Photochemistry orid Pliotobiology Vol. 54, No. 5, pp. 741-146. 1991 Printed in Great Britain. All rights reserved
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RELATIVE INDUCTION OF CYCLOBUTANE DIMERS AND CYTOSINE PHOTOHYDRATES IN DNA IRRADIATED in vitro AND in vivo WITH ULTRAVIOLETC AND ULTRAVIOLET-B LIGHT DAVID L. MITCHELL',JIN JEN' and JAMESE. CLEAVER'* 'University of Texas M.D. Anderson Cancer Center, Science ParkiResearch Division, Smithville, TX 78957. USA and 'Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143-0750, USA (Received 7 November 1990; accepted 12 April 1991)
DNA was irradiated in vitro and in vivo with UV-C (240-280 nm) and UV-B (28s320 nm) light, and damaged sites sensitive to digestion with Escherichia coli endonuclease 111 (endo 111) and bacteriophage T4 endonuclease V (endo V) were quantified. The frequency of endo 111-sensitive sites (primarily cytosine photohydrates) induced was 1-2% of the frequency of endo Vsensitive sites (cyclobutane dimers) in both purified SV40 DNA and intracellular episomal SV40 DNA. Endo 111- and endo V-sensitive sites in DNA were induced in the same relative proportion at both UV-C and UV-B wavelengths. We found no evidence to support earlier inferences that intracellular conditions enhance the formation of cytosine photohydrates or other monobasic forms of DNA damage.
Abstract-SV40
INTRODUCTION
There is some uncertainty about the rates of formation of various photoproducts in purified D N A and in intracellular D N A at various wavelengths of U V light. Using an indirect, degradative assay, Hariharan and Cerutti (1977) generated action spectra for the induction of thymine glycols in human cell D N A and calculated that these photoproducts were induced at only 6% of the rate of cyclobutane dimers at wavelengths < 280 nm, whereas they were inducted at nearly 75% of the rate of cyclobutane dimers at 313 nm. In contrast, subsequent analysis of putative thymine glycols with Escherichia coli endonuclease 111 (endo 1II)t showed low levels of induction relative to cyclobutane dimers in purified D N A irradiated with high fluences of UV-C and UV-B light (313 and 325 nm) (Demple and Linn, 1980). More recently, the wavelength dependence of endo 111-sensitive site induction at the sequence level showed a much reduced efficiency of photoproduct formation at and around 313 nm (Weiss and Duker, 1987; Doetsch et a / . , 1988). In both qualitative studies, the peak efficiency for induction of endo 111-sensitive sites occurred at -280 nm. These data suggest that the induction of pyrimidine photohydrates in D N A irradiated in vitro has a wavelength dependence resembling that of the major dimeric photoproducts, cyclobutane dimers and pyrimidine-pyrimidone[&4] photoproducts. *To whom correspondence should be addressed. +Ahhrevations: bp, base pairs; endo 111, Escherichia coli endonuclease 111; endo V, bacteriophage T4 endonuclease V.
Although procedural differences may account for the divergent results obtained by the acid-base degradation and enzymatic assays, cellular factors (e.g. sensitizers) could contribute to the enhanced induction of thymine photohydrates after UV-B irradiation in vivo. Until this issue is resolved, the biological importance of thymine hydrates (especially those resulting from solar radiation) remains an open question. Escherichia coli endo TI1 is a broadly specific enzyme that recognizes D N A damage produced by both ionizing and non-ionizing radiations as well as chemical oxidation (Radman, 1976; Gates and Linn, 1977). Activities of the purified enzyme include a D N A N-glycosylase that recognizes various D N A base modifications and a D N A apuriniciapyrimidinic endonuclease that cleaves resultant apyrimidinic sites by p-elimination leaving a modified terminal deoxyribose (Wallace, 1988). Endo I11 recognizes a variety of monobasic damage in D N A (and alternating copolymers) irradiated with high fluences of U V light, including 6-hydroxy-5,6dihydrocytosine and 6-hydroxy-5,6-dihydrouracil (Doetsch e t a / . , 1986; Boorstein ef al., 1989; Ganguly et al., 1990), cis and trans 6-hydroxy-5,6-dihydrothymine (Ganguly et al., 1990), and purine photoproducts occurring at guanine (Helland et al., 1986; Doetsch et al., 1988) and adenine (Doetsch et a/.. 1988). N o thymine glycol (5,6-dihydroxy-5,6dihydrothymine) was released by endo 111 from poly(dA-dT).poly(dA-dT) irradiated with high U V fluences (Ganguly et a / . , 1990). These lesions are presumably repaired in E. coli by endo I11 and in mammalian cells by enzymes with similar substrate
74 1
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DAVIDL.
MITCHELL
specificites (Higgins et al., 1987, Doetsch et af., 1987; Boorstein et al., 1989). Analysis of endo I11 cleavage patterns at the DNA sequence level has yielded variable results on the spectrum of monobasic damage induced by UV light. On the one hand, endo I11 recognizes cytosine damage exclusively in a UV-irradiated segment of the human alphoid sequence (Weiss and Duker, 1986; Gallagher et al., 1989); on the other hand, although cytosine photoproducts predominated as cleavage targets for a human redoxyendonuclease analogous to endo 111, a thymine cleavage site was a "hotspot" for photoproduct formation in a SaZI-PvuI restriction fragment from plasmid pUC18 (Doetsch et af., 1988). To further elucidate the significance of photohydrates in biological systems exposed to solar UV wavelengths to evalute the early results of Hariharan and Cerutti (1977), we have quantified the induction of endo 111-sensitive sites in the same DNA molecule (SV40) suspended in aqueous solution (in vitro) and occurring episomally in human cells (in vivo) after irradiation with UV-C and UVB light. MATERIALS AND METHODS
DNA substrates. For in vitro determinations, form I supercoiled SV40 DNA (5243 base pairs [bp]) was purchased from Bethesda Research Laboratories and suspended in 10 mM Tris HCI, 1 mM EDTA, pH 7.5, at a concentration of -50 pg/mL. To quantify endonucleasesensitive sites in SV40 DNA irradiated in vivo, we grew transformed human fibroblasts (GM637) that carry SV40 as an episome in modified Eagle's medium containing fetal calf serum (lo%), streptomycin, and penicillin. This SV40 molecule carries a small deletion (322 bp) in the early region resulting in a slightly reduced size (4921 bp) that permits the viral DNA to replicate episomally and to reach high copy number in confluent cells (104 viral DNA molecules per cell) (Cleaver et al., 1990). Cells were seeded at a density of lo7 cells per 100 mm plate in medium containing 0.15 pCi/mL [14C]thymidine (51.5 rnCiimmol) and allowed to grow for 3 additional days as confluent cells. Cultures were washed twice with ice-cold phosphate-buffered saline before irradiation and irradiated in 5 mL of this same buffer on a bed of ice water. Immediately after irradiation, cells were harvested by centrifugation. Supercoiled, episomal DNA was isolated by alkaline lysis and ion-exchange chromatography (Qiagen Corp.) (Lutze and Winegar, 1990). Ultraviolet irradiation. Ultraviolet-C (240-280 nm) irradiations were carried out with six 8 W General Electric germicidal lamps emitting predominantly 254 nm light at a fluence rate of 1.3 J/m*/s. Ultraviolet-B (280-320 nm) irradiations were carried out under four Westinghouse FS 20 sunlamps filtered through cellulose acetate (a gift of W. Carrier, Oak Ridge National Laboratory) emitting 300 nm light at 3.1 J/m*ls. These UV-B irradiation conditions have a lower wavelength cut-off of 295-300 nm and closely approximate those of the solar spectrum reaching the earth's surface (Caldwell et al., 1983). The fluence rates at 254 and 300 nm were determined with a Spectroline radiometer (Spectronics Corp., Westbury, NY) equipped with DM-254N and DM300N photodetectors. Enzyme digestion. Form I DNA that was UV-irradiated in aqueous solution or isolated from irradiated human cells was incubated with T4 endo V (a gift of S. Lloyd,
et a/.
