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Originally published In Press as doi:10.1074/jbc.M107696200 on September 25, 2001

J. Biol. Chem., Vol. 276, Issue 48, 44688-44694, November 30, 2001
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Cyclobutane Pyrimidine Dimers Are Responsible for the Vast Majority of Mutations Induced by UVB Irradiation in Mammalian Cells*

Young-Hyun YouDagger §, Dong-Hyun LeeDagger , Jung-Hoon YoonDagger , Satoshi Nakajima, Akira Yasui, and Gerd P. PfeiferDagger ||

From the Dagger  Department of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010 and the  Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-77, Japan

Received for publication, August 10, 2001, and in revised form, September 18, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The most prevalent DNA lesions induced by UVB are the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). It has been a long standing controversy as to which of these photoproduct is responsible for mutations in mammalian cells. Here we have introduced photoproduct-specific DNA photolyases into a mouse cell line carrying the transgenic mutation reporter genes lacI and cII. Exposure of the photolyase-expressing cell lines to photoreactivating light resulted in almost complete repair of either CPDs or (6-4)PPs within less than 3 h. The mutations produced by the remaining, nonrepaired photoproducts were scored. The mutant frequency in the cII gene after photoreactivation by CPD photolyase was reduced from 127 × 10-5 to 34 × 10-5 (background, 8-10 × 10-5). Photoreactivation with (6-4) photolyase did not lower the mutant frequency appreciably. In the lacI gene the mutant frequency after photoreactivation repair of CPDs was reduced from 148 × 10-5 to 28 × 10-5 (background, 6-10 × 10-5). Mutation spectra obtained with and without photoreactivation by CPD photolyase indicated that the remaining mutations were derived from background mutations, unrepaired CPDs, and other DNA photopoducts including perhaps a small contribution from (6-4)PPs. We conclude that CPDs are responsible for at least 80% of the UVB-induced mutations in this mammalian cell model.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The UV component of sunlight is responsible for the induction of skin tumors, most notably basal cell and squamous cell carcinomas and probably also melanomas (1, 2). Mutations in the p53 gene have been found in a large percentage of human skin malignancies (3-6). The most frequent p53 mutations in skin tumors are C to T or CC to TT mutations involving dipyrimidine sequences. These are considered characteristic mutational changes that can be ascribed to solar UV irradiation (7).

The most abundant UV-induced DNA photoproducts are the cis-syn cyclobutane pyrimidine dimers (CPDs)1 and the pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). Both lesions are produced by UVB (280-320 nm) and UVC (200-280 nm) irradiation in DNA (8, 9). Most of the mutagenic and carcinogenic action of sunlight has been ascribed to the UVB portion of the solar spectrum (10) with a possible role for UVA (320-400 nm) in the induction of melanoma (11). The absorption of UVA photons by DNA is rather weak, and it is thought that indirect damaging mechanisms may involve endogenous chromophores as radiation absorbing intermediates (12). These can generate reactive oxygen species, which may then damage DNA.

