Activation of alternative sites of replicon initiation in Chinese hamster cells exposed to ultraviolet light

39

Mutation Research, 184 (1987) 39-46 DNA Repair Reports Elsevier MTR 06221

Activation of alternative sites of replicon initiation in Chinese hamster cells exposed to ultraviolet light T. Daniel Griffiths and Su Y. Ling Department of Biological Sciences, Northern Illinois University, DeKalb, 1L 60115 ( U.S. A.) (Received 15 July 1986) (Revision received 23 December 1986) (Accepted 29 December 1986)

Keywords: Replicon initiation; (Chinese hamster cells); UV light; DNA polymerases, blocking; Autoradiography.

Summary Exposure to UV light is known to produce lesions that block DNA polymerases at least on the leading strand. If several lesions are present in adjacent replicons, it is likely that sections of DNA would remain unreplicated because of the presence for blocking lesions. For cells to multiply and survive these areas must eventually be replicated. One mechanism that has been postulated to be involved in the replication of DNA between two blocking lesions is the activation of alternative sites of replicon initiation. To detect the existence of alternative sites of replicon initiation we employed the high specific/low specific activity labeling protocol first used by Huberman and Riggs (1968) for DNA fiber autoradiography. After development of the autoradiographs, the distances between adjacent sites of replicon initiation (inter-origin distances) were measured. In both wild-type Chinese hamster ovary (CHO) cells and UV-5 CHO cells, which exhibit no excision repair abilities, the inter-origin distances were, on average, shorter in cells exposed to UV, indicating that exposure to UV results in the activation of alternative sites of initiation. This activation appears to occur immediately after UV in both cell lines, but persist for a longer time in the excision-deficient line.

Many mutagens and carcinogens inhibit DNA replication by producing DNA lesions that block DNA fork progression (Dahle et al., 1979; Edenberg, 1976; Meneghini et al., 1981; Painter, 1985; Griffiths and Ling, 1985) a n d / o r inhibit the activation of replicon clusters (Watanabe, 1974; Painter and Young, 1975; Makino and Okada, 1975; Park and Cleaver, 1979; Dahle et al., 1980; Painter, 1985). Although many mutagens and/or Correspondence: Dr. T. Daniel Griffiths, Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115

(U.S.A.).

carcinogens have been examined for their effects on DNA replication, ionizing radiation and 254 nm ultraviolet radiation have been studied most extensively. The primary effect after exposure to ionizing radiation is the temporary inhibition of initiation of entire replicon clusters (Watanabe, 1974; Painter and Young, 1975; Dahle et al., 1979), while exposure to ultraviolet radiation produces effects on both DNA fork progression (Edenberg, 1976; Park and Cleaver, 1979; Dahle et al., 1980; Meneghini et al., 1981, Painter, 1985; Griffiths and Ling, 1985; Berger and Edenberg, 1986) and replicon initiation (Kaufmann et al.,

0167-8817/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

40 1980; K a u f m a n n and Cleaver, 1981). Although there is still some debate, most data suggest that at least for the first few hours after exposure to UV, pyrimidine dimers block fork progression on the leading, but not the lagging strand (Meneghini et al., 1981; Berger and Edenberg, 1986). After the initial depression in the rate of D N A replication, most mammalian cells eventually recover normal rates of D N A replication. For rodent cells exposed to UV, this recovery occurs despite the retention of m a n y pyrimidine dimers (Meechan et al., 1986). Pyrimidine dimers are thought to be the principal lesion responsible for blocking D N A fork progression. Fig. 1 shows a hypothetical model for the effects of pyrimidine dimers on D N A fork progression. Similar models have been proposed previously (Park and Cleaver, 1979; Dahle et al., 1980; Painter, 1985). If pyrimidine dimers (or other D N A lesions) block D N A fork progression on the leading strand, and if there were a sufficient number of lesions between two normal sites of initiation (i~ and i2: Fig. 1) areas of D N A may remain unreplicated. Specifically, areas of D N A that are bounded both upstream and downstream by lesions that block the leading strand (Ba and B2: Fig. 1) and do not contain a normal site of initiation would remain unreplicated. Exposure to 5.0 J / m 2 produces between 5 and 10 pyrimidine dimers per replicon in rodent cells (Ahmed and Setlow, 1977). Thus, exposure to 5.0 J / m 2 should produce m a n y areas within replicons that would il

