257
Mutation Research, DNA Repair, 255 (1991) 257-264
© 1991 ElsevierScience Publishers B.V. All rights reserved 0921-8777/91/$03.50 ADONIS 092187779100101Q MUTDNA 06464
Effect of U V light on sister-chromatid exchanges, activation of alternative sites of replicon initiation and thymidine incorporation in CHO AA8, UV61 and UV5 cells Sharon A. Taft, Su Y. Ling and T. Daniel Griffiths Department of Biological Sciences and Plant Molecular Biology Center, Northern Illinois University, DeKalb, IL 60115 (U.S.A.)
(Received23 January 1991) (Revision received 13 May 1991) (Accepted 15 May 1991)
Keywords: DNA repair; DNA replication; SCE induction;CHO cells; UV; Thymidineincorporation
Summary The relative importance of the UV-induced pyrimidine(5-6)pyrimidine and the pyrimidine(6-4)pyrimidone lesions in sister-chromatid exchanges (SCEs), activation of alternative sites of replicon initiation and thymidine incorporation were examined using wild-type Chinese hamster ovary (CHO) AA8 cells which remove both lesions, mutant CHO UV61 cells which remove only the (6-4) lesion and mutant CHO UV5 cells which remove neither lesion. Our data suggest that both lesions play a role in each end point examined. The relative importance of these lesions is dependent on the end point studied as well as the fluence used. For SCE induction and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to play a predominant role, while for the thymidine incorporation studies the (6-4) lesion appears to play the predominant role at low fluences while the role of the (5-6) lesion increases at higher fluences.
There has been much debate concerning the relative roles of the two predominant UV-induced lesions, the pyrimidine(5-6)pyrimidine or (5-6) lesion, and the pyrimidine(6-4)pyrimidone or (6-4) lesion, in cytotoxicity, inhibition of DNA synthesis and mutation induction. Work performed with )t phage indicates that the (5-6) dimer can be lethal but it is not the principal
Correspondence: Dr. T. Daniel Griffiths,Professor, Department of Biological Sciences and Plant Molecular Biology Center, Northern Illinois University, DeKalb, IL 60115 (U.S.A.).
pre-mutagenic lesion (Wood, 1985). By using Escherichia coli photolyase in the presence of visible light, Wood was able to show that removal of the (5-6) dimer resulted in little change in the mutation frequency of the )t phage, but did increase the ability to form plaques. In E. coli, enzymatic photoreactivation (EPR) has been used to remove the (5-6) dimer and to observe the effect of this action on cell survival and mutagenesis. Brash et al. (1985) found that the (5-6) dimer is the principal cytotoxic lesion. They also determined that the (6-4) lesion is most likely the pre-mutagenic lesion. The (5-6) dimer appeared to correlate to mutation fie-
258 quency, but Brash et al. pointed out that this response is probably the result of decreased SOS induction resulting from the removal of the (5-6) dimer. Previously, Howard-Flanders et al. (1966) showed that E. coli mutants unable to remove the (5-6) lesion were hypersensitive to killing and mutation. These results may have been due to the induction of the SOS response, as suggested by Brash et al. Studies with lower eukaryotes containing the EPR system have focused on DNA synthesis and repair. Regan and Cook (1967) showed that E P R increased the growth of UV-irradiated fish cells and also reduced the extent of the UV-induced depression in the rate of DNA replication. This increase in growth rate and reduction in the depression of DNA replication did not return to control levels indicating that while the (5-6) lesion was important, other lesions were probably also important. Chick embryo cells showed little recovery of overall DNA synthetic rates after EPR (Lehmann and Stevens, 1975). Also, based on alkaline sucrose gradient studies, it was shown that EPR increased the size of newly synthesized DNA strands in UV-exposed cells, indicating that there were fewer gaps or there was less chain blockage. This result is similar to the results observed with photoreactivable insect cells. Styer et al. (1989) showed that the (5-6) dimer was the principal blocking lesion to DNA chain elongation in these insect cells. At fluences below 10 J / m 2, EPR removed most of the blocks. Above 10 J / m 2, EPR did not completely alleviate the blockage of fork progression. In addition, E P R did not completely reverse the UV-induced depression in the rate of DNA synthesis. Work completed with mammalian cells has been highly confusing. In shuttle vector DNA transfected into monkey cells, E P R by E. coli photolyase and visible light in vitro decreased the mutation rate by 80%. Interestingly, many of the mutations that remained after E P R were at sites where only (5-6) lesions would be expected to occur. This indicated that the (5-6) dimer was mutagenic in D N A replicated in monkey cells (Protie-Sabljic et al., 1986). Cleaver et at. (1987, 1988) showed that an XP 'revertant' which removes only the (6-4) lesion, exhibited normal
levels of survival and mutation suggesting that the (5-6) lesion is neither toxic nor mutagenic in human cells. In contrast, Broughton et al. (1990) recently came to the opposite conclusion. In that study, they reported that a class of trichothiodystrophy which removed the (5-6) but not the (6-4) lesion exhibited normal survival following exposure to UV. This difference might be explained by recent observations that the XP revertant removes (5-6) dimers from actively transcribed sequences (Lommel and Hanawalt, personal communication). Another study which examined mutations in a shuttle vector transfected into human cells reported that neither the (5-6) nor the (6-4) lesion frequencies correlated to mutation frequency even in the absence of excision repair (Brash et al., 1987). It was suggested that the mutation hot and cold spots observed were related to structural features of the DNA. Because of some of the confusion that has arisen from studies using EPR and because it has recently been claimed that in some cases EPR enhances indirectly the removal of lesions other than (5-6) cyclobutane dimers (Mitchell et al., 1990), we have been using UV61 cells, which remove only the (6-4) lesion (Thompson et al., 1989; Regan et al., 1990), to help elucidate the relative roles of both lesions. Previously, we (Griffiths et al., 1990) reported that although UV61 cells exhibited a more extensive blockage in DNA fork progression when compared to wild-type AA8 cells, the duration of blockage was shorter than that observed in UV5 cells which remove neither (5-6) nor (6-4) lesions. This suggested that while both lesions block DNA fork progression immediately after exposure, the (6-4) lesion is responsible for the prolonged blockage. In this study we wished to extend these studies to determine the role of the (6-4) lesion in sisterchromatid exchanges (SCEs) and the activation of alternative sites of replicon initiation. It has been proposed that since SCEs appear to be formed during the S phase, long-term blocks to replication are sites for SCEs (Painter, 1980). If this is the case, the (6-4) lesion, which we found to produce long-term replication blocks (Griffiths et al., 1990), should play a major role in UV-induced SCE induction. Likewise, we have observed a correlation between the extent and dura-
259 tion of blockage to DNA fork progression and the activation of alternative sites of replicon initiation (Griffiths and Ling, 1985, 1987, 1989). This would suggest that the (6-4) lesion should also play a major role in the activation of alternative sites of replicon initiation. Activation of alternative sites of replicon initiation allow blocking lesions which had been encountered by the leading strand, to be replicated by the lagging strand (Painter, 1985; Griffiths and Ling, 1985). It is thought that, during DNA replication, lesions encountered by the leading are capable of blocking fork progression, while lesions encountered by the lagging strand do not block replication (Meneghini et al., 1981; Berger and Edenberg, 1986). Materials and methods
Cell line maintenance The 3 Chinese hamster ovary (CHO) cell lines used in this study (AA8, UV61, and UV5) were kindly supplied by Dr. L.H. Thompson, Cells were routinely maintained in Falcon 75-cm 2 flasks. Cultures were grown in Ham's F10 supplemented with 10% calf serum, 5% fetal bovine serum and kanamycin (Gibco). Ceils were incubated at 37°C in a water-saturated environment with 5% CO 2 and were trypsinized (trypsinEDTA, 0.05%) every 3-4 days in order to maintain the cultures in a subconfluent state. Cells were routinely checked for contamination by mycoplasma using the Hoechst 33258 staining assay (Adams, 1980).