Vanderbilt University) in 20 mM Tris, pH 8.0, 50 mM NaC1, 1 mM EDTA, 100 pg/mL bovine serum albumin, or with E. coli endo I11 (a gift of R. Cunningham, State University of New York, Albany) in 40 mM KH,PO,, pH 7.4, 1 mM EDTA, for 3 h at 37°C. Sample DNA (0.5 pg) was incubated with 3 units of T4 endo V for 3 h at 37°C (1 unit = amount of enzyme that incises 1 pg of DNA containing 25 cyclobutane dimers in 30 min at 37°C). Endo 111 was diluted 1/50; 1 pL was incubated with 0.5 pg DNA for 3 h at 37°C in 20 p L reaction volume. DNA preparations were not further purified before gel electrophoresis. Quantification of endonuclease-sensitive sites. After incubation, samples irradiated in vitro were mixed with loading buffer, and unnicked form I and nicked form I1 molecules were separated by electrophoresis on 0.8% neutral agarose gels in Tris borate buffer. After electrophoresis, the gels were stained with ethidium bromide and photographed with Polaroid Type 55 film. The negatives were scanned with an LKB 2222-020 Ultrascan XL laser densitometer (Pharmacia). Bands representing forms I and I1 DNA were analyzed, and the percentage of unnicked molecules remaining after UV irradiation and digestion with endo V or endo 111 was determined. The form I DNA was multiplied by 1.42 in these calculations to correct for topologically restricted reduction in ethidium bromide binding (Lloyd et al., 1978). For in vivo determinations, 14C-labeled form I and I1 bands were separated on 1.O% low-melting agarose (BRL), cut from the gel with a Nr.4 cork borer, and melted in 0.5 mL H,O in a microwave oven. The I4C in each band was quantified by liquid scintillation spectrometry, and the percentage of form I DNA in each sample was determined. On the assumption that photoproducts are distributed according to Poisson statistics, the UV fluence that reduces the number of form I molecules to 37% of the unirradiated control (D37) induces an average of one endonuclease-sensitive site per molecule (Table 1). RESULTS
Form I SV40 DNA that was irradiated with UVC light in vitro was converted to open circular form I1 DNA by digestion with endo V and endo 111 (Fig. 1). The loss of form I DNA with increasing UV fluences was exponential and can be analyzed by Poisson statistics. The D3, values calculated from these lines by linear regression indicated that, on average, one endo V-sensitive site (cyclobutane dimer) was induced per plasmid after 12.6 JimZUVC light measured at 254 nm. From the size of the plasmid and the D3, value, the induction rate was calculated to be one cyclobutane dimeri5243 bp x 660 Dai12.6 Jim2 or 229 cyclobutane dimersi10'" Da/J/m2. This value is in close agreement with similar determinations in other plasmids irradiated with UV-C light in vitro, such as pSV2catSVgpt (ProticSabljic and Kraemer, 1985), pRSVPgal (Mitchell et al., 1989), and pUC19 (Mitchell et al., 1990). Similar calculations showed that after in vitro irradiation with UV-C light, endo 111-sensitive sites were induced at a much reduced frequency (5.4 lesionsil0'" Da/J/m*) compared with cyclobutane dimers. Form I SV40 DNA that was irradiated with UV-B light in vitro was likewise converted to form I1 DNA by enzyme digestion (Fig. 2). Induction of both endo V- and endo 111-sensitive sites was
Cyclobutane dimers and photohydrates
743
Tablc 1. Photoproduct induction in purified and intraccllular SV40 DNA irradiated with UV-C or UV-B light
Irradiation conditions
In vitro
Light source
Enzyme
Endo I11
UV-C
Endo V UV-B
Endo Ill Endo V
In vivo
UV-C
Endo Ill Endo V
UV-B
Endo Ill Endo V
In vivoirn
Induction rate (breakdl 0"' Da/J/mZ)
D17 (J/m*)
531 (8)* 0.99827 12.6 (10) 0.9959 4376 (8) 0.9961 95 ( 5 ) 0.9900 1432 (12) 0.9678 13.7 (12) 0.9928 10,248 (15) 0.9197 111 (20) 0.9374
Endo IIIi Endo V
UV-B/ UV-C
0.024
0.122
0.022
0.133
0.010
0.139
~~~
vilro
~
Endo 111 Endo V
5.4 229
0.40
0.66 30.4
0.98
2.15
0.45
225 0.30
0.91
0.123 0.01 1
27.7
*Number in parcnthescs is number of data points used in linear rcgrcssion ?Correlation coefficient.
considerably lower than that observed for irradiation with UV-C light, but the ratio of endo IIIto endo V-sensitive sites was the same (Table 1). Induction of endo V- and endo 111-sensitive sites by
UV-C and UV-B irradiation of intracellular SV40 D N A in human cells gave results similar to those found in vitro (Figs. 3 and 4; Table 1). Sequence analysis of D N A strand scissions in a 450 bp hamster Alu I sequence (Fig. 5 ) indicated that modified cytosine residues were the primary 100
In Vitro
80
60 u)
-W3 V
2 4c I
-
c
LL 1
C
W
2 2c
L 0.2
0.4
0.6
0.0
'OO
UVC ( k J / m 2 )
Figure 1. Induction of endo V-sensitive sites (0)and endo 111-sensitive sites (0)in purified SV40 DNA irradiated with UV-C light. The loss of form I molecules by nicking with T4 endo V and E. coli endo I11 is shown for increasing fluences of UV-C light. D,, values for each type of photodamage were calculated from regression lines; correlation coefficients were 0.9959 ( N = 10) for endo V-sensitive sites and 0.9982 ( N = 8) for endo 111-sensitive sites.