Of all lesions formed in DNA after UVB irradiation, the CPD is considered one of the most important ones based on its relatively high abundance, slow repair, and known mutagenicity (8, 9, 13). However, a strong case can be made that the (6-4)PPs are equally if not more important than the CPDs for inducing mutations. C to T transitions can be induced by both CPDs and (6-4)PPs in mutagenesis studies using site-specific photolesions, and (6-4)PPs appear to be much more mutagenic than CPDs in these experiments. Plasmid constructs containing defined UV photoproducts have been used to study the mutagenic specificities of CPDs and (6-4)PPs. The mutation frequency obtained with site-specific 5'-TT-CPDs is generally very low (14-17). This is consistent with the infrequent recovery of mutations at 5'-TT sequences in UV-irradiated cells and is due to the likely involvement of DNA polymerase eta  in correct bypass of these lesions (18-20). DNA polymerase eta  is encoded by the RAD30 gene in yeast and by the XPV gene in humans. Further, the mutagenicity of a site-specific CPD containing the 5'-TC sequence also is very low, with >95% accurate lesion bypass (21), and this is consistent with the proposal that DNA polymerase eta  correctly bypasses this lesion (22). In fact, CPDs may become highly mutagenic only after deamination of cytosine or 5-methylcytosine has occurred within the lesion (23-27). A (6-4)PP at a 5'-TT site was shown to be highly mutagenic in Escherichia coli (28, 29). Most of the mutations were T to C transitions at the 3'-T. In mammalian cells, an unusual type of mutation, semi-targeted to the base flanking the 5'-T was observed (17). Another report described a similar specificity in mammalian cells as in yeast, i.e. T to C transitions at the 3' base (30). Both types of mutations are, however, not very common in UV-irradiated mammalian cells. Consequently, the (6-4)PPs at 5'-TT sites may not be very frequent after UV exposure, or they may be repaired too rapidly to become mutagenic. The 5'-TC (6-4)PP was found to be less mutagenic than the 5'-TT (6-4) product but much more mutagenic than a CPD at the sequence 5'-TT (31). 80% of the mutations were C to T transitions at the 3' base. Because a change from 5'-TC to 5'-TT is a typical change frequently observed in UV mutagenesis experiments, the 5'-TC (6-4)PP is a candidate for a strongly premutagenic UV-induced lesion. Studies with site-specific 5'-CT and 5'-CC lesions have not yet been reported. In summary, although (6-4)PPs are less frequent than CPDs and are repaired more efficiently (32), they may nonetheless be highly mutagenic. This is perhaps a consequence of the inability of DNA polymerase eta  to correctly bypass this lesion (33), creating either a mutation or leaving the lesion more susceptible to bypass by more error-prone DNA polymerases.

To dissect the individual contributions of CPDs and (6-4)PPs to UV mutagenesis, we have introduced foreign photolyase genes into a mouse cell line that carries two transgenic mutation reporter genes. We have studied the mutations produced after photoproduct-specific photoreactivation and show that the CPD is responsible for a great majority of the mutations induced by UVB irradiation.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transfection of Photolyase Genes-- The mouse embryonic fibroblast cell line carrying 40 copies of the Big Blue® lambda LIZ shuttle vector was purchased from Stratagene (La Jolla, CA). The cells were grown in Dulbecco's modified Eagle's medium containing 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum at 37 °C. The CPD photolyase gene from the rat kangaroo Potorous tridactylis (34) and a neomycin resistance gene were cloned into the mammalian expression vector pCY4B to form the construct pCY4Bneo3ptkCPD. The pCY4B expression vector contains the CMV-IE enhancer and the chicken beta -actin promoter for high level expression in mammalian cells (35). The (6-4) photolyase gene from Arabidopsis thaliana (36), without its N-terminal signal sequences for mitochondrial and chloroplast targeting, was cloned into the vector pCY4B to form the construct pCY4B/At6-4(neo31). These plasmids were transfected into Big Blue® mouse cells by electroporation. Empty vector was transfected to produce a control cell line. After selection in 800 µg/ml G418 (Life Technologies, Inc.), single colonies were picked and expanded. The cloned cell lines were maintained in the presence of 200 µg/ml G418.

Western Blot Analysis-- To verify the expression of the transfected photolyases in stable clones, total cellular proteins were analyzed by Western blotting. The cloned cells were harvested and homogenized in buffer containing 20 mM HEPES, pH 7.5, 2 mM dithiothreitol, 20 mM NaCl, 5 mM EDTA, 1 mM EGTA, 5 µg/ml aprotinin, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 10 mM benzamidine chloride, and 0.2% (v/v) Triton X-100. Total lysates from cells were denatured in gel loading buffer and subjected to electrophoresis in 10% SDS-polyacrylamide gels. Antibodies specific for P. tridactylis CPD photolyase and A. thaliana (6-4) photolyases, respectively, were raised in rabbits against the recombinant proteins and were used in Western blotting at dilutions of 1:2000. Immunoblot analysis after transfer of the proteins onto nitrocellulose membranes was performed using enhanced chemiluminescence (Amersham Pharmacia Biotech).