UV irradiation Prior to exposure, attached monolayer cultures were rinsed 2 times with sterile prewarmed phosphate-buffered saline (PBS). After adding approximately 1 ml of sterile prewarmed PBS, cells were sham exposed or exposed to the appropriate fluence. The source of radiation was 2 G15T8 germicidal lamps (General Electric) with a fluence rate of 0.1 W/s, as measured by a germicidal photometer (International Light Model IL 1500). Immediately following exposure, the PBS was removed and the appropriate amount of prewarmed medium was added to each dish.
Sister-chromatid exchanges Slide preparation. Approximately 24 h prior to exposure, cells were plated at a density of 7.5 x 105 cells/100-mm 2 dish. Following irradiation, medium containing 10 /~m 5-bromodeoxyuridine was added and the plates were incubated in the dark for 30 h. Chromosomes were then prepared according to Griffiths and Carpenter (1979). Differential staining. Slides were stained with 0.5/xg/ml Hoechst 33258 in 0.1 M sodium phosphate buffer (pH 6.8) for 20 rain and then rinsed with distilled water. PBS and a coverslip were placed on each slide prior to exposure to 366-nm light to fade the fluorescent Hoechst stain. After 10 rain, coverslips were removed and the slides were placed in 2 × SSC at 60 ° C for 60 min. After the slides were rinsed with distilled water, they were stained in 4% Giemsa for 20 rain and then rinsed and allowed to dry. The slides were scored for the number of SCEs. Only cells with 18-21 differentially stained chromosomes were used. The SCEs scored were reciprocal exchanges. DNA fiber autoradiography Slides were prepared according to Huberman and Riggs (1968) and Dahle et al. (1978). Approximately 2 x 104 cells were plated into each waxringed dish. 48 h after plating, the cells were rinsed with sterile prewarmed PBS, then sham or UV exposed. A high/low specific activity protocol was followed (Huberman and Riggs, 1968; Dahle et al., 1980). At 0, 2.5, or 5 h after exposure, high-specific-activity [3H]thymidine (3.7 MBq/ml; 3.11 TBq/mmole) was added to each dish for 25 rain and was then chased by a 25-rain pulse of unlabeled thymidine in sufficient amounts to reduce the specific activity to 0.62 TBq/mmole. Termination of labeling, transfer of cells to slides, lysis of cells and the addition of NTB3 (Kodak) photographic emulsion have been described previously (Griffiths and Ling, 1984, 1985). The slides were stored at 4°C for 6-9 months prior to development and fixation. The slides were scored using a single blind method. Replication segments (post-pulse figures) with dark, 'hot' centers and light, 'warm' trails were
260
used to measure the inter-origin distances. Measurements were taken from the middle of the dark area of one origin to the middle of the dark region in the adjacent origin using the Bioquant I1 system and digitizing pad (Griffiths and Ling, 1984, 1985). Data was analyzed using Student's t-test and the M a n n - W h i t n e y U-test.
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Thymidine incorporation assay Thymidine incorporation was measured according to Griffiths and Ling (1984, 1985). Triplicate cultures containing 3.5 x 10 4 cells were seeded into wax-ringed 35-mm ~ plates. The next day, cells were prelabeled with [14C]thymidine (1.85 kBq/ml; 1.85 M B q / m o l e ) in order to help detect and control for cell loss. Cells were exposed to UV 18-20 h later. At various times after exposure, [SH]thymidine (185 k B q / m l ; 3.7 T B q / m m o l e ) was added for 30 min. Labeling was terminated by rinsing the plates twice with 4°C PBS and then incubating the plates at 4°C for at least 2 h with 2% perchloric acid. Plates were rinsed with saline, and the cells were removed with a rubber policeman and vacuum filtered onto glass fiber filters. The filters were then rinsed with 95% ethanol, dried, and prepared for liquid scintillation counting. Results
Sister-chromatid exchanges Because SCEs are thought to arise from blockage of D N A replication (Painter, 1980), we were interested in determining whether there was a correlation between the extent of blockage induced by a lesion and its SCE potential. From our previous data (Griffiths et al., 1990), which showed that the (6-4) lesion produces more extensive and prolonged blockage than the (5-6) lesion, and from the initiation data presented below, we suspected that the (6-4) lesion was the lesion responsible for the majority of SCEs formed. Fig. 1 shows the results of SCE induction by UV light. AA8 cells showed a very small increase in SCEs over the range of fluences used. UV61 cells had more SCEs at each fluence compared to the AA8 cells, but SCE levels were much lower in UV61 ceils compared to UV5 cells suggesting
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that the (6-4) lesion is more efficient than the (5-6) lesion in producing SCEs. The plateau seen in UV5 cells was probably due to the inability to distinguish and count the total number of exchanges observed above 2.5 J / m e. The plateaus might also be due to our choice of sampling times. Specifically, at the higher fluences more heavily damaged cells might not have had time to reach the second mitosis by the time mitotic collections were made.