1c
2
4 6 UVB (kJ/rn2)
8
Figure 2. Induction of endo V-sensitive sites (0)and endo in purified SVl0 DNA irradiated 111-sensitive sites (0) with UV-B light. D,, values were calculated as described in the legend to Fig. 1; correlation coefficients were 0.9900 ( N = 5 ) for endo V-sensitive sites and 0.9961 ( N = 8) for endo 111-sensitive sites.
DAVIDL. MITCHELL et al.
144
In Vivo
Endo V
d
”0
0.3
0.6
0.9
1.2
1.5
UVC ( k J / m 2 )
3
Figure 3. Induction of endo V-sensitive sites (0)and endo 111-sensitive sites (0)in SV40 episomal DNA purified from UV-C-irradiated human host cells. D,, values were calculated as described in the legend to Fig. 1; correlation coefficients were 0.9928 ( N = 12) for endo V-sensitive sites and 0.9678 ( N = 12) for endo 111-sensitive sites.
UV-induced photoproducts we could detect by endo 111. Strand breaks were not observed at thymine bases in UV-C-irradiated DNA after endo 111 digestion, even though this sequence contained three sites where thymine was flanked by purines at which monobasic thymine photoproducts could have been detected. Endo I11 does cleave thymine glycols in DNA treated with reducing agents such as hydrogen peroxide (Demple and Linn, 1982); hence, these lesions were not detectably induced by 1.5 kJ UVC light in our particular sequence. DISCUSSION
Previous reports indicated a significantly greater induction of thymine glycols (5,6-dihydroxydihydrothymine) relative to cyclobutane dimers after 313 nm irradiation of cellular DNA than after 254 nm irradiation (Hariharan and Cerutti, 1977). Sites sensitive to endo I11 were therefore expected to be detected in relatively greater proportions after irradiation by UV-B light than by UV-C, since thymine glycols are suitable enzyme substrates (Demple and Linn, 1982). Demple and Linn tested this hypothesis by irradiating purified PM2 supercoiled DNA with 254 nm germicidal and monochromatic 313 and 325 nm light, using enzyme digestion with T4 endo V and E. coli endo 111 to calculate rates of photoproduct induction. They concluded that after all exposures, the cyclobutane dimer “greatly predominated over 5,6-hydrated thymine”.
6
9
12
UVB ( k J l m 2 )
Figure 4. Induction of endo V-sensitive sites (0)and endo 111-sensitive sites (0)in SV40 episomal DNA purified from UV-B-irradiated human host cells. D,, values were calculated as described in the legend to Fig. 1; correlation coefficients were 0.9374 ( N = 20) for endo V-sensitive sites and 0.9197 ( N = 15) for endo 111-sensitive sites.
To resolve these disparate results, Demple (personal communication) suggested that sensitizers that specifically enhance induction of thymine hydrates in DNA may come into play during irradiation of cells. Sensitization of photohydrate induction by cellular factors does not, however, reconcile the results of Hariharan and Cerutti (1977) with subsequent data generated by us and others. Two major differences between our protocol and that of Hariharan and Cerutti are noteworthy: (1) A monochromatic 313 nm light was used as UV source in the earlier work, whereas in our studies a broad-spectrum UV-B light source with a wavelength cutoff >295 nm was used. Mechanisms of DNA damage induction by wavelengths 310 nm (Doetsch et al., 1988; Gallagher et al., 1989). (2) The earlier work used an acid-base degradation assay to quantify thymine photohydrates in DNA; we used a well-characterized enzyme (endo 111) to measure these photoproducts. It is conceivable that thymine photoproducts other than photohydrates, which have been shown to occur with enhanced frequency by wavelengths between 310 and 320 nm, were detected by the acid-base degra-
Cyclobutane dimers and photohydrates
dation assay. One such candidate would be the Dewar photoisomer of the pyrimidine-pyrimidone ( 6 4 ) photoproduct, which may occur at a greater frequency than other dimeric photoproducts, such as the cyclobutane dimer, after high UV-B fluences (Mitchell and Clarkson, 1984; Taylor and Cohrs, 1987; Taylor, 1990). Considering the broad specificity of the acid-base degradation assay (Becker and Wang, 1984), the results of Hariharan and Cerutti (1977) may reflect the enhanced induction of Dewar pyrimidones, for instance, rather than thymine photohydrates, after high UV-B fluences. Using these broad-spectrum light sources, we have investigated the wavelength dependence of the induction of endo V- and endo 111-sensitivesites in DNA irradiated in vitro and in vivo. The ratio of both types of photodamage induced by UV-B and UV-C light was the same (i.e. UV-BIUV-C = 12-1470) whether the DNA was irradiated in aqueous solution or in human cells (Table 1). These results approximate those of Weiss and Duker (1987), who showed endo 111-sensitive site production by 313 nm monochromatic light to be about 10% of the frequency of that observed after 254 nm irradiation. Our experimental approach also allowed us to assess the effects of cellular shielding and absorption on the induction of endo 111-sensitive sites and cyclobutane dimers (Table 1). Endo 111-sensitive sites were induced in vivo at about one-half the frequency of those induced in vitro. This is consistent with previous measurements of endo V-sensitive sites in DNA irradiated in vitro and in vivo; the induction of these lesions was 25-50% lower in cellular DNA than in purified DNA (Lippke et a l . , 1981). Reduced photoproduct induction in vivo is presumed to result from shielding by cellular organelles and/or the presence of absorbing compounds within the irradiated cells. Surprisingly, endo V-sensitive site induction by UV-C and UVB light was the same in SV40 DNA irradiated either in vivo and in vitro. Although we cannot offer a conclusive explanation for these results, the biochemical milieu encompassing SV40 episomal DNA may be significantly different from that associated with genomic DNA and may affect the efficiency of photodamage induction. These results are complicated by the observation that the mitigation of DNA damage by cellular factors appears to depend on the type of damage induced. Sequence analysis of UV-C- and UV-B irradiated DNA indicates that the spectrum of damage recognized by endo 111 is dominated by cytosine photoproducts, with thymine photohydrates playing a secondary role (Weiss and Duker, 1986; Doetsch et al., 1988). In addition to cytosine photohydrates, recent data from our laboratory have identified a heat-stable endo 111-sensitive site occurring preferentially at the 3' cytosine of a (6-4) photoproduct (J. Jen, J.E. Cleaver, and D.L. Mitchell, manuscript
745
Figure 5. Incision of UV-C-irradiated DNA by E. coli endo 111. The DNA fragment was 3ZP-end-labeled at the Eco RI site, irradiated with 1.5 kJ UV-C light (lane 4) or unirradiated (lane 3), digested with endo 111, and analyzed on sequencing gels. Lane 1, A + G ; lane 2, C+T.