UV Irradiation and Photoreactivation-- The mouse cell clones expressing either CPD photolyase, (6-4)PP photolyase, or the vector only controls were expanded and grown on 100-mm cell culture dishes (5 × 105 cells/dish). After removal of the medium and washing in phosphate buffered saline, the cells at 30-40% confluence were irradiated with a UVB source (a Philips TL 20W/12RS lamp filtered through cellulose acetate (peak emission 312 nm; lower wavelength cut-off, 295-300 nm) at a dose of 500 J/m2 (about 30 s). The UV dose was determined with a UVX radiometer and a UVB sensor (Ultraviolet Products, Upland, CA). After UVB exposure, the cells were put into Hanks' solution without phenol red. They were exposed to photoreactivating 360-nm UVA light from two black lights (Sylvania 15W F15T8) through the bottoms of the dishes for different time periods at 37 °C. To block shorter wavelengths and to avoid heat production, the black lights were filtered through two glass plates with a thickness of 4 mm each. The distance from the lamp to the cells was about 5 cm. After photoreactivation, either the cells were harvested and cellular DNA was isolated immediately (for the DNA repair experiments), or regular growth medium was returned to the cells for further incubation and mutation fixation. Genomic DNA was isolated by standard procedures (37).

DNA Damage Detection by Immuno-Dot-blot-- The amount of thymine dimers and (6-4)PPs in the DNA was measured by an immuno-Dot-blot assay (38) using the CPD-specific monoclonal antibody TDM-2 and the (6-4)PP-specific monoclonal antibody 64M-2 (39). Cellular DNA was denatured in TE buffer (10 mM Tris-CL and 1 mM EDTA, pH 7.5) by boiling for 5 min. The samples were dot-blotted in triplicate onto a Hybond N+ membrane using 60 ng of DNA for the CPD assay and 1 µg of DNA for the (6-4)PP assay. DNA was fixed to the membrane for 20 min on 3MM paper soaked in 0.4 N NaOH. The membranes were blocked overnight in phosphate-buffered saline, 0.2% Tween 20 (PBS-T) containing 5% (w/v) skim milk. After washing in PBS-T, the membranes were incubated for 2 h at room temperature with monoclonal antibody 64M-2 (anti (6-4)PP antibody) and TDM-2 (anti CPD monoclonal antibody) using a dilution of 1:2000 in phosphate-buffered saline. After washing, they were incubated for 1 h with anti-mouse immunoglobulin monoclonal antibody diluted 1:4000 in phosphate-buffered saline. Signals were detected with a chemiluminescence kit (Amersham Pharmacia Biotech). The repair of CPDs after photoreactivation was also measured by digestion of DNA with T4 endonuclease V and alkaline-agarose gel electrophoresis as described previously (40, 41). In a similar assay, the repair of (6-4)PPs was measured by first removing CPDs with E. coli CPD photolyase in vitro followed by digestion of the DNA with the UV damage endonuclease (42).

cII and lacI Assays-- After photoreactivation, the cells were grown for 5 days to allow mutation fixation. The cells were passaged once after 3 days. DNA was isolated as described previously (37, 43). For the lacI mutation assay, the lambda LIZ shuttle vector containing the lacI target gene was rescued from total genomic DNA by mixing 0.5 µg/µl DNA aliquots with lambda  phage packaging extract (TranspackTM; Stratagene, La Jolla, CA) as described in the Big Blue® manual (Stratagene). Mutations were detected as blue phage plaques on E. coli K-12 lawns (SCS-8 strain: recA--, McrA-, McrBC-, Mrr-, and HsdR-; Stratagene) on 25-cm NZY agar plates containing 1.5 mg/ml of 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside (X-gal). The plates were incubated overnight at 37 °C, and the mutant blue plaques were counted. The lacI mutant frequency was calculated by dividing the number of mutant blue plaques, excluding sectored and pinpoint plaques, by the calculated number of total clear plaques. The numbers shown are the averages of three to four independent experiments.

