Immunoprecipitation of pyrimidine(6-4)pyrimidone photoproducts and cyclobutane pyrimidine dimers in uv-irradiated DNA

Biological studies suggest that a significant proportion of the cytotoxicity observed in mammalian cells after uv irradiation may be due to damage other than cyclobutane dimers in DNA. Although pyrimidine-pyrimidone (6-4) photoproducts have been implicated as major contributors to cell lethality, their induction has been measured at considerably less than cyclobutane pyrimidine dimers when measured by chromatographic techniques. Because the yield of (6-4) photoproducts may be reduced by their lability to extreme heat and pH, we have advised an alternative, immunological quantification which does not require DNA hydrolysis. Affinity-purified rabbit antisera were used to precipitate low molecular weight 32P-labeled PM2 DNA irradiated with increasing fluences of uv light. DNA of known molecular weight was used to determine rates of induction for antibody-binding sites associated with (6-4) photoproducts and cyclobutane dimers. These rates were calculated to be 0.6 (6-4) photoproducts and 1.2 cyclobutane dimers/10(8) Da/J/m2. At low uv fluences (6-4) photoproducts were induced at one-half the rate of cyclobutane dimers, whereas at higher fluences (6-4) photoproducts predominated.     

Cyclobutane pyrimidine dimers are responsible for the vast majority of mutations induced by UVB irradiation in mammalian cells

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 x 10(-5) to 34 x 10(-5) (background, 8-10 x 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 x 10(-5) to 28 x 10(-5) (background, 6-10 x 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.     

Photoreactivation implicates cyclobutane dimers as the major promutagenic UVB lesions in yeast.

Previously we compared the mutational specificities of polychromatic UVB (285-320 nm) and UVC (254 nm) light in the SUP4-o gene of the yeast Saccharomyces cerevisiae. Striking similarities in the types and distributions of induced SUP4-o mutations were consistent with roles for cyclobutane dimers and pyrimidine(6-4)pyrimidone photoproducts in mutation induction by UVB. To assess the relative importance of cyclobutane dimers, we have now examined the effect of photoreactivation (PR), which specifically reverses these lesions, on UVB and UVC induction of SUP4-o mutations. PR reduced the frequencies of both UVB and UVC mutagenesis by approximately 75%. Collections of 138 and 158 SUP4-o mutants induced by treatment with UVB plus PR or UVC plus PR, respectively, were characterized by DNA sequencing and the results were compared to those for 208 UVB and 211 UVC-induced mutants analyzed earlier. PR decreased the frequency of UVB-induced G.C----A.T transitions by 85%, diminished the substitution frequencies at individual sites by 64% on average, and reduced the mutation frequencies at the five UVB hotspots by 87%. A more detailed examination revealed that the transition frequencies at the 3' base of 5'-TC-3' and 5'-CC-3' sequences were decreased by 90% and 72%, respectively. Finally, PR appeared to occur to the same extent on both the transcribed and non-transcribed strands of SUP4-o. Similar results were obtained for PR following UVC irradiation. Our findings indicate that cyclobutane dimers are responsible for the majority of UVB mutagenesis in yeast.     

UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells.

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.     

The role of the (6-4) photoproduct in ultraviolet light-induced transition mutations in E. coli