Actiuation of alternative sites of initiation As mentioned above, UV61 cells do not exhibit the extended blockage of fork progression that is observed in UV5 cells (Griffiths and Ling, 1985, 1987; Griffiths et al., 1990), which suggests that the (6-4) lesion is responsible for the extended blockage. In the past we observed a correlation between the extent of blockage of fork progression and the extent of activation of alternative sites of replicon initiation. Activation of alternative sites of initiation allows for the replication of D N A that contain blocking lesions (Painter, 1985; Griffiths and Ling, 1985). Thus, we next wished to determine if UV61 cells exhibited prolonged activation of alternative sites of initiation. Distances between adjacent sites of replicon initiation were visualized and measured through the use of DNA fiber autoradiography (Huberman and Riggs, 1968; Dahle et al., 1980; Griffiths and Ling, 1984). By using a high-specific-activity pulse and a chase with cold, unlabeled thymidine, the activation of alternative sites of initiation was
261
detected as a decrease in the inter-origin distance (IOD) of adjacent post-pulse figures. (Post-pulse figures have a dark center which represents incorporation that occurred during the incubation with the high-specific-activity label and low-density tails on either side which occurred during the chase with lower specific activity.) As is shown in Fig. 2, immediately after exposure to 5 J / m 2 or 10 J / m 2, AA8 cells showed a significant decrease in the IOD of UV-exposed cells. This indicated a significant activation of alternative sites of initiation. AA8 cells had recovered to control IOD by 2.5 or 5 h after either exposure, indicating that at these times alternative sites were no longer being activated. As is shown in Fig. 3, UV61 cells also exhibited a significant decrease in IOD levels immediately after exposure to 5 or 10 J / m 2. In contrast to the
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Fig. 2. Distribution of inter-origin distances in AA8 cells after exposure to 5 J / m 2 (left panel) or 10 J / m 2 (right panel). Cells were labeled for 25 min with high-specific-activity label followed by 25 min in low-specific-activity label as described in Materials and methods either immediately (top panel), 2.5 h (middle panel) or 5 h (lower panel) after exposure. The histograms for the control cells are represented by the hatched areas and are bounded by the dashed lines, while the histograms for treated cells are bounded by the solid lines.
results obtained for AA8 cells, however, UV61 cells still exhibited significant reduction in IOD values at both fluences 2.5 h after exposure. By 5 h after exposure, neither cell line exhibited a significant reduction of IOD values at either fluence. The extent of decrease in IODs at both fluences and at the 3 time intervals is summarized in Table 1. These results indicated that the (5-6) lesion could activate alternative sites of initiation up to 2.5 h after exposure. However, this lesion was not responsible for the extended activation (up to 5 h) observed previously in UV5 cells (Griffiths and Ling, 1985, 1987).
Thymidine incorporation Due to the fact that UV61 cells exhibited activation of alternative sites of replication 2.5 h
262 TABLE 1 E F F E C T O F U V ON T H E R E L A T I V E I N T E R - O R I G I N D I S T A N C E S IN AA8 A N D UV61 CELLS Time (h)
Fluence ( J / m 2)
Relative inter-origin distances ~' (/xm) AA8
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The relative inter-origin distances were obtained by dividing the values obtained in UV-exposed cells to the value obtained in sham-exposed cells. * Significant according to Student's t-test, P < 0.05.
ously concluded that this decrease in inter-origin distances represented an activation of alternative sites of replicon initiation (Griffiths and Ling, 1984, 1985, 1987). A second explanation is that UV irradiation selectively inhibits replicons with longer than average inter-origin distances. The results, shown in Fig. 4, suggest that selective inhibition is not likely since at 5 h after exposure of UV61 cells to 10 J / m 2 there is still an extensive depression in thymidine incorporation, yet there is no significant decrease in inter-origin distances. In addition, the extent of depression in UV5 cells is the same as that exhibited by the UV61 cells. This indicated that the (5-6) lesion was playing a greater role at higher fluences than was seen at lower fluences (Griffiths et al., 1990) in inhibiting the rate of thymidine incorporation.
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but not at 5 h after exposure to UV, we wanted to examine overall DNA synthesis at these times. In previous studies in repair-deficient cells there was a correlation between the extent of depression in D N A replication and the extent of decrease in inter-origin distances. We had previKinetics
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Discussion
In previous studies, either the (5-6) or (6-4) lesion has been implicated as having a predominant or sole role in various UV-induced end points (e.g., Brash et al., 1985; Cleaver et al., 1988; Broughton et al., 1990). The results reported here indicate that both the (5-6) and (6-4) lesions play a role in the effects of UV on DNA replication, SCE induction and the activation of alternative sites of replicon initiation. The results from the initiation studies with UV61 cells showed that the (5-6) lesion does play a role in the activation of alternative sites. However, this activation is not extended up to 5 h. UV5 cells were previously shown to exhibit significant activation at 2.5 and 5 J / m 2 (Griffiths and Ling, 1985, 1987) for up to 5 h after exposure. Thus, the prolonged activation of alternative sites observed in UV5 cells must be due to the (6-4) lesion. Our earlier work also showed that the (6-4) lesion is responsible for the prolonged blockage of fork progression observed in UV5 cells (Griffiths and Ling, 1984, 1985). Thus, there appears to be a strong correlation between the extent of D N A chain blockage and the activation of alternative sites of replicon initiation. The SCE results clearly indicate a greater role of the (6-4) lesion in the activation or induction of SCE events since the extent of UV-induced SCEs in the UV61 cell was closer to that exhib-
263 ited by AA8 cells. Similar results were observed by Cleaver et al. (1987) in XP variant revertants. The SCE data also correlates with the fiber data in which the (6-4) lesion apparently causes prolonged blockage of fork progression and activation of alternative sites of replication. These resuits support the theory of blocking lesions as instigators or sites of SCE induction (Painter, 1980), as both lesions can form blocks to D N A chain elongation (Styer et al., 1989; Griffiths and Ling, 1984, 1985; Griffiths et al., 1990). The greater role of (6-4) lesions in C H O cells appears to be due to the fact that they block replication for a longer period of time. If these lesions serve as sites for SCE formation, a recombinational exchange, they may also be stimuli for homologous recombination. It should be noted, however, that similar work with ICR-2A frog cells showed a decrease in SCEs after EPR, suggesting the (5-6) dimer is the critical lesion in SCE formation for these cells (Chao et al., 1985). One problem in comparing the relative roles of these two lesions is that the (5-6) lesion more abundant than the (6-4) lesion and that E P R may indirectly enhance the removal of (6-4) lesions (Mitchell et al., 1990). Previously, we (Griffiths et al. 1990) reported that after exposure to 2.5 J / m 2 the kinetics of thymidine incorporation in UV61 cells was close to that exhibited by AA8 cells and was quite different from that observed in UV5 cells. Specifically, UV5 cells never recovered normal rates of incorporation while AA8 cells and UV61 cells recovered normal rates within 5 and 7 h, respectively. Although the response of UV61 was still intermediate when the fluence was raised to 5 J / m 2, UV61 cells behaved more like UV5 cells. In the thymidine incorporation assay reported here, the UV61 curve was indistinguishable from the UV5 curve after exposure to 10 J / m 2. The increasing numbers of (5-6) lesions induced in the D N A at this fluence may increase the number of dimers becoming blocking lesions. It may become increasingly difficult for the UV61 cells (and to some extent AA8 cells) to complete D N A synthesis even with the activation of alternative sites of replicon initiation. Although the (6-4) lesion is being removed in the UV61 cells (Thompson et al., 1989), the sheer numbers of
(5-6) lesions may be perturbing the metabolic process that normally allows for bypass or modification of these lesions at low fluences. Thus, the relative roles of the (5-6) and (6-4) lesions appear not only to depend on the end point and organism studied, but also on the fluence employed. For SCE induction, and for the prolonged effects of U V light on D N A chain growth and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to be more important. For the effects of UV on D N A replication, both lesions a p p e a r to be important at high fluences (>/5 j / m 2 ) , while at lower fluences the (6-4) lesion appears to be more important in inhibiting thymidine incorporation.