For the cII mutation assay, the lambda LIZ shuttle vector was rescued from genomic DNA by mixing 0.5 µg/µl DNA aliquots with lambda  phage packaging extract. The cII mutation assay was performed as described in the lambda Select-cII mutation assay manual (Stratagene) using the G1250 hfl- E. coli host strain (a specialized hfl- version of Stratagene's XL1-Blue MRA cells, as described by Jakubczak et al. (44)). The mutation assay was done as described in You and Pfeifer (43). For mutant selection, 100 µl of the packaged phage was mixed with 200 µl of the G1250 strain, plated on TB1 plates, and incubated at 24 °C for 48 h. After incubation at 24 °C, lambda  phage bearing nonmutant cII genes will undergo lysogenic growth, but phage with mutant cII genes will undergo lytic growth and give rise to plaques. Upon 37 °C incubation, non-cII-mutants also undergo a lytic cycle and form plaques. The cII mutant frequency was calculated by dividing the number of mutant plaques by the calculated number of total plaques. For sequencing analysis, putative mutant plaques were replated at low density to verify the mutant phenotype and to isolate plaques. Single-well isolated plaques were picked, placed into 25 µl of TE buffer, and boiled for 5 min. A 433-base pair segment containing the cII gene and flanking regions was amplified by PCR with two primers: 5'-CCACACCTATGGTGTATG (positions -68 to -50) and 5'-CCTCTGCCGAAGTTGAGTAT (positions +345 to +365) using conditions described previously (43). The PCR products were purified using PCR purification kits (Qiagen, Chatsworth, CA) and were sequenced with the Big DyeTM Terminator Cycle Sequencing Ready Reaction DNA sequencing kit (ABI Prism, PerkinElmer Applied BioSystems, Foster City, CA) on an ABI 377 DNA sequencer. Each cII mutant was sequenced in its entirety with PCR primers as mentioned above. Each mutation was confirmed by sequencing the same region on the opposite strand. It may be argued that some of the mutational hot spots seen after UV irradiation are clonally expanded mutants. However, "jackpots" of transgene mutations are not a concern for induced mutations because only a minute fraction of all cII mutants after a limited number of cell divisions are rescued and eventually packaged into phage (45).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to determine the relative contributions of CPDs and (6-4)PPs to UVB-induced mutations in mammalian cells. Rather than using shuttle vectors that may not entirely recapitulate the UV mutagenesis process in mammalian chromatin, we devised a strategy to selectively remove one type of photoproduct after UV irradiation and then measure the mutations produced by the remaining lesions in two chromosomal reporter genes (Fig. 1). A permanent mouse embryonic fibroblast cell line carrying the lacI and cII transgenes as mutation reporters was transfected with genes encoding two different DNA photolyases. DNA photolyases are enzymes that use light harvesting chromophores and energy transfer to photorepair photoproducts in DNA (46). There are photolyases specific for either CPDs or (6-4)PPs (reviewed in Refs. 47-50). To repair CPDs, the CPD photolyase gene of P. tridactylis (34) was introduced, and for repairing (6-4)PPs, the (6-4) photolyase gene from A. thaliana (36) was introduced into the mouse embryonic fibroblast cell line.


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  		Fig. 1.   Schematic representation of the experimental approach used to determine the relative contributions of CPDs and (6-4)PPs to UVB-induced mutagenesis in a mammalian cell system.

Stable G418-resistant clones were selected and expanded. The presence and expression of the photolyases was confirmed by Western blot analysis using antibodies specific for the two proteins. Two clones with relatively high expression levels of the photolyase proteins were selected for further study (Fig. 2).


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  		Fig. 2.   Western blot analysis of expression of CPD and (6-4) photolyases in transfected mouse fibroblast clones. Two negative clones (co) and two positive ones are shown. The positive clones were used for further analysis of DNA repair rates and UV mutagenesis.

