51

Mutation Research, 236 (1990) 51-58 DNA Repair

Elsevier MUTDNA 06393

Effect of UV light on D N A replication and chain elongation in Chinese hamster UV61 cells T. Daniel Griffiths, Sharon A. Taft and Su Y. Ling Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115 (U.S.A.)

(Received 22 August 1989) (Revision received12 January 1990) (Accepted 19 February 1990) Keywords: Replication; Damage; Repair; Post-replicationrepair; Chain elongation; DNA fork progression

Summary Exposure of eukaryotic cells to ultraviolet light results in a temporary inhibition of D N A replication as well as a temporary blockage of D N A fork progression. Recently there has been considerable debate as to whether the (5-6)cyclobutane pyrimidine dimer, the pyrimidine(6-4)pyrimidone lesion or both are responsible for these effects. Using cell fines that repair both of these lesions (CHO AA8), only (6-4) lesions (CHO UV61) or neither (CHO UV5), we have shown that in rodent cells both lesions appear to play a role in both the inhibition of thymidine incorporation and the blockage of D N A fork progression. Specifically, after exposure to 2.5 J / m 2, AA8 cells recover normal rates of D N A replication within 5 h after exposure, while UV5 cells exhibit a greater depression in thymidine incorporation for at least 10 h. UV61 cells, on the other hand, show an intermediate response, both with respect to the extent of the initial depression and the rate of recovery of thymidine incorporation. UV61 cells also exhibit an intermediate response with respect to blockage of D N A fork progression. In previous publications we have shown that UV5 cells exhibit extensive blockage of D N A fork progression and only limited recovery of this effect within the first 5 h after exposure to UV. In this report we show that UV61 cells exhibit a more extensive blockage of fork progression than is observed in AA8 cells. These blocks also appear to be removed (or overcome) more slowly than in the AA8 cells, but more rapidly than in UV5 cells. Taken together we conclude that both lesions appear to be involved in the initial depression in thymidine incorporation and the initial blockage of D N A fork progression in rodent cells. These data also indicate that (6-4) lesions may be responsible for the prolonged depression in thymidine incorporation and the prolonged blockage of DNA fork progression observed in UV5 cells.

Exposure of cells to UV fight can have several effects including cell killing, mutagenesis, altered gene expression and alterations in D N A repli-

Correspondence: T. Daniel Griffiths (Ph.D.), Professor, Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115 (U.S.A.).

cation. The principal D N A lesions produced by 254-nm UV [(5-6)cyclobutane dimers and pyrimidine(6-4)pyrimidone products] disrupt the normal base pairing so that these lesions are gen: erally considered to be non-instructive. Because of the proposed non-coding nature of these lesions, there have been numerous studies examining how the D N A replicative process handles them. Since

0921-8777/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)

52 the late 1960s it has been realized that in bacteria gaps are left in newly replicated DNA across from DNA lesions. These gaps are later filled in by recombination (Rupp et al., 1968, 1971). In other systems such as ~X174 polymerase stops one base prior to the lesion (Moore et al., 1981). Several mechanisms appear to allow mammalian cells to tolerate UV-induced lesions. It has been shown that although gaps are left in newly replicated DNA, they are filled in by de novo synthesis rather than by recombination (Lehmann, 1972, 1974; Buhl et al., 1972). In addition, the work of Edenberg (1976) and Dahle et al. (1980) showed that UV-induced lesions can block replication at least temporarily. The extent of blockage appears to be fluence dependent with blockage playing a more predominant role at higher fluences (Meechan et al., 1986). Finally, the work of Meneghini (1981) and Berger and Edenberg (1986) suggests that in mammalian cells and simian virus 40, lesions encountered by the leading strand result in blocks, while lesions encountered by the lagging strand result in gaps. For some time it has been assumed that the (5-6)cyclobutane pyrimidine dimer is the lesion responsible for blocking D N A fork progression. This conclusion is based on the fact that this lesion is the predominant lesion formed following exposure to 254 nm UV and from the observation that in some non-mammalian eukaryotes, enzymatic photoreactivation partially reverses the effects of UV light on DNA replication (Buhl et al., 1974; Rosenstein and Setlow, 1980). More recently we (Styer et al., 1989) have shown a similar response in insect cells. In addition, we showed that while exposure to 10 or 20 J / m 2 produced significant blockage to D N A fork progression in these cells, the blockage is removed by subsequent exposure to photoreactivating light (Styer et al., 1989). Recently, however, there have been several studies which suggest that the pyrimidine(64)pyrimidone photoproducts may be the principal lesion responsible for UV-induced blockage of D N A replication in mammalian cells. First, it has been realized for some time that the extent of blockage observed after exposure to UV is much less than would be predicted if every dimer blocked DNA fork progression (Dahle et al., 1980;