Acknowledgements This work was supported by USPHS grant CA32579 awarded by the National Cancer Institute. The authors wish to thank Drs. Paul Meechan and Linda Yasui for their comments concerning this work.
References Adams, R.L.P. (1980) Cell Culture for Biochemists, Elsevier Science Publishers, New York, 292 pp. Berger, C.A., and H.J. Edenberg (1986) Pyrimidine dimers block simian virus 40 replication forks, Mol. Cell. Biol., 6, 3443-3450. Brash, D.E., W.A. Franklin, G.B. Sancar and W.A. Hazeltine (1985) Escherichia coli DNA photolyase reverses cyclobutane pyrimidine dimers but not pyrimidine-pyrimidone (6-4) photoproducts, J. Biol. Chem., 26, 1!438-11441. Brash, D.E., S. Seetharam, K.H. Kraemer, M.M. Seidman and A. Bredberg (1987) Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells, Proc. Natl. Acad. Sci. (U.S.A.), 84, 3782-3786. Broughton, B.C., A.R. Lehmann, S.A. Harcourt, C.E. Arlett, A. Sarasin, W.J. Klajer, F.A. Beemer, R. Nairen and D.L. Mitchell (1990) Relationship between pyrimidine dimers 6-4 photoproducts, repair synthesis and cell survival: Studies using patients with trichothiodystrophy, Mutation Res., 235, 33-40. Chao, C.C.-K., R.B. Rosenstein and B.S. Rosenstein (1985) Induction of sister-chromatid exchanges in ICR 2A frog cells exposed to 265-313 nm monochromatic ultraviolet wavelengths and photoreactivating light, Mutation Res., 149, 443-450. Cleaver, J.E., F. Cortes, L.. Lutze, W.F. Morgan, A.N. Player and D.L. Mitchell (1987) Unique repair properties of a
264 xeroderma pigmentosum revertant, Mol. Cell. Biol., 7, 3353-3357. Cleaver, J.E., F. Cortes, D. Karentz, L.H. Lutze, W.F. Morgan, A.N. Player, L. Vuksanovic and D.L. Mitchell (1988) The relative biological importance of cyclobutane and (6-4) pyrimidine-pyrimidone dimer photoproducts in human cells: evidence from a xeroderma pigmentosum revertant, Photochem. Photobiol., 48, 41-49. Dahle, D.B., T.D. Griffiths and J.G. Carpenter (1978) Inhibition of deoxyribonucleic acid synthesis and replicon elongation in mammalian cells exposed to methyl methanesulfonate, Mol. Pharmacol., 14, 278-289. Dahle, D., T.D. Griffiths and J.G. Carpenter (1980) Inhibition and recovery of DNA synthesis in UV-irradiated Chinese hamster V-79 cells, Photochem. Photobiol., 32, 157-165. Griffiths, T.D., and J.G. Carpenter (1979) Premature chromosome condensation following X-irradiation of mammalian cells: Expression time and dose response, Radiat. Res., 79, 187-202. Griffiths, T.D., and S.Y. Ling (1984) Effect of ultraviolet light on DNA replication in excision-deficient mammalian cells, Mutation Res., 132, 119-127. Griffiths, T.D., and S.Y. Ling (1985) Effect of ultraviolet light on thymidine incorporation, DNA chain elongation and replicon initiation in wild-type and excision-deficient Chinese hamster ovary cells, Biochim. Biophys. Acta, 826, 121-128. Griffiths, T.D., and S.Y. Ling (1987) Activation of alternative sites of replicon initiation in Chinese hamster cells exposed to ultraviolet light, Mutation Res., 184, 39-46. Griffiths, T.D., and S.Y. Ling (1989) Effects of UV light on DNA chain growth and replicon initiation in human cells, Mutation Res., 218, 87-94. Griffiths, T.D., S.A. Taft and S.Y. Ling (1990) Effect of UV light on DNA replication and chain elongation in Chinese hamster UV61 cells, Mutation Res., 236, 51-58. Howard-Flanders, P., R.P. Boyce and R. Theriot (1966) Three loci in Escherichia coli K-12 that control the excision of thymidine dimers and certain other mutagen products from host phage DNA, Genetics, 53, 1119-1136. Huberman, J.A., and A.D. Riggs (1968) On the mechanism of DNA replication in mammalian chromosomes, J. Mol. Biol., 32, 327-341.
Lehmann, A.R., and S. Stevens (1975) Post replication repair of DNA in chick cells: studies using photoreactivation, Biochim. Biophys. Acta, 402, 179-187. Meneghini, R., C.F.M. Menck and R.I. Schumacher (1981) Mechanism of tolerance to DNA lesions in mammalian cells, Q. Rev. Biophys., 14, 381-432. Mitchell, D.L., L.A. Applegate, R.S. Nairn and R.D. Ley (1990) Photoreactivation of cyclobutane dimers and (6-4) photoproducts in the epidermis of the marsupial Monodelphis domestica, Photochem. Photobiol., 51,653-658. Painter, R.B. (1980) A replication model for sister-chromatid exchange, Mutation Res., 70, 337-341. Painter, R.B. (1985) Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light, Mutation Res., 145, 63-69. Protic-Sabljic, M., and K.H. Kraemer (1986) Reduced repair of non-dimer photoproducts in a gene transfected into xeroderma pigmentosum cells, Photochem. Photobiol., 43, 509-513. Protic-Sabljic, M., N. Tuteja, P.J. Munson, J. Hauser, K.H. Kraemer and K. Dixon (1986) UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells, Mol. Cell. Biol., 6, 3349-3356. Regan, J.D., and J.S. Cook (1967) Photoreactivation in an established vertebrate cell line, Proc. Natl. Acad. Sci. (U.S.A.), 58, 2274-2279. Regan, J.D., L.H. Thompson, W.L. Carrier, C.A. Weber, A.A. Frances and M.Z. Zdzienicka (1990) Cyclobutane-pyrimidine dimer excision in UV-sensitive CHO mutants and the effect of the human ERCC2 repair gene, Mutation Res., 235, 157-163. Styer, S.C., P.J. Meechan and T.D. Griffiths (1989) Effects of ultraviolet light on thymidine incorporation and DNA chain elongation in photoreactivable insect cells, Photochem. Photobiol., 50, 557-562. Thompson, L.H., D.L. Mitchell, J.D. Regan, S.D. Bouffler, S.A. Stewart, W.L. Carrier, R.S. Nairn and R.T. Johnson (1989) CHO mutant UV61 removes (6-4) photoproducts but not cyclobutane dimers, Mutagenesis, 4, 140-146. Wood, R.D. (1985) Pyrimidine dimers are not the principal premutagenic lesions induced in lambda phage DNA by ultraviolet light, J. Mol. Biol., 185, 577-585.