We next established the conditions under which the introduced photolyases repair UVB-damaged DNA in vivo. The cells were grown in plastic dishes, the medium was removed, and the cells were washed with phosphate-buffered saline and then exposed to 500 J/m2 of UVB irradiation. Under these conditions, approximately one CPD is formed in every 10 kilobases of DNA. After UVB irradiation, the cells were exposed to 360-nm UVA light through the bottoms of the dishes to provide the necessary wavelength for enzymatic photoreactivation. After exposure of the cells to UVA, we analyzed repair of the two photoproducts using specific anti-photoproduct antibodies and an immuno-Dot-blot procedure (Fig. 3). After photoreactivation in the CPD photolyase expressing cells, repair of CPDs is almost (about 90%) complete after 3 h, whereas there is no repair in the vector only transfected cells or in the same cells without photoreactivation (not shown). For (6-4)PPs, repair is virtually complete in 3 h, and there is partial repair in the control cells (vector-transfected). This difference in endogenous genomic repair of CPDs versus (6-4)PPs is consistent with the known preferential repair of (6-4)PPs in mammalian cells (32).


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  		Fig. 3.   Antibody assay to measure repair of DNA photoproducts. Cell lines expressing CPD photolyase (CPD-PL) or (6-4) photolyase ((6-4)-PL) and vector only controls were irradiated with 500 J/m2 of UVB. The cells were then exposed to 360-nm photoreactivating light (PR) for 1 and 3 h. The removal of CPDs from the genome was monitored with a monoclonal antibody specific for CPDs. The removal of (6-4)PPs from the genome was monitored with a monoclonal antibody specific for (6-4)PPs. For the dose response assay, UVC (254 nm) was used.

We also measured the removal of CPDs after photoreactivation by alkaline-agarose gel analysis of T4 endonuclease V digestion products. After 3 h of photoreactivation in the cell line expressing CPD photolyase (CPD-PL), the size distribution of the DNA fragments was similar to that of nonirradiated DNA (not shown). In contrast, the cells that were transfected with the vector only exhibited no repair of CPDs after 3 h. The repair of (6-4)PPs including that of any Dewar valence isomers was measured by UV damage endonuclease digestion of the DNA after removal of CPDs with E. coli CPD photolyase in vitro. Both DNA repair assays showed that there was very efficient repair of the photoproducts after 3 h. Because the doubling time of these mouse cell lines is in the order of 30-36 h, and repair is virtually completed after 3 h, we proceeded to use the cell lines for studying mutations induced in the lacI and cII transgenes.

The three cell lines transfected with CPD photolyase, (6-4) photolyase, or vector only were exposed to 500 J/m2 of UVB and then exposed to photoreactivating light for 3 h. Controls included cells not exposed to UVB, either exposed to photoreactivating light or not, and cells exposed to UVB but not subjected to photoreactivation. After UVB irradiation and photoreactivation treatment, the cells were incubated for 5 days to allow mutation fixation. During this time period, the cell numbers increased by ~10-15-fold for all cell lines, and the cells were split after 3 days. After 5 days, DNA was isolated, and the lambda LIZ shuttle vector was rescued from total genomic DNA by packaging into phage particles.

Mutations were first scored in the cII gene (Fig. 4A). The mutant frequency in nonirradiated cells, with or without exposure to photoreactivating UVA light, was in the order of 6-20 × 10-5. Compared with the other two cell lines, the cell line expressing (6-4) photolyase seemed to have a slightly elevated background mutant frequency. Similar data for background mutant frequencies in the cII gene have been reported by us and by others (43, 51, 52). After UVB irradiation, the mutant frequency increased 10-15-fold to values ranging from ~110 × 10-5 to 140 × 10-5. However, after photoreactivation in the cell line expressing CPD photolyase, there was a striking reduction of mutant frequency, from 127 × 10-5 down to 34 × 10-5 (Fig. 4A). In contrast, photoreactivation in the cell line expressing (6-4) photolyase did not reduce the mutant frequency to a significant extent. These results indicate that the majority of the mutations in the UVB-irradiated cells were derived from cyclobutane pyrimidine dimers.