Meneghini et al., 1981). Even if it assumed that only lesions encountered by the leading strand produce blocks (Meneghini et al., 1981), the degree of blockage that is observed is still much less than what is expected. It is also possible that the genomic location of lesions may also influence the ability of lesions to block replication. Second, as noted by Mitchell (1989) the rate of removal of (6-4) lesions in Chinese hamster ovary (CHO) cells corresponds to the rate of recovery of DNA replication that we have reported previously for CHO cells (Meechan et al., 1986). Third, a xeroderma pigmentosum (XP) revertant that is capable of removing (6-4), but not (5-6) photoproducts has been reported to exhibit near normal UV responses in cell survival, mutagenesis and DNA replication (Cleaver et al., 1987, 1988). It should be noted, however, that while the replication patterns in these cells were similar to control cells at early times after UV exposure, incorporation levels were depressed (compared to control cells) at later times (Cleaver et al., 1988). Finally, it has been recently reported that a CHO mutant that also selectively removes (6-4) products exhibited little or no alterations in the long-term maturation of D N A after exposure to UV light (Thompson et al., 1989). This CHO mutant (UV61) removes (6-4) photoproducts at a normal rate, but is unable to remove (5-6) photoproducts. Unlike the XP variant which exhibits near normal levels of survival and mutagenesis following exposure to UV, UV61 cells exhibit intermediate survival and mutagenesis values (Thompson et al., 1989). Because of these unique properties of UV61 cells, we examined the effects of UV light on DNA replication and D N A fork progression on these cells as well as wild-type AA8 cells and a mutant (UV5) that is unable to remove either (5-6) lesions or (6-4) lesions. We report here that UV61 cells also exhibit an intermediate response with respect to the effects of UV light on thymidine incorporation and DNA-chain elongation suggesting that in mammalian cells both types of lesions are potential blocks to D N A fork progression. Materials and methods Cells and culture conditions

The cell fines used in this study CHO AA8, CHO UV5 and CHO UV61 were supplied by Dr.

53 L. Thompson. Cells were routinely cultured in 75-cm2 tissue culture flasks (Falcon) in Ham's F10 nutrient medium (Gibco) supplemented with 10% calf, 5% fetal bovine serum and kanamycin. Cultures were maintained at 37 ° C in a water saturated environment of 5% CO 2 and 95% air. Randomly dividing cultures were obtained by trypsinizing (0.025% trypsin-EDTA: Gibco) exponentially growing stock cultures and seeding identical 1-ml inocula containing approx. 2 × 104 cells for fiber autoradiography and 3.5 × 104 cells for thymidine incorporation studies into 35-mm petri dishes which had their inside diameter ringed with a wax pencil in order to confine the cells to the center of the dish. Approx. 15-20 h after trypsinization, another 1 ml of fresh medium was added to each petri dish. Cultures were routinely checked for mycoplasma contamination by fluorescence observation of cells stained with Hoechst 33528.

UV-Irradiation Cultures were always exposed to UV 40-45 h after the 35-mm petri dishes were set up. Before exposure to UV the culture medium was aspirated from each petri dish and the cells were rinsed twice with sterile, prewarmed phosphate-buffered saline (PBS). After adding 1 ml sterile, prewarmed PBS the petri dishes and attached cells were irradiated at room temperature using a custom built irradiator containing 2 G15T8 germicidal lamps (General Electric) at a fluence rate of 0.1 W / m 2 as measured by a germicidal photometer (International Light Model IL 1500). Immediately following exposure to UV, 1 ml of fresh prewarmed medium was added and the petri dishes were returned to the incubator. In preliminary experiments, exposure of cells to fluorescent light after UV treatment did not alter survival or thymidine incorporation values (results not shown). For this reason, all experiments reported in this paper were carried out under fluorescent