in preparation). Since these lesions occur infrequently relative to the major dimeric photoproducts (i.e. cyclobutane dimers and (6-4) photoproducts) after UV-B (solar) radiation, their importance as molecular determinants in disorders associated with exposure to sunlight may be minimal. However, to evaluate the effects of these lesions on such pathological endpoints as skin cancer and aging, it is necessary to assess their stability and
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DAVIDL. MITCHELL et al.
repair in normal and UV-hypersensitive cell lines, as well as their relative cytotoxicity and mutagenicity compared with other UV-B-induced photoproducts. Until the effects of these photoproducts on molecular processes such as DNA replication and gene transcription can be assessed, their ultimate influence on tumorigenesis and sunlight-induced human skin cancers remains unresolved. Acknowledgements-We thank Dr. Bruce Demple for helpful discussions and Drs. Steven Lloyd and Richard Cunningham for providing enzymes without which this study would have been impossible. In addition, we gratefully acknowledge the editorial advice of Mary McKenney. This research was supported in part by DLM’s appointment to the Alexander Hollaender Distinguished Postdoctoral Fellowship Program sponsored by the U.S. Department of Energy, Office of Health and Environmental Research, administered by the Oak Ridge Associated Universities, by U.S. Department of Energy contract DEAC03-76-SF01012, and by National Institutes of Health National Research Service Award 5-T32-ES07106 from the National Institute of Environmental Health Sciences. REFERENCES
Becker. M. M. and J. C. Wang (1984) Use of light for footprinting DNA in vivo. Nature 309, 682-687. Boorstein, R. J . , T. P. Hilbert, J. Cadet, R. P. Cunningham and G. W. Teebor (1989) UV-induced pyrimidine hydrates in DNA are repaired by bacterial and mamallian DNA glycosylase activities. Biochemistry 28, 6164-6 170. Caldwell, M. M., W. G. Gold, G. Harris and C. W. Ashurst (1983) A modulated lamp system for solar UVB (280-320 nm). Supplementation studies in the field. Photochem. Photobiol. 37, 47S485. Cleaver, J. E., R. Rose and D. L. Mitchell (1990) Replication of chromosomal and episomal DNA in X-raydamaged human cells: A cis- or trans-acting mechanism? Radiat. Res. 124, 294-299. Demple, B. and S. Linn (1980) DNA N-glycosylases and UV repair. Nature 287, 203-208. Demple, B. and S. Linn (1982) 5,6-Saturated thymine lesions in DNA: production by ultraviolet light or hydrogen peroxide. Nucl. Acids Res. 10, 3781-3789. Doetsch, P. W., D. E. Helland and W. A. Haseltine (1986) Mechanism of action of a mammalian DNA repair endonuclease. Biochemistry 25, 2212-2220. Doetsch, P. W., D. E. Helland and K. Lee (1988) Wavelength dependence for human redoxy-endonucleasemediated DNA cleavage at sites of UV-induced photoproducts. Radiat. Res. 113, 543-549. Doetsch, P. W., W. D . Henner, R. P. Cunningham, J. H. Toney and D. E. Helland (1987) A highly conserved endonuclease activity present in Escherichia coli, bovine, and human cells recognizes oxidative DNA damage at sites of pyrimidines. Mol. Cell. Biol. 7, 26-32. Gallagher, P. E., R. B. Weiss, T. P. Brent and N. J. Duker (1989) Wavelength dependence of DNA incision by a human ultraviolet endonuclease. Photochem. Photobiol. 49, 363-367. Ganguly, T., K. M. Weems and N. J. Duker (1990) Ultraviolet-induced thymine hydrates in DNA are excised by bacterial and human DNA glycosylase activities. Biochemistry 29, 7222-7228.
Gates, F. T., 111, and S. Linn (1977) Endonuclease from Escherichia coli that acts specifically upon duplex DNA damaged by ultraviolet light, osmium tetroxide, or Xrays. J. Biol. Chem. 252, 2802-2807. Hariharan, P. V. and P. A. Cerutti (1977) Formation of products of the 5,6-dihydroxydihydrothyminetype by ultraviolet light in HeLa cells. Biochemistry 16, 2791-2795. Helland, D. E., P. W. Doetsch and W. A. Haseltine (1986) Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage. Mol. Cell. Biol. 6, 1983-1990. Higgins, S. A., K. Frenkel, A. Cummings and G. W. Teebor (1987) Definitive characterization of human thymine glycol N-glycosylase activity. Biochemistry 26, 1683-1688. Lippke, J. A., L. K. Gordon, D. E. Brash and W. A. Haseltine (1981) Distribution of UV light-induced damage in a defined sequence of human DNA: detection of alkaline-sensitive lesions at pyrimidine nucleosidecytidine sequences. Proc. Natl. Acad. Sci. U.S.A. 78, 3388-3392. Lloyd, R. S . , C. W. Haidle and D. L. Robberson (1978) Bleomycin-specific fragmentation of double-stranded DNA. Biochemistry 17, 1890-1896. Lutze, L. H. and R. A. Winegar (1990) A quick and efficient method for the recovery of plasmid or viral DNA from mammalian cells. Nucleic Acids Res. 18, 6150. Mitchell, D. L., D. E. Brash and R. S. Nairn (1990) Rapid repair kinetics of pyrimidine(6-4)pyrimidone photoproducts in human cells are due to excision rather than conformational change. Nucl. Acids Res. 18, 963-971. Mitchell, D. L. and J . M. Clarkson (1984) Use of synthetic polynucleotides to characterise an antiserum made against UV-irradiated DNA. Photochem. Photobiol. 40, 743-748. Mitchell, D . L., J . E. Vaughan and R. S. Nairn (1989) Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA. Plasmid 21, 21-30. Protic-Sabljic, M. and K. H. Kraemer (1985) One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. Proc. Natl. Acad. Sci. U.S.A. 82, 6622-6626. Radman, M. (1976) An endonuclease from Escherichia coli that introduces single polynucleotide chain scissions in ultraviolet-irradiated DNA. J. Biol. Chem. 251, 1438-1445. Taylor, J . 4 . (1990) A combined chemical, physical and biological approach to obtaining precise DNA photoproduct structure-activity relationships (abstract), 18th Ann. Meeting Am. SOC. Photobiol., Vancouver, B.C., Canada, 1 6 2 0 June 1990, pp. 6Os-61s. Taylor, J.-S. and M. P. Cohrs (1987) DNA, light, and Dewar pyrimidinones: The structure and biological significance of TpT3. J . Am. Chem. SOC.109, 2834-2835. Wallace, S. S. (1988) AP endonucleases and DNA glycosylases that recognize oxidative DNA damage. Environ. Mol. Mutagen. 12, 431-477. Weiss, R. B. and N. J. Duker (1986) Photoalkylated DNA and ultraviolet-irradiated DNA are incised at cytosines by endonuclease 111. Nucl. Acids Res. 14, 6621-6631. Weiss, R. B. and N. J. Duker (1987) Endonucleolytic incision of UVB-irradiated DNA. Photochem. Phorobiol. 45, 763-768.