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  		Fig. 4.   Mutant frequencies in the cII and lacI transgenes in cell lines expressing DNA photolyases. The cell lines were a control (vector only) cell line, a cell line expressing (6-4) photolyase ((6-4)PL), and a cell line expressing CPD photolyase (CPD-PL). Mutant frequencies were determined in the cell lines in the absence of irradiation (No UV), with (+PR) and without (-PR) photoreactivating 360-nm light. The same cell lines were exposed to 500 J/m2 of UVB, with and without photoreactivation, and the mutant frequencies were determined. The mutant frequency numbers are the average of three to four independent experiments (error bars, ± S.D). A, cII gene; B, lacI gene.

The more widely used lacI gene is another mutation reporter present in the genome of the lambda -transgenic mouse cells. We determined mutant frequencies for lacI in the three cell lines using the same conditions and controls as used for the cII data (Fig. 4B). The background frequency for lacI mutations was generally between 6 × 10-5 and 20 × 10-5. Again, the background frequency was somewhat higher in the cell line expressing (6-4) photolyase. The reason for this increased spontaneous mutant frequency in this cell line is not known. UVB irradiation led to mutant frequencies between 134 × 10-5 and 192 × 10-5, an increase of up to 15-fold over background (Fig. 4B). After exposure of the cell line expressing CPD photolyase to photoreactivating light, the lacI mutant frequency dropped to about 28 × 10-5. This is a reduction of more than 6-fold when the background mutant frequency is subtracted. In contrast to photoreactivation by CPD photolyase, exposure of the cell line expressing (6-4) photolyase to photoreactivating light did not result in an appreciable change in the lacI mutant frequency. Thus, the results with lacI are consistent with those obtained for cII and point to a major role of the CPD photoproduct in mammalian UV mutagenesis.

We also determined the nature of the mutations that remained after photoreactivation in the cell line expressing CPD photolyase. Because cII is a smaller gene, and the assay is less expensive, we analyzed cII mutations after UVB irradiation, with and without photoreactivation, in the cell line expressing CPD photolyase. Mutations in the absence of UVB treatment were also sequenced. We sequenced 61 mutations in untreated cells, 97 mutations in UVB-exposed cells without photoreactivation, and 108 mutations in UVB-exposed cells with photoreactivation (Table I and Fig. 5). Many different types of mutations were present in nonirradiated cells with no clear pattern of specificity. After UVB irradiation, 65% of the mutations were C to T transitions, and more than 95% of the UV-induced mutations were at dipyrimidine sites. Nine of 97 mutations (9.3%) after UVB treatment were CC to TT tandem mutations. After photoreactivation of UVB-treated cells, the majority of mutations (59%) still were C to T transitions. CC to TT mutations were reduced to 4 of 108 (3.7%). Tandem changes still present after photoreactivation of CPDs often involved two base changes within a trinucleotide (e.g. CTC to TTT). It is unclear whether these mutations arose through dimerization of nonadjacent pyrimidines or other unknown photolesions. Although the frequency of mutations at some hot spots was reduced after photoreactivation (Fig. 5), mutations at other hot spot sites still remained.

                              
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Table I
Types of mutations induced in CPD photolyase expressing cells