light. Determination of the rate of DNA synthesis DNA synthesis was measured by [3H]thymidine incorporation into acid-precipitable material. At various times after exposure or sham exposure to UV, triplicate cultures were labeled for 30 rain with [3H]thymidine (185 k B q / m l ; 3.7 T B q /

mmole). Labeling was terminated by rinsing the plates twice with 4 ° C PBS and incubating the plates for at least 2 h with 2% perchloric acid at 4 ° C. Plates were then rinsed with saline and the cells were removed with the help of a rubber policeman and vacuum filtered onto glass fiber filters. The filters were then treated with 95% ethanol, dried in a drying oven (60 o C) and then placed in scintillation cocktail for liquid scintillation counting. Plates were pre-labeled with [laC]thymidine (1.85 k B q / m l ; 1.85 M B q / m o l e ) for 18 h prior to exposure or sham exposure to UV in order to help control for any cell loss. For each cell line and at every time point, the incorporation in the 3 exposed cultures was compared to the incorporation in the 3 sham-exposed cultures. In most cases the results of three experiments were pooled and the average relative rate of incorporation _ SEM were recorded. In one case (UV-5 cells exposed to 5.0 J / m 2) the result of 2 Expts. were pooled and the average relative incorporation value and the range were plotted.

Effects of UV on DNA chain growth D N A fiber autoradiography was employed to examine the effects of UV on D N A chain growth. The specific details of how D N A fiber autoradiographs are prepared can be found in our previous publications (Griffiths et al., 1978; Dahle et al., 1980; Griffiths and Ling, 1984). Briefly, cells were pulse-labeled for 40 min with high specific activity [3H]thymidine (3.7 M B q / m l ; 3.11 T B q / m m o l e ) either immediately, 2.5 or 5.0 h after exposure or sham exposure to UV. After termination of labeling, cells were removed from the petri dishes, placed on glass slides, lysed, spread across the slide and prepared for autoradiography. Following standard autoradiographic procedures, cells were stored in the dark at 4 ° C for 4 - 6 months. After developing, areas on the slide where D N A fibers were well spread were located. The lengths of internal segments were then measured using a computerized system ( R & M Biometrics, Nashville) described previously (Griffiths and Ling, 1984). For all studies at least 6 slides were scored for each cell line and exposure condition. On average 15-20 fiber segments were scored for each slide. All slides were randomly coded and scored according to a single-blind experimental design.

54 Statistical analysis involved the M a n n - W h i t n e y U test as described previously (Griffiths and Ling, 1984).

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Results Figs. 1 and 2 show the effect of 2.5 and 5.0 J / m E on thymidine incorporation, respectively in C H O AA8, UV61 and UV5 cells. As we reported previously (Griffiths and Ling, 1984, 1985), UV5 cells exposed to 2.5 J / m E exhibited a m u c h more p r o n o u n c e d depression in D N A synthesis than did wild-type A A 8 cells. In addition, UV5 cells exhibited little if any recovery in synthetic abilities for several hours after exposure to UV. The extent of the depression in thymidine incorporation of UV61 cells exposed to 2.5 J / m 2 was only slightly greater than that observed for AA8. Also, unlike UV5 cells, UV61 cells exhibited a recovery, but at a slightly slower rate than that observed in the A A 8 cells. W h e n the fluence was increased to 5.0 J / m 2, however, UV61 cells exhibited a m u c h greater depression in thymidine incorporation than was exhibited by A A 8 cells and also failed to show any recovery in synthetic rates during the time frame of the experiment. After exposure to 5.0 J / m 2, AA8 cells recovered normal rates of thymidine incorporation by 5 h after exposure. The reduction in the rate of thymidine incorporation following exposure to U V is the result of the interaction of inhibition of replicon initiation and inhibition of chain elongation. In order to determine the relative extent of blockage of D N A fork progression, D N A fiber autoradiographs were prepared either immediately, 2.5 h or 5.0 h after exposure to either 2.5 or 5.0 J / m 2. The results for 2.5 J / m E are shown in Fig. 3, while the results for 5.0 J / m E are shown in Fig. 4. F o r A A 8 cells exposed to 2.5 J / m 2 there was a slight (12%), but significant reduction in fiber lengths immediately after exposure to UV. Fiber lengths returned to control levels, however by 2.5 h after exposure. F o r the UV61 cells, a slightly greater reduction (20%) in fiber lengths was evident immediately after exposure to 2.5 J / m E. UV61 cells still exhibited a significant reduction in fiber lengths 2.5 h after exposure to 2.5 J / m E. By 5.0 h, however, fiber lengths in UV61 cells had returned to control levels. A somewhat similar picture was observed