Copyright
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0 1991 Pcrgdmon
RELATIVE INDUCTION OF CYCLOBUTANE DIMERS AND CYTOSINE PHOTOHYDRATES IN DNA IRRADIATED in vitro AND in vivo WITH ULTRAVIOLETC AND ULTRAVIOLET-B LIGHT DAVID L. MITCHELL',JIN JEN' and JAMESE. CLEAVER'* 'University of Texas M.D. Anderson Cancer Center, Science ParkiResearch Division, Smithville, TX 78957. USA and 'Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143-0750, USA (Received 7 November 1990; accepted 12 April 1991)
DNA was irradiated in vitro and in vivo with UV-C (240-280 nm) and UV-B (28s320 nm) light, and damaged sites sensitive to digestion with Escherichia coli endonuclease 111 (endo 111) and bacteriophage T4 endonuclease V (endo V) were quantified. The frequency of endo 111-sensitive sites (primarily cytosine photohydrates) induced was 1-2% of the frequency of endo Vsensitive sites (cyclobutane dimers) in both purified SV40 DNA and intracellular episomal SV40 DNA. Endo 111- and endo V-sensitive sites in DNA were induced in the same relative proportion at both UV-C and UV-B wavelengths. We found no evidence to support earlier inferences that intracellular conditions enhance the formation of cytosine photohydrates or other monobasic forms of DNA damage.
Abstract-SV40
INTRODUCTION
There is some uncertainty about the rates of formation of various photoproducts in purified D N A and in intracellular D N A at various wavelengths of U V light. Using an indirect, degradative assay, Hariharan and Cerutti (1977) generated action spectra for the induction of thymine glycols in human cell D N A and calculated that these photoproducts were induced at only 6% of the rate of cyclobutane dimers at wavelengths < 280 nm, whereas they were inducted at nearly 75% of the rate of cyclobutane dimers at 313 nm. In contrast, subsequent analysis of putative thymine glycols with Escherichia coli endonuclease 111 (endo 1II)t showed low levels of induction relative to cyclobutane dimers in purified D N A irradiated with high fluences of UV-C and UV-B light (313 and 325 nm) (Demple and Linn, 1980). More recently, the wavelength dependence of endo 111-sensitive site induction at the sequence level showed a much reduced efficiency of photoproduct formation at and around 313 nm (Weiss and Duker, 1987; Doetsch et a / . , 1988). In both qualitative studies, the peak efficiency for induction of endo 111-sensitive sites occurred at -280 nm. These data suggest that the induction of pyrimidine photohydrates in D N A irradiated in vitro has a wavelength dependence resembling that of the major dimeric photoproducts, cyclobutane dimers and pyrimidine-pyrimidone[&4] photoproducts. *To whom correspondence should be addressed. +Ahhrevations: bp, base pairs; endo 111, Escherichia coli endonuclease 111; endo V, bacteriophage T4 endonuclease V.
Although procedural differences may account for the divergent results obtained by the acid-base degradation and enzymatic assays, cellular factors (e.g. sensitizers) could contribute to the enhanced induction of thymine photohydrates after UV-B irradiation in vivo. Until this issue is resolved, the biological importance of thymine hydrates (especially those resulting from solar radiation) remains an open question. Escherichia coli endo TI1 is a broadly specific enzyme that recognizes D N A damage produced by both ionizing and non-ionizing radiations as well as chemical oxidation (Radman, 1976; Gates and Linn, 1977). Activities of the purified enzyme include a D N A N-glycosylase that recognizes various D N A base modifications and a D N A apuriniciapyrimidinic endonuclease that cleaves resultant apyrimidinic sites by p-elimination leaving a modified terminal deoxyribose (Wallace, 1988). Endo I11 recognizes a variety of monobasic damage in D N A (and alternating copolymers) irradiated with high fluences of U V light, including 6-hydroxy-5,6dihydrocytosine and 6-hydroxy-5,6-dihydrouracil (Doetsch e t a / . , 1986; Boorstein ef al., 1989; Ganguly et al., 1990), cis and trans 6-hydroxy-5,6-dihydrothymine (Ganguly et al., 1990), and purine photoproducts occurring at guanine (Helland et al., 1986; Doetsch et al., 1988) and adenine (Doetsch et a/.. 1988). N o thymine glycol (5,6-dihydroxy-5,6dihydrothymine) was released by endo 111 from poly(dA-dT).poly(dA-dT) irradiated with high U V fluences (Ganguly et a / . , 1990). These lesions are presumably repaired in E. coli by endo I11 and in mammalian cells by enzymes with similar substrate
74 1
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DAVIDL.
MITCHELL
specificites (Higgins et al., 1987, Doetsch et af., 1987; Boorstein et al., 1989). Analysis of endo I11 cleavage patterns at the DNA sequence level has yielded variable results on the spectrum of monobasic damage induced by UV light. On the one hand, endo I11 recognizes cytosine damage exclusively in a UV-irradiated segment of the human alphoid sequence (Weiss and Duker, 1986; Gallagher et al., 1989); on the other hand, although cytosine photoproducts predominated as cleavage targets for a human redoxyendonuclease analogous to endo 111, a thymine cleavage site was a "hotspot" for photoproduct formation in a SaZI-PvuI restriction fragment from plasmid pUC18 (Doetsch et af., 1988). To further elucidate the significance of photohydrates in biological systems exposed to solar UV wavelengths to evalute the early results of Hariharan and Cerutti (1977), we have quantified the induction of endo 111-sensitive sites in the same DNA molecule (SV40) suspended in aqueous solution (in vitro) and occurring episomally in human cells (in vivo) after irradiation with UV-C and UVB light. MATERIALS AND METHODS
DNA substrates. For in vitro determinations, form I supercoiled SV40 DNA (5243 base pairs [bp]) was purchased from Bethesda Research Laboratories and suspended in 10 mM Tris HCI, 1 mM EDTA, pH 7.5, at a concentration of -50 pg/mL. To quantify endonucleasesensitive sites in SV40 DNA irradiated in vivo, we grew transformed human fibroblasts (GM637) that carry SV40 as an episome in modified Eagle's medium containing fetal calf serum (lo%), streptomycin, and penicillin. This SV40 molecule carries a small deletion (322 bp) in the early region resulting in a slightly reduced size (4921 bp) that permits the viral DNA to replicate episomally and to reach high copy number in confluent cells (104 viral DNA molecules per cell) (Cleaver et al., 1990). Cells were seeded at a density of lo7 cells per 100 mm plate in medium containing 0.15 pCi/mL [14C]thymidine (51.5 rnCiimmol) and allowed to grow for 3 additional days as confluent cells. Cultures were washed twice with ice-cold phosphate-buffered saline before irradiation and irradiated in 5 mL of this same buffer on a bed of ice water. Immediately after irradiation, cells were harvested by centrifugation. Supercoiled, episomal DNA was isolated by alkaline lysis and ion-exchange chromatography (Qiagen Corp.) (Lutze and Winegar, 1990). Ultraviolet irradiation. Ultraviolet-C (240-280 nm) irradiations were carried out with six 8 W General Electric germicidal lamps emitting predominantly 254 nm light at a fluence rate of 1.3 J/m*/s. Ultraviolet-B (280-320 nm) irradiations were carried out under four Westinghouse FS 20 sunlamps filtered through cellulose acetate (a gift of W. Carrier, Oak Ridge National Laboratory) emitting 300 nm light at 3.1 J/m*ls. These UV-B irradiation conditions have a lower wavelength cut-off of 295-300 nm and closely approximate those of the solar spectrum reaching the earth's surface (Caldwell et al., 1983). The fluence rates at 254 and 300 nm were determined with a Spectroline radiometer (Spectronics Corp., Westbury, NY) equipped with DM-254N and DM300N photodetectors. Enzyme digestion. Form I DNA that was UV-irradiated in aqueous solution or isolated from irradiated human cells was incubated with T4 endo V (a gift of S. Lloyd,
et a/.