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  		Fig. 5.   Mutation spectra of the CPD photolyase expressing cell line. Mutations were determined in cells not exposed to UV irradiation (lowercase letters below the sequence). Mutations produced by 500 J/m2 of UVB are shown as capital letters below the sequence. Mutations remaining after 500 J/m2 of UVB and enzymatic photoreactivation of CPDs are shown as capital letters above the sequence. Tandem mutations are underlined. Triangles, single base deletions (closed symbol in nonirradiated cells).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We established a mammalian mutagenesis assay system that can be used to dissect the biological roles of the two major DNA photoproducts produced by UVB irradiation, the CPDs and the (6-4)PPs. The rapid and almost complete repair of UV photoproducts indicates that most lesions are accessible and repaired by the foreign photolyases. Specific enzymatic removal of CPDs within cells resulted in a drastic reduction of the mutant frequency in two reporter genes. On the other hand, specific enzymatic removal of (6-4)PPs did not result in any appreciable change in the mutant frequency. These results are in line with previous data showing that photoreactivation repair of CPDs on UV-treated shuttle vectors lowers the mutation frequency (53-55). However, these studies did not examine the specific contribution from (6-4)PPs, and they did not analyze mutagenesis on a chromosomal gene. Using photoreactivation of UV-irradiated shuttle vectors with CPD photolyase or (6-4) photolyase, Otoshi et al. (56) showed that a substantial fraction (up to 50%) of the UV-induced mutations may have been derived from (6-4)PPs. This result is not quite consistent with our data and may have been due to the higher dose of 254 nm UVC irradiation (1000 J/m2) used in that study, which produces increased levels of (6-4)PPs (13), and to the use of an XP-A cell line. Another study using photoreactivation of CPDs and (6-4)PPs on a UV-irradiated supF shuttle vector found that both photoproducts are almost equally mutagenic in E. coli (57). This could be a consequence of the much faster cell division rate in E. coli as compared with mammalian cells, where most of the (6-4)PPs may be removed before DNA replication. After photoreactivation of CPDs, the remaining mutations were predominantly T to C transitions at 5'TT sites (57), a type of mutation that is rare in UVB-irradiated mammalian cells, even after photoreactivation of CPDs (Table I and Fig. 5).

The results presented here provide clear-cut evidence that the CPD is responsible for the majority of UVB-induced mutations in repair-proficient mammalian cells. Experiments with a cell line that contains a transfected CPD photolyase gene have shown that the CPD is not only responsible for mutagenesis but also for triggering UV-induced apoptosis in human cells (58).

We have deliberately chosen a repair-proficient cell line to dissect the mutagenicity of the two types of photolesions in a normal cellular context. It is possible that (6-4)PPs may contribute more significantly to UV mutagenesis in cells that are completely deficient in nucleotide excision repair (56). The lacI and cII transgenes are not transcribed in mouse cells. It has been shown that repair of CPDs is faster in a transcribed gene (59), and this could diminish their mutagenic effect in transcribed sequences. However, the (6-4)PPs are also repaired preferentially by transcription-coupled repair (60), and the relative contribution of the two lesions should remain the same. Although mouse cells are often considered defective in global genome repair of CPDs, the mouse cell line we used here is proficient in removing CPDs from the genome over a time period of 24-48 h (37). This efficiency almost approaches that of human cells.

In our experiments, the mutant frequency after photoreactivation of CPDs was not reduced to background but was reduced by 80-90% after subtraction of the background mutant frequency. There could be several reasons for this. First, the remaining mutations may have been produced by (6-4)PPs. This may be a likely explanation, but we caution that activation of the (6-4) photolyase did not lower the mutant frequency to any significant extent, in spite of efficient removal of (6-4)PPs from the genome (Fig. 3). The types of mutations in the absence or presence of CPD photoreactivation were quite similar (Fig. 5 and Table I). Some mutational hot spots occurred at similar sequence positions. However, tandem CC to TT mutations, considered a likely consequence of CPD photoproducts at 5'CC sites (27), were reduced almost 3-fold after photoreactivation (Table I). Certain mutations may have been caused by non-CPD and non-(6-4) lesions such as the base changes between nucleotides +106 and +109 (5'-GATA). These may involve a photoproduct that specifically forms at 5'-AT sequences (61). The most likely explanation for the nature of most mutations remaining after CPD photoreactivation may be the persistence of some CPD lesions during and after the time course of photoreactivation. If during the photoreactivation process some cells replicated their DNA and the replication fork encountered a nonrepaired CPD, one would expect this to generate a CPD-induced mutation. As discussed in the introduction, signatures of mutations produced by the (6-4)PP could be changes from 5'-TT to 5'-TC, 5'-CT to 5'-CC, and 5'-TC to 5'-TT. The first two changes are virtually absent from the UVB spectrum (Fig. 5; note that one 5'-TT to 5'-TC change at position +154 is also present in nonirradiated cells). 5'-TC to 5'-TT mutations are, however, present after photoreactivation of CPDs, and we cannot exclude the possibility that some of these may be derived from (6-4)PPs or their Dewar valence isomers. However, given the dramatic reduction of mutant frequencies after CPD photoreactivation, the quantitative contribution of (6-4)PPs to UVB-induced mutations can be only minor.