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after exposure to 5.0 J / m 2. Immediately after exposure, UV61 cells exhibited a large (40%) reduction in average fiber lengths. Again, A A 8 cells exhibited a slight (15%) but significant reduction in average fiber lengths. As was observed after exposure to 2.5 J / m 2, fiber lengths returned to control levels in A A 8 cells by 2.5 h, but UV61 cells still exhibited a significant (20%) reduction in

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Fig. 4. Distribution of fiber lengths of CHO AA8 and UV61 cells exposed (or sham-exposed) to 5.0 J / m E. Cells were labeled for 40 min with [3H]thymidine either immediately, 2.5 or 5.0 h 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 the treated cells are bounded by the solid lines.

average fiber lengths. Although there was still an 8% reduction in fiber lengths in UV61 cells 5.0 h after exposure to 5.0 J / m 2, this reduction was not significant at the p < 0.025 level.

both these end points than are mutant UV5 cells (Thompson et al., 1989). Most interestingly, it was shown that while both UV5 cells and UV61 cells lack the ability to remove (5-6)cyclobutane dimers, UV61 cells exhibit a normal rate of removal of (6-4) products. The results reported in this paper indicate that, when compared to wild-type AA8 and excision-deficient UV5, UV61 cells have an intermediate response with respect to the effects of UV exposure on thymidine incorporation. Specifically, when exposed to 2.5 J / m 2 the kinetic response of UV61 cells is very close to that of AA8 cells, while after exposure to 5.0 J / m 2 the response is much closer to that exhibited by UV5

Discussion

UV61 mutants were initially isolated by Busch and co-workers (Busch et al., 1980) and were later assigned to complementation group 6 (Thompson et al., 1987). Recently it has been reported that this mutant is more sensitive to cell killing and mutation induction following exposure to UV than wild-type AA8 cells, but it is more resistant for

56 cells. Based on our previous work which shows that UV5 cells exhibit blocks to D N A fork progression 5 h after exposure to 5.0 J / m 2 (Griffiths and Ling, 1984, 1985; Meechan et al., 1986, unpublished observations) we also conclude that UV61 cells exhibit an intermediate response on the effects of UV fight on DNA fork progression. This would in turn suggest that since UV61 only removes (6-4) lesions (Thompson et al., 1989) that both the (6-4) and (5-6) lesions are involved in the post-UV depression in D N A replication as well as the UV-induced blockage of DNA fork progression. Thompson et al. (1989), concluded that UV61 cells had normal post-replication repair and therefore there was insignificant blockage of D N A forks. The technique they used for measuring defects in post-replication repair, D N A chromatography on benzoylated, naphthoylated DEAE cellulose (BND cellulose) is not specific for detecting blocks in D N A fork progression. This technique is based on the ability of BND cellulose to retain regions of D N A that are single-stranded (Strauss, 1981). Besides growing points, this technique has been used to detect 'gapped' DNA synthesis during DNA repair and excision repair. DNA fiber autoradiography, on the other hand, is specific for detecting blocks to DNA fork progression (Dahle et al., 1980). In addition, as the cells were labeled with [3H]thymidine for only 10 min in the Thompson et al. (1989) study, this would be insufficient time for most growing points to become blocked at DNA lesions (Edenberg, 1976; Dahle et al., 1980). The results of Thompson et al. (1989) do indicate that the long-term maturation of D N A is nearly normal in UV61 cells. Our data would support this conclusion since we show that although there are initial blocks to replication in the UV61 cells, these blocks are overcome within 5 h. The relative importance of (5-6) versus (6-4) lesions in various UV responses has been debated over the past few years (e.g., Brash and Hazeltine, 1982; Brash et al., 1987; Lawrence et al., 1985; Cleaver et al., 1987, 1988; Mitchell, 1988; Thompson et al., 1988). With regard, to the effects of these two lesions on D N A replication in eukaryotes the picture, at present, is somewhat confusing. In photoreactivable eukaryotes (Regan and Cook, 1967; Rosenstein and Setlow, 1980;