Vanderbilt University) in 20 mM Tris, pH 8.0, 50 mM NaC1, 1 mM EDTA, 100 pg/mL bovine serum albumin, or with E. coli endo I11 (a gift of R. Cunningham, State University of New York, Albany) in 40 mM KH,PO,, pH 7.4, 1 mM EDTA, for 3 h at 37°C. Sample DNA (0.5 pg) was incubated with 3 units of T4 endo V for 3 h at 37°C (1 unit = amount of enzyme that incises 1 pg of DNA containing 25 cyclobutane dimers in 30 min at 37°C). Endo 111 was diluted 1/50; 1 pL was incubated with 0.5 pg DNA for 3 h at 37°C in 20 p L reaction volume. DNA preparations were not further purified before gel electrophoresis. Quantification of endonuclease-sensitive sites. After incubation, samples irradiated in vitro were mixed with loading buffer, and unnicked form I and nicked form I1 molecules were separated by electrophoresis on 0.8% neutral agarose gels in Tris borate buffer. After electrophoresis, the gels were stained with ethidium bromide and photographed with Polaroid Type 55 film. The negatives were scanned with an LKB 2222-020 Ultrascan XL laser densitometer (Pharmacia). Bands representing forms I and I1 DNA were analyzed, and the percentage of unnicked molecules remaining after UV irradiation and digestion with endo V or endo 111 was determined. The form I DNA was multiplied by 1.42 in these calculations to correct for topologically restricted reduction in ethidium bromide binding (Lloyd et al., 1978). For in vivo determinations, 14C-labeled form I and I1 bands were separated on 1.O% low-melting agarose (BRL), cut from the gel with a Nr.4 cork borer, and melted in 0.5 mL H,O in a microwave oven. The I4C in each band was quantified by liquid scintillation spectrometry, and the percentage of form I DNA in each sample was determined. On the assumption that photoproducts are distributed according to Poisson statistics, the UV fluence that reduces the number of form I molecules to 37% of the unirradiated control (D37) induces an average of one endonuclease-sensitive site per molecule (Table 1). RESULTS
Form I SV40 DNA that was irradiated with UVC light in vitro was converted to open circular form I1 DNA by digestion with endo V and endo 111 (Fig. 1). The loss of form I DNA with increasing UV fluences was exponential and can be analyzed by Poisson statistics. The D3, values calculated from these lines by linear regression indicated that, on average, one endo V-sensitive site (cyclobutane dimer) was induced per plasmid after 12.6 JimZUVC light measured at 254 nm. From the size of the plasmid and the D3, value, the induction rate was calculated to be one cyclobutane dimeri5243 bp x 660 Dai12.6 Jim2 or 229 cyclobutane dimersi10'" Da/J/m2. This value is in close agreement with similar determinations in other plasmids irradiated with UV-C light in vitro, such as pSV2catSVgpt (ProticSabljic and Kraemer, 1985), pRSVPgal (Mitchell et al., 1989), and pUC19 (Mitchell et al., 1990). Similar calculations showed that after in vitro irradiation with UV-C light, endo 111-sensitive sites were induced at a much reduced frequency (5.4 lesionsil0'" Da/J/m*) compared with cyclobutane dimers. Form I SV40 DNA that was irradiated with UV-B light in vitro was likewise converted to form I1 DNA by enzyme digestion (Fig. 2). Induction of both endo V- and endo 111-sensitive sites was
Cyclobutane dimers and photohydrates
743
Tablc 1. Photoproduct induction in purified and intraccllular SV40 DNA irradiated with UV-C or UV-B light
Irradiation conditions
In vitro
Light source
Enzyme
Endo I11
UV-C
Endo V UV-B
Endo Ill Endo V
In vivo
UV-C
Endo Ill Endo V
UV-B
Endo Ill Endo V
In vivoirn
Induction rate (breakdl 0"' Da/J/mZ)
D17 (J/m*)
531 (8)* 0.99827 12.6 (10) 0.9959 4376 (8) 0.9961 95 ( 5 ) 0.9900 1432 (12) 0.9678 13.7 (12) 0.9928 10,248 (15) 0.9197 111 (20) 0.9374
Endo IIIi Endo V
UV-B/ UV-C
0.024
0.122
0.022
0.133
0.010
0.139
~~~
vilro
~
Endo 111 Endo V
5.4 229
0.40
0.66 30.4
0.98
2.15
0.45
225 0.30
0.91
0.123 0.01 1
27.7
*Number in parcnthescs is number of data points used in linear rcgrcssion ?Correlation coefficient.
considerably lower than that observed for irradiation with UV-C light, but the ratio of endo IIIto endo V-sensitive sites was the same (Table 1). Induction of endo V- and endo 111-sensitive sites by
UV-C and UV-B irradiation of intracellular SV40 D N A in human cells gave results similar to those found in vitro (Figs. 3 and 4; Table 1). Sequence analysis of D N A strand scissions in a 450 bp hamster Alu I sequence (Fig. 5 ) indicated that modified cytosine residues were the primary 100
In Vitro
80
60 u)
-W3 V
2 4c I
-
c
LL 1
C
W
2 2c
L 0.2
0.4
0.6
0.0
'OO
UVC ( k J / m 2 )
Figure 1. Induction of endo V-sensitive sites (0)and endo 111-sensitive sites (0)in purified SV40 DNA irradiated with UV-C light. The loss of form I molecules by nicking with T4 endo V and E. coli endo I11 is shown for increasing fluences of UV-C light. D,, values for each type of photodamage were calculated from regression lines; correlation coefficients were 0.9959 ( N = 10) for endo V-sensitive sites and 0.9982 ( N = 8) for endo 111-sensitive sites.