Available evidence rules out the possibility that cyclobutane dimers are the major premutagenic lesions responsible for point mutations at sites of adjacent pyrimidine residues in the experiment systems examined to date in sufficient detail, that is, UV-induced mutations in chromosome loci in E. coli and UV-induced mutations in the cI gene of phage lambda. However, it is likely that the major cytotoxic effects of UV irradiation can be attributed to the cyclobutane pyrimidine dimer, as these lesions occur at 10 times the frequency of other UV-induced photoproducts in the dose range of 0.1-100 J/m2. The evidence also suggests that cyclobutane pyrimidine dimers are the major lesions responsible for induction of the SOS response and that as such they play an important, though indirect role, in the formation of mutations in irradiated DNA. Cyclobutane dimers may also be the major lesions responsible for other types of UV-light-induced mutations such as deletions. None of the available evidence rules out (6-4) photoproducts as a major premutagenic lesion induced by UV irradiation using these experimental systems. On the contrary, the mutation spectrum induced both in the lacI gene and the cI gene of phage lambda is that predicted for mutations induced by (6-4) photoproducts. The observation that neither the premutagenic lesions nor the (6-4) photoproduct is subject to enzymatic photoreactivation also implies that the (6-4) photoproducts are premutagenic. As reviewed above, neither the photosensitization experiments nor the action spectrum of the (6-4) photoproducts rules out such a role. Might a lesion other than the (6-4) photoproduct be the major premutagenic lesion responsible for point mutations in these experimental systems? It cannot be ruled out that another as yet undefined minor photoproduct that occurs with the same sequence distribution specificity as that of the (6-4) photoproduct and that is also not subject to the reactivating treatments is more mutagenic than the (6-4) photoproduct itself. Candidates for such a lesion might include a photohydrate of the (6-4) photoproduct itself or as yet undefined photoproducts. However, we believe these alternative possibilities to be remote.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells

We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.     

T4 DNA polymerase (3''-5'') exonuclease, an enzyme for the detection and quantitation of stable DNA lesions: the ultraviolet light example.

Ultraviolet light irradiation of DNA results in the formation of two major types of photoproducts, cyclobutane dimers and 6-4' [pyrimidin-2'-one] -pyrimidine photoproducts. The enzyme T4 DNA polymerase possesses a 3' to 5' exonuclease activity and hydrolyzes both single and double stranded DNA in the absence of deoxynucleotide triphosphate substrates. Here we describe the use of T4 DNA polymerase associated exonuclease for the detection and quantitation of UV light-induced damage on both single and double stranded DNA. Hydrolysis of UV-irradiated single or double stranded DNA by the DNA polymerase associated exonuclease is quantitatively blocked by both cyclobutane dimers and (6-4) photoproducts. The enzyme terminates digestion of UV-irradiated DNA at the 3' pyrimidine of both cyclobutane dimers and (6-4) photoproducts. For a given photoproduct site, the induction of cyclobutane dimers was the same for both single and double stranded DNA. A similar relationship was also found for the induction of (6-4) photoproducts. These results suggest that the T4 DNA polymerase proofreading activity alone cannot remove these UV photoproducts present on DNA templates, but instead must function together with enzymes such as the T4 pyrimidine dimer-specific endonuclease in the repair of DNA photoproducts. The T4 DNA polymerase associated exonuclease should be useful for the analysis of a wide variety of bulky, stable DNA adducts.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation

DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.     

Excision Repair of Uv Radiation-Induced DNA Damage in Caenorhabditis Elegans

Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts [cyclobutane dimers and (6-4) photoproducts]. Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.     

UV-B-induced DNA damage and repair pathways in polar Pseudogymnoascus sp. from the Arctic and Antarctic regions and their effects on growth,pigmentation and conidiogenesis

Solar radiation regulates most biological activities on Earth. Prolonged exposure to solar UV radiation can cause deleterious effects by inducing two major types of DNA damage, namely, cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts. These lesions may be repaired by the photoreactivation (Phr) and nucleotide excision repair (NER) pathways; however, the principal UV-induced DNA repair pathway is not known in the fungal genus Pseudogymnoascus. In this study, we demonstrated that an unweighted UV-B dosage of 1.6 kJ m⁻² d⁻¹ significantly reduced fungal growth rates (by between 22% and 35%) and inhibited conidia production in a 10 d exposure. The comparison of two DNA repair conditions, light or dark, which respectively induced photoreactivation (Phr) and NER, showed that the UV-B-induced CPDs were repaired significantly more rapidly in light than in dark conditions. The expression levels of two DNA repair genes, RAD2 and PHR1 (encoding a protein in NER and Phr respectively), demonstrated that NER rather than Phr was primarily activated for repairing UV-B-induced DNA damage in these Pseudogymnoascus strains. In contrast, Phr was inhibited after exposure to UV-B radiation, suggesting that PHR1 may have other functional roles. We present the first study to examine the capability of the Arctic and Antarctic Pseudogymnoascus sp. to perform photoreactivation and/or NER via RT-qPCR approaches, and also clarify the effects of light on UV-B-induced DNA damage repair in vivo by quantifying cyclobutene pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts. Physiological response data, including relative growth rate, pigmentation and conidia production in these Pseudogymnoascus isolates exposed to UV-B radiation are also presented.     