Mutation Research, DNA Repair, 255 (1991) 257-264
© 1991 ElsevierScience Publishers B.V. All rights reserved 0921-8777/91/$03.50 ADONIS 092187779100101Q MUTDNA 06464
Effect of U V light on sister-chromatid exchanges, activation of alternative sites of replicon initiation and thymidine incorporation in CHO AA8, UV61 and UV5 cells Sharon A. Taft, Su Y. Ling and T. Daniel Griffiths Department of Biological Sciences and Plant Molecular Biology Center, Northern Illinois University, DeKalb, IL 60115 (U.S.A.)
(Received23 January 1991) (Revision received 13 May 1991) (Accepted 15 May 1991)
Keywords: DNA repair; DNA replication; SCE induction;CHO cells; UV; Thymidineincorporation
Summary The relative importance of the UV-induced pyrimidine(5-6)pyrimidine and the pyrimidine(6-4)pyrimidone lesions in sister-chromatid exchanges (SCEs), activation of alternative sites of replicon initiation and thymidine incorporation were examined using wild-type Chinese hamster ovary (CHO) AA8 cells which remove both lesions, mutant CHO UV61 cells which remove only the (6-4) lesion and mutant CHO UV5 cells which remove neither lesion. Our data suggest that both lesions play a role in each end point examined. The relative importance of these lesions is dependent on the end point studied as well as the fluence used. For SCE induction and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to play a predominant role, while for the thymidine incorporation studies the (6-4) lesion appears to play the predominant role at low fluences while the role of the (5-6) lesion increases at higher fluences.
There has been much debate concerning the relative roles of the two predominant UV-induced lesions, the pyrimidine(5-6)pyrimidine or (5-6) lesion, and the pyrimidine(6-4)pyrimidone or (6-4) lesion, in cytotoxicity, inhibition of DNA synthesis and mutation induction. Work performed with )t phage indicates that the (5-6) dimer can be lethal but it is not the principal
Correspondence: Dr. T. Daniel Griffiths,Professor, Department of Biological Sciences and Plant Molecular Biology Center, Northern Illinois University, DeKalb, IL 60115 (U.S.A.).
pre-mutagenic lesion (Wood, 1985). By using Escherichia coli photolyase in the presence of visible light, Wood was able to show that removal of the (5-6) dimer resulted in little change in the mutation frequency of the )t phage, but did increase the ability to form plaques. In E. coli, enzymatic photoreactivation (EPR) has been used to remove the (5-6) dimer and to observe the effect of this action on cell survival and mutagenesis. Brash et al. (1985) found that the (5-6) dimer is the principal cytotoxic lesion. They also determined that the (6-4) lesion is most likely the pre-mutagenic lesion. The (5-6) dimer appeared to correlate to mutation fie-
258 quency, but Brash et al. pointed out that this response is probably the result of decreased SOS induction resulting from the removal of the (5-6) dimer. Previously, Howard-Flanders et al. (1966) showed that E. coli mutants unable to remove the (5-6) lesion were hypersensitive to killing and mutation. These results may have been due to the induction of the SOS response, as suggested by Brash et al. Studies with lower eukaryotes containing the EPR system have focused on DNA synthesis and repair. Regan and Cook (1967) showed that E P R increased the growth of UV-irradiated fish cells and also reduced the extent of the UV-induced depression in the rate of DNA replication. This increase in growth rate and reduction in the depression of DNA replication did not return to control levels indicating that while the (5-6) lesion was important, other lesions were probably also important. Chick embryo cells showed little recovery of overall DNA synthetic rates after EPR (Lehmann and Stevens, 1975). Also, based on alkaline sucrose gradient studies, it was shown that EPR increased the size of newly synthesized DNA strands in UV-exposed cells, indicating that there were fewer gaps or there was less chain blockage. This result is similar to the results observed with photoreactivable insect cells. Styer et al. (1989) showed that the (5-6) dimer was the principal blocking lesion to DNA chain elongation in these insect cells. At fluences below 10 J / m 2, EPR removed most of the blocks. Above 10 J / m 2, EPR did not completely alleviate the blockage of fork progression. In addition, E P R did not completely reverse the UV-induced depression in the rate of DNA synthesis. Work completed with mammalian cells has been highly confusing. In shuttle vector DNA transfected into monkey cells, E P R by E. coli photolyase and visible light in vitro decreased the mutation rate by 80%. Interestingly, many of the mutations that remained after E P R were at sites where only (5-6) lesions would be expected to occur. This indicated that the (5-6) dimer was mutagenic in D N A replicated in monkey cells (Protie-Sabljic et al., 1986). Cleaver et at. (1987, 1988) showed that an XP 'revertant' which removes only the (6-4) lesion, exhibited normal
levels of survival and mutation suggesting that the (5-6) lesion is neither toxic nor mutagenic in human cells. In contrast, Broughton et al. (1990) recently came to the opposite conclusion. In that study, they reported that a class of trichothiodystrophy which removed the (5-6) but not the (6-4) lesion exhibited normal survival following exposure to UV. This difference might be explained by recent observations that the XP revertant removes (5-6) dimers from actively transcribed sequences (Lommel and Hanawalt, personal communication). Another study which examined mutations in a shuttle vector transfected into human cells reported that neither the (5-6) nor the (6-4) lesion frequencies correlated to mutation frequency even in the absence of excision repair (Brash et al., 1987). It was suggested that the mutation hot and cold spots observed were related to structural features of the DNA. Because of some of the confusion that has arisen from studies using EPR and because it has recently been claimed that in some cases EPR enhances indirectly the removal of lesions other than (5-6) cyclobutane dimers (Mitchell et al., 1990), we have been using UV61 cells, which remove only the (6-4) lesion (Thompson et al., 1989; Regan et al., 1990), to help elucidate the relative roles of both lesions. Previously, we (Griffiths et al., 1990) reported that although UV61 cells exhibited a more extensive blockage in DNA fork progression when compared to wild-type AA8 cells, the duration of blockage was shorter than that observed in UV5 cells which remove neither (5-6) nor (6-4) lesions. This suggested that while both lesions block DNA fork progression immediately after exposure, the (6-4) lesion is responsible for the prolonged blockage. In this study we wished to extend these studies to determine the role of the (6-4) lesion in sisterchromatid exchanges (SCEs) and the activation of alternative sites of replicon initiation. It has been proposed that since SCEs appear to be formed during the S phase, long-term blocks to replication are sites for SCEs (Painter, 1980). If this is the case, the (6-4) lesion, which we found to produce long-term replication blocks (Griffiths et al., 1990), should play a major role in UV-induced SCE induction. Likewise, we have observed a correlation between the extent and dura-
259 tion of blockage to DNA fork progression and the activation of alternative sites of replicon initiation (Griffiths and Ling, 1985, 1987, 1989). This would suggest that the (6-4) lesion should also play a major role in the activation of alternative sites of replicon initiation. Activation of alternative sites of replicon initiation allow blocking lesions which had been encountered by the leading strand, to be replicated by the lagging strand (Painter, 1985; Griffiths and Ling, 1985). It is thought that, during DNA replication, lesions encountered by the leading are capable of blocking fork progression, while lesions encountered by the lagging strand do not block replication (Meneghini et al., 1981; Berger and Edenberg, 1986). Materials and methods
Cell line maintenance The 3 Chinese hamster ovary (CHO) cell lines used in this study (AA8, UV61, and UV5) were kindly supplied by Dr. L.H. Thompson, Cells were routinely maintained in Falcon 75-cm 2 flasks. Cultures were grown in Ham's F10 supplemented with 10% calf serum, 5% fetal bovine serum and kanamycin (Gibco). Ceils were incubated at 37°C in a water-saturated environment with 5% CO 2 and were trypsinized (trypsinEDTA, 0.05%) every 3-4 days in order to maintain the cultures in a subconfluent state. Cells were routinely checked for contamination by mycoplasma using the Hoechst 33258 staining assay (Adams, 1980).