1c
2
4 6 UVB (kJ/rn2)
8
Figure 2. Induction of endo V-sensitive sites (0)and endo in purified SVl0 DNA irradiated 111-sensitive sites (0) with UV-B light. D,, values were calculated as described in the legend to Fig. 1; correlation coefficients were 0.9900 ( N = 5 ) for endo V-sensitive sites and 0.9961 ( N = 8) for endo 111-sensitive sites.
DAVIDL. MITCHELL et al.
144
In Vivo
Endo V
d
”0
0.3
0.6
0.9
1.2
1.5
UVC ( k J / m 2 )
3
Figure 3. Induction of endo V-sensitive sites (0)and endo 111-sensitive sites (0)in SV40 episomal DNA purified from UV-C-irradiated human host cells. D,, values were calculated as described in the legend to Fig. 1; correlation coefficients were 0.9928 ( N = 12) for endo V-sensitive sites and 0.9678 ( N = 12) for endo 111-sensitive sites.
UV-induced photoproducts we could detect by endo 111. Strand breaks were not observed at thymine bases in UV-C-irradiated DNA after endo 111 digestion, even though this sequence contained three sites where thymine was flanked by purines at which monobasic thymine photoproducts could have been detected. Endo I11 does cleave thymine glycols in DNA treated with reducing agents such as hydrogen peroxide (Demple and Linn, 1982); hence, these lesions were not detectably induced by 1.5 kJ UVC light in our particular sequence. DISCUSSION
Previous reports indicated a significantly greater induction of thymine glycols (5,6-dihydroxydihydrothymine) relative to cyclobutane dimers after 313 nm irradiation of cellular DNA than after 254 nm irradiation (Hariharan and Cerutti, 1977). Sites sensitive to endo I11 were therefore expected to be detected in relatively greater proportions after irradiation by UV-B light than by UV-C, since thymine glycols are suitable enzyme substrates (Demple and Linn, 1982). Demple and Linn tested this hypothesis by irradiating purified PM2 supercoiled DNA with 254 nm germicidal and monochromatic 313 and 325 nm light, using enzyme digestion with T4 endo V and E. coli endo 111 to calculate rates of photoproduct induction. They concluded that after all exposures, the cyclobutane dimer “greatly predominated over 5,6-hydrated thymine”.
6
9
12
UVB ( k J l m 2 )
Figure 4. Induction of endo V-sensitive sites (0)and endo 111-sensitive sites (0)in SV40 episomal DNA purified from UV-B-irradiated human host cells. D,, values were calculated as described in the legend to Fig. 1; correlation coefficients were 0.9374 ( N = 20) for endo V-sensitive sites and 0.9197 ( N = 15) for endo 111-sensitive sites.
To resolve these disparate results, Demple (personal communication) suggested that sensitizers that specifically enhance induction of thymine hydrates in DNA may come into play during irradiation of cells. Sensitization of photohydrate induction by cellular factors does not, however, reconcile the results of Hariharan and Cerutti (1977) with subsequent data generated by us and others. Two major differences between our protocol and that of Hariharan and Cerutti are noteworthy: (1) A monochromatic 313 nm light was used as UV source in the earlier work, whereas in our studies a broad-spectrum UV-B light source with a wavelength cutoff >295 nm was used. Mechanisms of DNA damage induction by wavelengths 310 nm (Doetsch et al., 1988; Gallagher et al., 1989). (2) The earlier work used an acid-base degradation assay to quantify thymine photohydrates in DNA; we used a well-characterized enzyme (endo 111) to measure these photoproducts. It is conceivable that thymine photoproducts other than photohydrates, which have been shown to occur with enhanced frequency by wavelengths between 310 and 320 nm, were detected by the acid-base degra-
Cyclobutane dimers and photohydrates
dation assay. One such candidate would be the Dewar photoisomer of the pyrimidine-pyrimidone ( 6 4 ) photoproduct, which may occur at a greater frequency than other dimeric photoproducts, such as the cyclobutane dimer, after high UV-B fluences (Mitchell and Clarkson, 1984; Taylor and Cohrs, 1987; Taylor, 1990). Considering the broad specificity of the acid-base degradation assay (Becker and Wang, 1984), the results of Hariharan and Cerutti (1977) may reflect the enhanced induction of Dewar pyrimidones, for instance, rather than thymine photohydrates, after high UV-B fluences. Using these broad-spectrum light sources, we have investigated the wavelength dependence of the induction of endo V- and endo 111-sensitivesites in DNA irradiated in vitro and in vivo. The ratio of both types of photodamage induced by UV-B and UV-C light was the same (i.e. UV-BIUV-C = 12-1470) whether the DNA was irradiated in aqueous solution or in human cells (Table 1). These results approximate those of Weiss and Duker (1987), who showed endo 111-sensitive site production by 313 nm monochromatic light to be about 10% of the frequency of that observed after 254 nm irradiation. Our experimental approach also allowed us to assess the effects of cellular shielding and absorption on the induction of endo 111-sensitive sites and cyclobutane dimers (Table 1). Endo 111-sensitive sites were induced in vivo at about one-half the frequency of those induced in vitro. This is consistent with previous measurements of endo V-sensitive sites in DNA irradiated in vitro and in vivo; the induction of these lesions was 25-50% lower in cellular DNA than in purified DNA (Lippke et a l . , 1981). Reduced photoproduct induction in vivo is presumed to result from shielding by cellular organelles and/or the presence of absorbing compounds within the irradiated cells. Surprisingly, endo V-sensitive site induction by UV-C and UVB light was the same in SV40 DNA irradiated either in vivo and in vitro. Although we cannot offer a conclusive explanation for these results, the biochemical milieu encompassing SV40 episomal DNA may be significantly different from that associated with genomic DNA and may affect the efficiency of photodamage induction. These results are complicated by the observation that the mitigation of DNA damage by cellular factors appears to depend on the type of damage induced. Sequence analysis of UV-C- and UV-B irradiated DNA indicates that the spectrum of damage recognized by endo 111 is dominated by cytosine photoproducts, with thymine photohydrates playing a secondary role (Weiss and Duker, 1986; Doetsch et al., 1988). In addition to cytosine photohydrates, recent data from our laboratory have identified a heat-stable endo 111-sensitive site occurring preferentially at the 3' cytosine of a (6-4) photoproduct (J. Jen, J.E. Cleaver, and D.L. Mitchell, manuscript
745
Figure 5. Incision of UV-C-irradiated DNA by E. coli endo 111. The DNA fragment was 3ZP-end-labeled at the Eco RI site, irradiated with 1.5 kJ UV-C light (lane 4) or unirradiated (lane 3), digested with endo 111, and analyzed on sequencing gels. Lane 1, A + G ; lane 2, C+T.