Structural determination of the ultraviolet light-induced thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct.

Ultraviolet light induces damage to DNA, with the majority of the damage expressed as the formation of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts. The (6-4) photoproducts have been implicated as important UV light-induced premutagenic DNA lesions. The most abundant of the (6-4) products is the thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct, or TC (6-4) product. The structure of the TC (6-4) product was deduced by proton NMR, IR, and fast atom bombardment mass spectroscopy, and the product was found to differ from the previously described photoadduct, Thy(6-4)Pyo, by the presence of an amino group at the 5 position of the 5' pyrimidine. The implications of this structure on DNA base pairing and the induction of ultraviolet light-induced mutations are discussed.     

Nonrandom induction of pyrimidine-pyrimidone (6-4) photoproducts in ultraviolet-irradiated human chromatin

Radioimmunoassays that detect pyrimidine-pyrimidone (6-4) photoproducts and cyclobutane dimers were used to determine the relative induction of these photoproducts in nucleosomal (core) and internucleosomal (linker) DNA in human cell chromatin irradiated with UV light. Cyclobutane dimers were formed in equal amounts/nucleotide in core and linker DNA, whereas (6-4) photoproducts occurred with 6-fold greater frequency/nucleotide in linker DNA.     

Photoreactivation reverses ultraviolet radiation induced premutagenic lesions leading to frameshift mutations in Escherichia coli

Summary The effect of photoreactivation of the ultraviolet radiation induced reversion of a trpE9777 frameshift mutation was studied in a uvrA6 derivative of Escherichia coli K12. Two different photoreactivation treatments were used, one providing a single flash of photoreactivating light and another providing 10 min of light from fluorescent lamps. The reversion frequency of the trpE9777 frameshift mutation was strongly reduced when subsequently exposed to visible light. The dose modification factor (the ratio of equally effective doses), for cells challenged with single-flash photoreactivation, for survival and induction of reversion to Trp⁺ was 3.6 and 3.4, respectively. UV induction of RecA protein synthesis was not reversed by a single flash of photoreactivation. The dose modification factor for 10 min of fluorescent lamp photoreactivation for survival and for induction of reversion to Trp⁺ was 6.5 and 6.3, respectively. The dose modification factor for 10 min of photoreactivation for induction of RecA protein was 1.7–2.5. Photoreactivation decreased the reversion of trpE9777 and increased survival to the same extent. We concluded that cyclobutyl pyrimidine dimers are the premutagenic lesions of UV mutagenesis of the trpE9777 allele in a uvrA6 background.     

UV mutagenesis, cytotoxicity and split-dose recovery in a human-CHO cell hybrid having intermediate (6-4) photoproduct repair

Somatic cell hybrids constructed between UV-hypersensitive Chinese hamster ovary cell line UV20 and human lymphocytes were used to examine the influence of a human DNA repair gene, ERCC1, on UV photoproduct repair, mutability at several drug-resistance loci, UV cytotoxicity and UV split-dose recovery. In hybrid cell line 20HL21-4, which contains human chromosome 19, UV-induced mutagenesis at the APRT, HPRT and Na+/K+-ATPase loci was comparable to that in repair-proficient CHO AA8 cells, whereas cell line 20HL21-7, a reduced human-CHO hybrid not containing human chromosome 19, exhibited a hypermutable phenotype at all 3 loci indistinguishable from that of UV20 cells. The response of 20HL21-4 cells to UV cytotoxicity reflected substantial but incomplete restoration of wild-type UV cytotoxic response, whereas responses of UV20 and 20HL21-7 cell lines to UV cytotoxicity were essentially the same, reflecting several-fold UV hypersensitivity. Repair of UV-induced (5-6) cyclobutane dimers and (6-4) photoproducts was examined by radioimmunoassay; (6-4) photoproduct repair was deficient in UV20 and 20HL21-7 cell lines, and intermediate in 20HL21-4 cells relative to wild-type CHO AA8 cells. UV split-dose recovery in 20HL21-4 cells was also intermediate relative to AA8 cells. These results show that the human ERCC1 gene on chromosome 19 is responsible for substantial restoration of UV survival and mutation responses in repair-deficient UV20 cells, but only partially restores (6-4) UV photoproduct repair and UV split-dose recovery.     

Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts.

ABC excinuclease of Escherichia coli removes 6-4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion. We describe in this communication a method, which utilizes DNA photolyase from E. coli, pyrimidine dimer endonucleases from M. luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment. On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct. Both the nature of the adduct (6-4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease. We show directly that photolyase stimulates the removal of pyrimidine dimers (but not 6-4 photoproducts) by the excinuclease. Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.     

Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions

Bipyrimidine photoproducts induced in DNA by UVB radiation include cyclobutane dimers, (6-4) photoproducts, and their related Dewar valence isomers. Even though these lesions have been extensively studied, their rate of formation within DNA is still not known for each possible bipyrimidine site (TT, TC, CT, and CC). Using a method based on the coupling of liquid chromatography to mass spectrometry, we determined the distribution of the 12 possible bipyrimidine photoproducts within isolated and cellular DNA. TT and TC were found to be the most photoreactive sequences, whereas lower amounts of damage were produced at CT and CC sites. In addition to this quantitative aspect, sequence effects were observed on the relative yield of (6-4) adducts with respect to cyclobutane pyrimidine dimers. Another interesting result is the lack of formation of Dewar valence isomers in detectable amounts within the DNA of cells exposed to low doses of UVB radiation. The photoproduct distribution obtained does not fully correlate with the UV mutation spectrum. A major striking observation deals with the low yield of cytosine-cytosine photoproducts which are likely to be associated with the UV-specific CC to TT tandem mutation.     

Thermal resistance to photoreactivation of specific mutations potentiated in E. coli B/r ung by ultraviolet light

Summary Mutagenesis by ultraviolet light was studied in a strain of E. coli ung, which lacks uracil-DNA glycosylase activity. Mutation potentiated by UV in cells already induced by nalidixic acid treatment was still photoreversible suggesting that pyrimidine dimers act directly as premutational photoproducts. Secondly, irradiated cells were held in buffer at 48°C for 0 to 135 min to allow for deamination of cytosines in pyrimidine dimers. The mutation frequencies for class 2 de novo suppressor mutation, for class 2 converted suppressor mutation and for backmutation were individually determined, before and after photoreactivation, as a function of this thermal treatment. Backmutation remained sensitive to photoreactivation throughout the treatment but de novo and converted suppressor mutations rapidly developed resistance to photoreactivation. This resistance was not seen in an ung ⁺ control. A model is proposed to account for the selective resistance based on the hypothesis that class 2 de novo and converted suppressor mutations normally result from UV by GC to AT transitions at T=C dimers. The model describes deamination of the cytosine residues in these dimers to become uracil residues. In consequence, monomerization by photoreactivation in cells that can not repair uracils in DNA no longer reverses mutation and GC to AT transitions are established at the sites of uracils.     

Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scanning confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.     

Repair of (6-4)photoproducts correlates with split-dose recovery in UV-irradiated normal and hypersensitive rodent cells

Chinese hamster ovary cells and two UV-hypersensitive derivatives were used to determine the importance of DNA excision repair for split-dose recovery. In the wild-type cells 75% of the maximum theoretical recovery was observed when the fractions were delivered at 2-h intervals. Very little recovery was evident in the two hypersensitive cell lines. Using radioimmunoassays specific for (6-4)photoproducts and cyclobutane dimers, the ability of UV-irradiated repair-deficient cells representing 5 complementation groups to repair these 2 photoproducts was determined. Removal of antibody-binding sites specific for (6-4)photoproducts was 80% complete in 6 h and was defective in the UV-sensitive cells. In contrast, only 20-60% of antibody-binding sites specific for cyclobutane dimers were removed 18 h post-irradiation, and the extent of removal was the same in normal and defective cell lines. We conclude that repair of (6-4)photoproducts accounts for split-dose recovery. In addition, we conclude that a consequence of DNA repair in CHO cells is modification rather than removal of cyclobutane dimers.