UV irradiation Prior to exposure, attached monolayer cultures were rinsed 2 times with sterile prewarmed phosphate-buffered saline (PBS). After adding approximately 1 ml of sterile prewarmed PBS, cells were sham exposed or exposed to the appropriate fluence. The source of radiation was 2 G15T8 germicidal lamps (General Electric) with a fluence rate of 0.1 W/s, as measured by a germicidal photometer (International Light Model IL 1500). Immediately following exposure, the PBS was removed and the appropriate amount of prewarmed medium was added to each dish.
Sister-chromatid exchanges Slide preparation. Approximately 24 h prior to exposure, cells were plated at a density of 7.5 x 105 cells/100-mm 2 dish. Following irradiation, medium containing 10 /~m 5-bromodeoxyuridine was added and the plates were incubated in the dark for 30 h. Chromosomes were then prepared according to Griffiths and Carpenter (1979). Differential staining. Slides were stained with 0.5/xg/ml Hoechst 33258 in 0.1 M sodium phosphate buffer (pH 6.8) for 20 rain and then rinsed with distilled water. PBS and a coverslip were placed on each slide prior to exposure to 366-nm light to fade the fluorescent Hoechst stain. After 10 rain, coverslips were removed and the slides were placed in 2 × SSC at 60 ° C for 60 min. After the slides were rinsed with distilled water, they were stained in 4% Giemsa for 20 rain and then rinsed and allowed to dry. The slides were scored for the number of SCEs. Only cells with 18-21 differentially stained chromosomes were used. The SCEs scored were reciprocal exchanges. DNA fiber autoradiography Slides were prepared according to Huberman and Riggs (1968) and Dahle et al. (1978). Approximately 2 x 104 cells were plated into each waxringed dish. 48 h after plating, the cells were rinsed with sterile prewarmed PBS, then sham or UV exposed. A high/low specific activity protocol was followed (Huberman and Riggs, 1968; Dahle et al., 1980). At 0, 2.5, or 5 h after exposure, high-specific-activity [3H]thymidine (3.7 MBq/ml; 3.11 TBq/mmole) was added to each dish for 25 rain and was then chased by a 25-rain pulse of unlabeled thymidine in sufficient amounts to reduce the specific activity to 0.62 TBq/mmole. Termination of labeling, transfer of cells to slides, lysis of cells and the addition of NTB3 (Kodak) photographic emulsion have been described previously (Griffiths and Ling, 1984, 1985). The slides were stored at 4°C for 6-9 months prior to development and fixation. The slides were scored using a single blind method. Replication segments (post-pulse figures) with dark, 'hot' centers and light, 'warm' trails were
260
used to measure the inter-origin distances. Measurements were taken from the middle of the dark area of one origin to the middle of the dark region in the adjacent origin using the Bioquant I1 system and digitizing pad (Griffiths and Ling, 1984, 1985). Data was analyzed using Student's t-test and the M a n n - W h i t n e y U-test.
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Thymidine incorporation assay Thymidine incorporation was measured according to Griffiths and Ling (1984, 1985). Triplicate cultures containing 3.5 x 10 4 cells were seeded into wax-ringed 35-mm ~ plates. The next day, cells were prelabeled with [14C]thymidine (1.85 kBq/ml; 1.85 M B q / m o l e ) in order to help detect and control for cell loss. Cells were exposed to UV 18-20 h later. At various times after exposure, [SH]thymidine (185 k B q / m l ; 3.7 T B q / m m o l e ) was added for 30 min. Labeling was terminated by rinsing the plates twice with 4°C PBS and then incubating the plates at 4°C for at least 2 h with 2% perchloric acid. Plates were rinsed with saline, and the cells were removed with a rubber policeman and vacuum filtered onto glass fiber filters. The filters were then rinsed with 95% ethanol, dried, and prepared for liquid scintillation counting. Results
Sister-chromatid exchanges Because SCEs are thought to arise from blockage of D N A replication (Painter, 1980), we were interested in determining whether there was a correlation between the extent of blockage induced by a lesion and its SCE potential. From our previous data (Griffiths et al., 1990), which showed that the (6-4) lesion produces more extensive and prolonged blockage than the (5-6) lesion, and from the initiation data presented below, we suspected that the (6-4) lesion was the lesion responsible for the majority of SCEs formed. Fig. 1 shows the results of SCE induction by UV light. AA8 cells showed a very small increase in SCEs over the range of fluences used. UV61 cells had more SCEs at each fluence compared to the AA8 cells, but SCE levels were much lower in UV61 ceils compared to UV5 cells suggesting
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that the (6-4) lesion is more efficient than the (5-6) lesion in producing SCEs. The plateau seen in UV5 cells was probably due to the inability to distinguish and count the total number of exchanges observed above 2.5 J / m e. The plateaus might also be due to our choice of sampling times. Specifically, at the higher fluences more heavily damaged cells might not have had time to reach the second mitosis by the time mitotic collections were made.
