)Tb 
CTC T(T)Tb 
TTC C(T)Tb 
SiteMutationNo UV+UV+UV-CPDs+UV-6-4PPs+UV-CPDs/6-4PPs
CC TT 17 
 TA 
 GG 
 AT 
 Ta 
TT AA 
CT TA 
 TG 
 AC 
 AA 
CCC TTT 
TCC GTT 
 GGT 
TCCC GTTa 
CTTC AA(T)Tb 
CTC T(T)Tb 
TTC C(T)Tb 
a

The base was deleted.

b

Unchanged base was shown in parenthesis.

We thank Dr. Ciaren Morrison for critical reading of the manuscript and for helpful comments.

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