Our results are consistent with a model for mammalian UV mutagenesis that involves the CPD as the principal lesion. Overall, the mutation data are consistent with the higher levels of formation, slower repair, and increased mutagenicity of CPDs produced in mammalian cells by UV irradiation. In previous work, we found that the amounts of (6-4)PPs relative to CPDs were smallest when a solar UV simulator was used for irradiation as compared with other UV sources (13). This would indicate that the mutagenic contribution of (6-4)PPs produced by natural sunlight would be even less. The low mutagenicity of (6-4)PPs in mammalian cells may be a consequence of their low level of induction, efficient repair, and possibly also a bypass tolerance by DNA polymerase eta  for the most abundant (6-4)PP, the one which forms at 5'-TC sequences (13, 62, 63). DNA polymerase eta  preferentially incorporates a guanine opposite the 3' base of 5'-TT (6-4)PPs, although it is unable to extend from the inserted nucleotide (33). If, because of structural features of the lesion or because of deamination of the 3' base, this polymerase would have a similar incorporation specificity opposite the 3'-C of a 5'-TC (6-4)PP, then no mutation would be produced. This idea is consistent with the genetic data obtained by Yu et al. (22) in yeast.

What may be the mutagenic mechanism involving CPDs? TT dimers, although induced at high levels, are not very mutagenic, probably because of their correct replication bypass by DNA polymerase eta . CPDs containing cytosines, and in particular 5-methylcytosines (64), are also very abundant. We and others have proposed that most UV-induced transition mutations at dipyrimidines containing cytosine may result from correct DNA polymerase bypass of CPDs containing deaminated cytosine or 5-methylcytosine (27, 43, 65, 66). Deamination of cytosine or 5-methylcytosine may occur more rapidly within a CPD as opposed to within normal double-stranded DNA (67, 68). Deamination of C in TC or CC dimers leads to formation of TU or UU dimers, respectively. Adenines are incorporated with high specificity during bypass of site-specific TT, TU, or UU dimers in vivo (16, 65). After deamination of cytosine or 5-methylcytosine in CPDs, DNA polymerase eta  is probably bypassing these CPDs in an error-free manner (18, 19, 69). It is not known how these damage-tolerant DNA polymerases bypass CPDs containing nondeaminated cytosines or 5-methylcytosines. One possibility is that they incorporate adenines (70). However, our expectation is that they incorporate guanines in a mostly error-free pathway and that a mutation occurs only after deamination. There is in fact genetic evidence from studies in yeast that DNA polymerase eta  bypasses CPDs containing cytosine correctly (22). Deamination of cytosine and 5-methylcytosine in CPDs does occur at significant rates in vivo (27) and could contribute significantly to UVB mutagenesis in mammalian cells.

    ACKNOWLEDGEMENTS

We thank Aziz Sancar (University of North Carolina at Chapel Hill) for kindly providing E. coli photolyase, Stephen Lloyd (University of Texas Medical Branch, Galveston) for T4 endonuclease V, and Jun-ichi Miyazaki (Osaka University Medical School) for the pCY4B vector. Steven Bates is acknowledged for assistance with cell culture work.

    FOOTNOTES

* This work was supported by Grant ES06070 from the NIEHS, National Institutes of Health (to G. P. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Lawrence Berkeley National Laboratory, Berkeley, CA 94720.

|| To whom correspondence should be addressed. Tel.: 626-301-8853; Fax: 626-930-5366; E-mail: gpfeifer@coh.org.

Published, JBC Papers in Press, September 25, 2001, DOI 10.1074/jbc.M107696200

    ABBREVIATIONS

The abbreviations used are: CPD, cyclobutane pyrimidine dimer; (6-4)PP, pyrimidine (6-4) pyrimidone photoproduct; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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