Actiuation of alternative sites of initiation As mentioned above, UV61 cells do not exhibit the extended blockage of fork progression that is observed in UV5 cells (Griffiths and Ling, 1985, 1987; Griffiths et al., 1990), which suggests that the (6-4) lesion is responsible for the extended blockage. In the past we observed a correlation between the extent of blockage of fork progression and the extent of activation of alternative sites of replicon initiation. Activation of alternative sites of initiation allows for the replication of D N A that contain blocking lesions (Painter, 1985; Griffiths and Ling, 1985). Thus, we next wished to determine if UV61 cells exhibited prolonged activation of alternative sites of initiation. Distances between adjacent sites of replicon initiation were visualized and measured through the use of DNA fiber autoradiography (Huberman and Riggs, 1968; Dahle et al., 1980; Griffiths and Ling, 1984). By using a high-specific-activity pulse and a chase with cold, unlabeled thymidine, the activation of alternative sites of initiation was
261
detected as a decrease in the inter-origin distance (IOD) of adjacent post-pulse figures. (Post-pulse figures have a dark center which represents incorporation that occurred during the incubation with the high-specific-activity label and low-density tails on either side which occurred during the chase with lower specific activity.) As is shown in Fig. 2, immediately after exposure to 5 J / m 2 or 10 J / m 2, AA8 cells showed a significant decrease in the IOD of UV-exposed cells. This indicated a significant activation of alternative sites of initiation. AA8 cells had recovered to control IOD by 2.5 or 5 h after either exposure, indicating that at these times alternative sites were no longer being activated. As is shown in Fig. 3, UV61 cells also exhibited a significant decrease in IOD levels immediately after exposure to 5 or 10 J / m 2. In contrast to the
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Fig. 2. Distribution of inter-origin distances in AA8 cells after exposure to 5 J / m 2 (left panel) or 10 J / m 2 (right panel). Cells were labeled for 25 min with high-specific-activity label followed by 25 min in low-specific-activity label as described in Materials and methods either immediately (top panel), 2.5 h (middle panel) or 5 h (lower panel) after exposure. The histograms for the control cells are represented by the hatched areas and are bounded by the dashed lines, while the histograms for treated cells are bounded by the solid lines.
results obtained for AA8 cells, however, UV61 cells still exhibited significant reduction in IOD values at both fluences 2.5 h after exposure. By 5 h after exposure, neither cell line exhibited a significant reduction of IOD values at either fluence. The extent of decrease in IODs at both fluences and at the 3 time intervals is summarized in Table 1. These results indicated that the (5-6) lesion could activate alternative sites of initiation up to 2.5 h after exposure. However, this lesion was not responsible for the extended activation (up to 5 h) observed previously in UV5 cells (Griffiths and Ling, 1985, 1987).
Thymidine incorporation Due to the fact that UV61 cells exhibited activation of alternative sites of replication 2.5 h
262 TABLE 1 E F F E C T O F U V ON T H E R E L A T I V E I N T E R - O R I G I N D I S T A N C E S IN AA8 A N D UV61 CELLS Time (h)
Fluence ( J / m 2)
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The relative inter-origin distances were obtained by dividing the values obtained in UV-exposed cells to the value obtained in sham-exposed cells. * Significant according to Student's t-test, P < 0.05.
ously concluded that this decrease in inter-origin distances represented an activation of alternative sites of replicon initiation (Griffiths and Ling, 1984, 1985, 1987). A second explanation is that UV irradiation selectively inhibits replicons with longer than average inter-origin distances. The results, shown in Fig. 4, suggest that selective inhibition is not likely since at 5 h after exposure of UV61 cells to 10 J / m 2 there is still an extensive depression in thymidine incorporation, yet there is no significant decrease in inter-origin distances. In addition, the extent of depression in UV5 cells is the same as that exhibited by the UV61 cells. This indicated that the (5-6) lesion was playing a greater role at higher fluences than was seen at lower fluences (Griffiths et al., 1990) in inhibiting the rate of thymidine incorporation.
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but not at 5 h after exposure to UV, we wanted to examine overall DNA synthesis at these times. In previous studies in repair-deficient cells there was a correlation between the extent of depression in D N A replication and the extent of decrease in inter-origin distances. We had previKinetics
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Discussion
In previous studies, either the (5-6) or (6-4) lesion has been implicated as having a predominant or sole role in various UV-induced end points (e.g., Brash et al., 1985; Cleaver et al., 1988; Broughton et al., 1990). The results reported here indicate that both the (5-6) and (6-4) lesions play a role in the effects of UV on DNA replication, SCE induction and the activation of alternative sites of replicon initiation. The results from the initiation studies with UV61 cells showed that the (5-6) lesion does play a role in the activation of alternative sites. However, this activation is not extended up to 5 h. UV5 cells were previously shown to exhibit significant activation at 2.5 and 5 J / m 2 (Griffiths and Ling, 1985, 1987) for up to 5 h after exposure. Thus, the prolonged activation of alternative sites observed in UV5 cells must be due to the (6-4) lesion. Our earlier work also showed that the (6-4) lesion is responsible for the prolonged blockage of fork progression observed in UV5 cells (Griffiths and Ling, 1984, 1985). Thus, there appears to be a strong correlation between the extent of D N A chain blockage and the activation of alternative sites of replicon initiation. The SCE results clearly indicate a greater role of the (6-4) lesion in the activation or induction of SCE events since the extent of UV-induced SCEs in the UV61 cell was closer to that exhib-
263 ited by AA8 cells. Similar results were observed by Cleaver et al. (1987) in XP variant revertants. The SCE data also correlates with the fiber data in which the (6-4) lesion apparently causes prolonged blockage of fork progression and activation of alternative sites of replication. These resuits support the theory of blocking lesions as instigators or sites of SCE induction (Painter, 1980), as both lesions can form blocks to D N A chain elongation (Styer et al., 1989; Griffiths and Ling, 1984, 1985; Griffiths et al., 1990). The greater role of (6-4) lesions in C H O cells appears to be due to the fact that they block replication for a longer period of time. If these lesions serve as sites for SCE formation, a recombinational exchange, they may also be stimuli for homologous recombination. It should be noted, however, that similar work with ICR-2A frog cells showed a decrease in SCEs after EPR, suggesting the (5-6) dimer is the critical lesion in SCE formation for these cells (Chao et al., 1985). One problem in comparing the relative roles of these two lesions is that the (5-6) lesion more abundant than the (6-4) lesion and that E P R may indirectly enhance the removal of (6-4) lesions (Mitchell et al., 1990). Previously, we (Griffiths et al. 1990) reported that after exposure to 2.5 J / m 2 the kinetics of thymidine incorporation in UV61 cells was close to that exhibited by AA8 cells and was quite different from that observed in UV5 cells. Specifically, UV5 cells never recovered normal rates of incorporation while AA8 cells and UV61 cells recovered normal rates within 5 and 7 h, respectively. Although the response of UV61 was still intermediate when the fluence was raised to 5 J / m 2, UV61 cells behaved more like UV5 cells. In the thymidine incorporation assay reported here, the UV61 curve was indistinguishable from the UV5 curve after exposure to 10 J / m 2. The increasing numbers of (5-6) lesions induced in the D N A at this fluence may increase the number of dimers becoming blocking lesions. It may become increasingly difficult for the UV61 cells (and to some extent AA8 cells) to complete D N A synthesis even with the activation of alternative sites of replicon initiation. Although the (6-4) lesion is being removed in the UV61 cells (Thompson et al., 1989), the sheer numbers of
(5-6) lesions may be perturbing the metabolic process that normally allows for bypass or modification of these lesions at low fluences. Thus, the relative roles of the (5-6) and (6-4) lesions appear not only to depend on the end point and organism studied, but also on the fluence employed. For SCE induction, and for the prolonged effects of U V light on D N A chain growth and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to be more important. For the effects of UV on D N A replication, both lesions a p p e a r to be important at high fluences (>/5 j / m 2 ) , while at lower fluences the (6-4) lesion appears to be more important in inhibiting thymidine incorporation.