Styer et al., 1989) it is clear that although photoreactivation reverses a portion of the nost-UV depression in D N A replication, it is not completely reversed. This suggests that some other lesion or process in addition to (5-6)cyclobutane pyrimidine dimers is involved in the depression of DNA replication that is observed after exposure to UV. As for the effects of UV exposure on DNA fork progression, we have recently reported (Styer et al., 1989) that for photoreactivable cultured insect cells, exposure to photoreactivating light reverses virtually all blockage of fork progression. Thus from the insect work, we would conclude that the (5-6)cyclobutane pyrimidine dimer is the principal blocking lesion. A different conclusion has been arrived at by Cleaver et al. (1988) working with an XP revertant (XP-129). Like the mutant reported here, XP-129 cells lack the ability to excise (5-6)cyclobutane dimers but are able to repair (6-4) lesions. The authors claim that XP-129 cells respond like the excision-proficient human cell (GM 637) with respect to the effects of UV on D N A synthesis. Examination of their data shows that while XP-129 cells exhibit an initial depression that is similar to G M 637 cells, by 8 h after exposure the responses differ. Specifically, by this time, G M 637 cells have near normal rates of thymidine incorporation, while XP-129 cells are still inhibited by approx. 40%. The extent of depression in the XP12RO, the parent of XP-129, shows a much larger and extended depression in thymidine incorporation than do either G M 637 or XP-129. Therefore, we conclude that the XP-129 cells, like UV-61, exhibit an intermediate response with respect to the effects of UV on DNA replication. Work reported here with a mutant CHO UV61 that has the same repair deficient phenotype as the XP revertant suggests that both (5-6) and (6-4) lesions are capable of blocking DNA fork progression. In fact the data for these CHO UV61 cells suggests that (5-6)pyrimidine dimers may be the principal blocking lesion for the first few hours after exposure, while (6-4) lesions may account for the long-term blockage observed in UV5. One criticism of this suggestion is that the blockage to fork progression in rodent cells is removed much earlier than are (5-6) lesions. This observation has been discussed in the past and

57

various explanations have been put forward to account for this discrepancy (Meyn et al., 1971, 1976). One possibility is that C H O cells may contain a repair or tolerance mechanism that allows for the bypass of (5-6), but not (6-4) lesions. It is also possible that genomic location of lesions plays a role. Specifically it is known that actively transcribed regions are preferentially repaired in C H O cells (Bohr et al., 1985; Mellon et al., 1987). However, since only approx. 3-5% of the D N A is actively transcribed (Lewin, 1987) selective repair probably plays only a minor role in this recovery process. From our data and the data of others, it appears that the relative importance of (5-6) versus (6-4) lesions may be species-specific. Specifically, our work with insect cells (Styer et al., 1989) indicates that for insect cells (5-6)cyclobutane dimers play a critical role in the response of cells to UV. The recent work of Cleaver et al. (1987, 1988), however, indicates that in human cells (6-4) lesions are the critical lesion for survival and mutagenesis and cyclobutane dimers play only a minor role. In conclusion, the work reported here and by Thompson et al. (1989) suggests that for rodent cells both (5-6) and (6-4) lesions are involved in the response of cells to UV damage. The relative importance of these lesions may vary when different end points are studied and when different species are examined. Further work with other cell lines as well as the investigation of the heterogeneity of repair of (6-4) lesions should help clarify the relative importance of these D N A lesions in various UV responses.

Acknowledgments This work was supported by U.S.P.H.S. grant CA 32579 awarded by the National Cancer Institute. The authors wish to thank Drs. Paul Meechan and Linda Yasui for their comments concerning this work.

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