in preparation). Since these lesions occur infrequently relative to the major dimeric photoproducts (i.e. cyclobutane dimers and (6-4) photoproducts) after UV-B (solar) radiation, their importance as molecular determinants in disorders associated with exposure to sunlight may be minimal. However, to evaluate the effects of these lesions on such pathological endpoints as skin cancer and aging, it is necessary to assess their stability and
746
DAVIDL. MITCHELL et al.
repair in normal and UV-hypersensitive cell lines, as well as their relative cytotoxicity and mutagenicity compared with other UV-B-induced photoproducts. Until the effects of these photoproducts on molecular processes such as DNA replication and gene transcription can be assessed, their ultimate influence on tumorigenesis and sunlight-induced human skin cancers remains unresolved. Acknowledgements-We thank Dr. Bruce Demple for helpful discussions and Drs. Steven Lloyd and Richard Cunningham for providing enzymes without which this study would have been impossible. In addition, we gratefully acknowledge the editorial advice of Mary McKenney. This research was supported in part by DLM’s appointment to the Alexander Hollaender Distinguished Postdoctoral Fellowship Program sponsored by the U.S. Department of Energy, Office of Health and Environmental Research, administered by the Oak Ridge Associated Universities, by U.S. Department of Energy contract DEAC03-76-SF01012, and by National Institutes of Health National Research Service Award 5-T32-ES07106 from the National Institute of Environmental Health Sciences. REFERENCES
Becker. M. M. and J. C. Wang (1984) Use of light for footprinting DNA in vivo. Nature 309, 682-687. Boorstein, R. J . , T. P. Hilbert, J. Cadet, R. P. Cunningham and G. W. Teebor (1989) UV-induced pyrimidine hydrates in DNA are repaired by bacterial and mamallian DNA glycosylase activities. Biochemistry 28, 6164-6 170. Caldwell, M. M., W. G. Gold, G. Harris and C. W. Ashurst (1983) A modulated lamp system for solar UVB (280-320 nm). Supplementation studies in the field. Photochem. Photobiol. 37, 47S485. Cleaver, J. E., R. Rose and D. L. Mitchell (1990) Replication of chromosomal and episomal DNA in X-raydamaged human cells: A cis- or trans-acting mechanism? Radiat. Res. 124, 294-299. Demple, B. and S. Linn (1980) DNA N-glycosylases and UV repair. Nature 287, 203-208. Demple, B. and S. Linn (1982) 5,6-Saturated thymine lesions in DNA: production by ultraviolet light or hydrogen peroxide. Nucl. Acids Res. 10, 3781-3789. Doetsch, P. W., D. E. Helland and W. A. Haseltine (1986) Mechanism of action of a mammalian DNA repair endonuclease. Biochemistry 25, 2212-2220. Doetsch, P. W., D. E. Helland and K. Lee (1988) Wavelength dependence for human redoxy-endonucleasemediated DNA cleavage at sites of UV-induced photoproducts. Radiat. Res. 113, 543-549. Doetsch, P. W., W. D . Henner, R. P. Cunningham, J. H. Toney and D. E. Helland (1987) A highly conserved endonuclease activity present in Escherichia coli, bovine, and human cells recognizes oxidative DNA damage at sites of pyrimidines. Mol. Cell. Biol. 7, 26-32. Gallagher, P. E., R. B. Weiss, T. P. Brent and N. J. Duker (1989) Wavelength dependence of DNA incision by a human ultraviolet endonuclease. Photochem. Photobiol. 49, 363-367. Ganguly, T., K. M. Weems and N. J. Duker (1990) Ultraviolet-induced thymine hydrates in DNA are excised by bacterial and human DNA glycosylase activities. Biochemistry 29, 7222-7228.
Gates, F. T., 111, and S. Linn (1977) Endonuclease from Escherichia coli that acts specifically upon duplex DNA damaged by ultraviolet light, osmium tetroxide, or Xrays. J. Biol. Chem. 252, 2802-2807. Hariharan, P. V. and P. A. Cerutti (1977) Formation of products of the 5,6-dihydroxydihydrothyminetype by ultraviolet light in HeLa cells. Biochemistry 16, 2791-2795. Helland, D. E., P. W. Doetsch and W. A. Haseltine (1986) Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage. Mol. Cell. Biol. 6, 1983-1990. Higgins, S. A., K. Frenkel, A. Cummings and G. W. Teebor (1987) Definitive characterization of human thymine glycol N-glycosylase activity. Biochemistry 26, 1683-1688. Lippke, J. A., L. K. Gordon, D. E. Brash and W. A. Haseltine (1981) Distribution of UV light-induced damage in a defined sequence of human DNA: detection of alkaline-sensitive lesions at pyrimidine nucleosidecytidine sequences. Proc. Natl. Acad. Sci. U.S.A. 78, 3388-3392. Lloyd, R. S . , C. W. Haidle and D. L. Robberson (1978) Bleomycin-specific fragmentation of double-stranded DNA. Biochemistry 17, 1890-1896. Lutze, L. H. and R. A. Winegar (1990) A quick and efficient method for the recovery of plasmid or viral DNA from mammalian cells. Nucleic Acids Res. 18, 6150. Mitchell, D. L., D. E. Brash and R. S. Nairn (1990) Rapid repair kinetics of pyrimidine(6-4)pyrimidone photoproducts in human cells are due to excision rather than conformational change. Nucl. Acids Res. 18, 963-971. Mitchell, D. L. and J . M. Clarkson (1984) Use of synthetic polynucleotides to characterise an antiserum made against UV-irradiated DNA. Photochem. Photobiol. 40, 743-748. Mitchell, D . L., J . E. Vaughan and R. S. Nairn (1989) Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA. Plasmid 21, 21-30. Protic-Sabljic, M. and K. H. Kraemer (1985) One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. Proc. Natl. Acad. Sci. U.S.A. 82, 6622-6626. Radman, M. (1976) An endonuclease from Escherichia coli that introduces single polynucleotide chain scissions in ultraviolet-irradiated DNA. J. Biol. Chem. 251, 1438-1445. Taylor, J . 4 . (1990) A combined chemical, physical and biological approach to obtaining precise DNA photoproduct structure-activity relationships (abstract), 18th Ann. Meeting Am. SOC. Photobiol., Vancouver, B.C., Canada, 1 6 2 0 June 1990, pp. 6Os-61s. Taylor, J.-S. and M. P. Cohrs (1987) DNA, light, and Dewar pyrimidinones: The structure and biological significance of TpT3. J . Am. Chem. SOC.109, 2834-2835. Wallace, S. S. (1988) AP endonucleases and DNA glycosylases that recognize oxidative DNA damage. Environ. Mol. Mutagen. 12, 431-477. Weiss, R. B. and N. J. Duker (1986) Photoalkylated DNA and ultraviolet-irradiated DNA are incised at cytosines by endonuclease 111. Nucl. Acids Res. 14, 6621-6631. Weiss, R. B. and N. J. Duker (1987) Endonucleolytic incision of UVB-irradiated DNA. Photochem. Phorobiol. 45, 763-768.