Acknowledgements This work was supported by USPHS grant CA32579 awarded by the National Cancer Institute. The authors wish to thank Drs. Paul Meechan and Linda Yasui for their comments concerning this work.
References Adams, R.L.P. (1980) Cell Culture for Biochemists, Elsevier Science Publishers, New York, 292 pp. Berger, C.A., and H.J. Edenberg (1986) Pyrimidine dimers block simian virus 40 replication forks, Mol. Cell. Biol., 6, 3443-3450. Brash, D.E., W.A. Franklin, G.B. Sancar and W.A. Hazeltine (1985) Escherichia coli DNA photolyase reverses cyclobutane pyrimidine dimers but not pyrimidine-pyrimidone (6-4) photoproducts, J. Biol. Chem., 26, 1!438-11441. Brash, D.E., S. Seetharam, K.H. Kraemer, M.M. Seidman and A. Bredberg (1987) Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells, Proc. Natl. Acad. Sci. (U.S.A.), 84, 3782-3786. Broughton, B.C., A.R. Lehmann, S.A. Harcourt, C.E. Arlett, A. Sarasin, W.J. Klajer, F.A. Beemer, R. Nairen and D.L. Mitchell (1990) Relationship between pyrimidine dimers 6-4 photoproducts, repair synthesis and cell survival: Studies using patients with trichothiodystrophy, Mutation Res., 235, 33-40. Chao, C.C.-K., R.B. Rosenstein and B.S. Rosenstein (1985) Induction of sister-chromatid exchanges in ICR 2A frog cells exposed to 265-313 nm monochromatic ultraviolet wavelengths and photoreactivating light, Mutation Res., 149, 443-450. Cleaver, J.E., F. Cortes, L.. Lutze, W.F. Morgan, A.N. Player and D.L. Mitchell (1987) Unique repair properties of a
264 xeroderma pigmentosum revertant, Mol. Cell. Biol., 7, 3353-3357. Cleaver, J.E., F. Cortes, D. Karentz, L.H. Lutze, W.F. Morgan, A.N. Player, L. Vuksanovic and D.L. Mitchell (1988) The relative biological importance of cyclobutane and (6-4) pyrimidine-pyrimidone dimer photoproducts in human cells: evidence from a xeroderma pigmentosum revertant, Photochem. Photobiol., 48, 41-49. Dahle, D.B., T.D. Griffiths and J.G. Carpenter (1978) Inhibition of deoxyribonucleic acid synthesis and replicon elongation in mammalian cells exposed to methyl methanesulfonate, Mol. Pharmacol., 14, 278-289. Dahle, D., T.D. Griffiths and J.G. Carpenter (1980) Inhibition and recovery of DNA synthesis in UV-irradiated Chinese hamster V-79 cells, Photochem. Photobiol., 32, 157-165. Griffiths, T.D., and J.G. Carpenter (1979) Premature chromosome condensation following X-irradiation of mammalian cells: Expression time and dose response, Radiat. Res., 79, 187-202. Griffiths, T.D., and S.Y. Ling (1984) Effect of ultraviolet light on DNA replication in excision-deficient mammalian cells, Mutation Res., 132, 119-127. Griffiths, T.D., and S.Y. Ling (1985) Effect of ultraviolet light on thymidine incorporation, DNA chain elongation and replicon initiation in wild-type and excision-deficient Chinese hamster ovary cells, Biochim. Biophys. Acta, 826, 121-128. Griffiths, T.D., and S.Y. Ling (1987) Activation of alternative sites of replicon initiation in Chinese hamster cells exposed to ultraviolet light, Mutation Res., 184, 39-46. Griffiths, T.D., and S.Y. Ling (1989) Effects of UV light on DNA chain growth and replicon initiation in human cells, Mutation Res., 218, 87-94. Griffiths, T.D., S.A. Taft and S.Y. Ling (1990) Effect of UV light on DNA replication and chain elongation in Chinese hamster UV61 cells, Mutation Res., 236, 51-58. Howard-Flanders, P., R.P. Boyce and R. Theriot (1966) Three loci in Escherichia coli K-12 that control the excision of thymidine dimers and certain other mutagen products from host phage DNA, Genetics, 53, 1119-1136. Huberman, J.A., and A.D. Riggs (1968) On the mechanism of DNA replication in mammalian chromosomes, J. Mol. Biol., 32, 327-341.
Lehmann, A.R., and S. Stevens (1975) Post replication repair of DNA in chick cells: studies using photoreactivation, Biochim. Biophys. Acta, 402, 179-187. Meneghini, R., C.F.M. Menck and R.I. Schumacher (1981) Mechanism of tolerance to DNA lesions in mammalian cells, Q. Rev. Biophys., 14, 381-432. Mitchell, D.L., L.A. Applegate, R.S. Nairn and R.D. Ley (1990) Photoreactivation of cyclobutane dimers and (6-4) photoproducts in the epidermis of the marsupial Monodelphis domestica, Photochem. Photobiol., 51,653-658. Painter, R.B. (1980) A replication model for sister-chromatid exchange, Mutation Res., 70, 337-341. Painter, R.B. (1985) Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light, Mutation Res., 145, 63-69. Protic-Sabljic, M., and K.H. Kraemer (1986) Reduced repair of non-dimer photoproducts in a gene transfected into xeroderma pigmentosum cells, Photochem. Photobiol., 43, 509-513. Protic-Sabljic, M., N. Tuteja, P.J. Munson, J. Hauser, K.H. Kraemer and K. Dixon (1986) UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells, Mol. Cell. Biol., 6, 3349-3356. Regan, J.D., and J.S. Cook (1967) Photoreactivation in an established vertebrate cell line, Proc. Natl. Acad. Sci. (U.S.A.), 58, 2274-2279. Regan, J.D., L.H. Thompson, W.L. Carrier, C.A. Weber, A.A. Frances and M.Z. Zdzienicka (1990) Cyclobutane-pyrimidine dimer excision in UV-sensitive CHO mutants and the effect of the human ERCC2 repair gene, Mutation Res., 235, 157-163. Styer, S.C., P.J. Meechan and T.D. Griffiths (1989) Effects of ultraviolet light on thymidine incorporation and DNA chain elongation in photoreactivable insect cells, Photochem. Photobiol., 50, 557-562. Thompson, L.H., D.L. Mitchell, J.D. Regan, S.D. Bouffler, S.A. Stewart, W.L. Carrier, R.S. Nairn and R.T. Johnson (1989) CHO mutant UV61 removes (6-4) photoproducts but not cyclobutane dimers, Mutagenesis, 4, 140-146. Wood, R.D. (1985) Pyrimidine dimers are not the principal premutagenic lesions induced in lambda phage DNA by ultraviolet light, J. Mol. Biol., 185, 577-585.