i2

remain unreplicated if pyrimidine dimers were absolute blocks on the leading strand and if there were no alternative sites of replicon initiation available to the cell. Using the model of Painter (1985), it is possible to predict that the probability that such an unreplicatable region will occur in any inter-origin region after exposure of rodent cells to 5.0 J / m 2 is between 0.83 and 0.99. Since exposure to 5.0 J / m 2 kills only 10-20% of the cells, it is obvious that these areas are eventually replicated. Replication could be completed by 3 mechanisms: (1) bypass of the lesion leaving a gap in the newly synthesized strand, (2) translesion synthesis directly across from the lesion, and (3) the use of alternative sites of replicon initiation (e.g., Fig. 1). We and others have published data previously supporting the existence of the first 2 mechanisms (Park and Cleaver, 1979; Dahle et al., 1980; Menegnini et al., 1981; Griffiths and Ling, 1985; Meechan et al., 1986). Taylor and Hozier (1976), using D N A fiber autoradiography, have shown that C H O cells contain sites of replicon initiation that under normal conditions are not used. It is also known that exposure of m a m malian cells to caffeine (Lehmann, 1972; Griffiths et al., 1978; Tatsumi and Strauss, 1979) or flourodeoxyuridine (Taylor and Hozier, 1976; Ockey and Safhill, 1976) results in the activation of aberrant or alternative sites of replicon initiation. We (Griffiths and Ling, 1985) have recently published preliminary data suggesting that exposure of mammalian cells to UV also results in the activation of alternative sites of replicon initiation. In this communication we extend these observations. Materials and methods

Fig. 1. Hypothetical model of the activation of alternative sites of replicon initiation after insult with UV. i 1 and i 2 represent 'normal' sites of replicon initiation. When cells are exposed to UV, lesions (e.g. pyrimidine dimers) are formed which block DNA fork progression if they are encountered by the leading strand (solid wiggled line). Thus in this figure the solid triangles represent DNA lesions and the lesions labeled B1 and B2 would produce blocks since they would be encountered by the leading strand. Thus, if B1 and B2 were permanent blocks to DNA fork progression, the area between Bx and B2 would remain unreplicated unless an alternative site of initiation (ia) were activated. [A similar model has been proposed previously by Painter (1985).]

Cell and culture conditions Wild-type C H O line (AA8) and an excision-deficient line (UV-5) derived from the AA8 line were routinely cultured in 80-cm 2 tissue culture flasks (Nunc) in H a m ' s F10 nutrient medium (Gibco) supplemented with 10% calf, 5% fetal bovine serum and kanamycin. Cultures were maintained at 37 o C in a water-saturated atmosphere of 5% CO 2 and 95% air. Cultures were screened routinely for mycoplasma contamination (Schneider et al., 1974). Asynchronous cultures were obtained by

41 trypsinizing (0.05% trypsin-EDTA; Gibco) exponentially growing stock cultures and plating 1-ml aliquots each containing approx. 2 × 104 cells into 35-mm petri dishes (Falcon) which had had their inside diameter ringed with wax in order to confine the cells to the center of the dish. After 15-20 h another 1 ml of fresh medium was added to each petri dish.

Ultraviolet irradiation Cells were allowed to grow for at least 48 h prior to exposure to UV. Immediately prior to exposure the culture medium was aspirated from each petri dish and the attached monolayers of cells were rinsed twice with prewarmed sterile phosphate-buffered saline (PBS). After adding 1 ml sterile PBS to the petri dishes the plates were irradiated at room temperature using a custombuilt irradiator containing two 615T8 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 after exposure, 1 ml of fresh, prewarmed medium was added to each petri dish and the dishes were returned to the incubators. Experiments designed to detect photoreactivation capability in the cells were negative and all procedures were carried under fluorescent light. DNA fiber autoradiography To measure distances between origins of replication (inter-origin distances) a high specific activity-low specific activity labeling protocol was used (Huberman and Riggs, 1968; Hand, 1975). Cells were first labeled for 25 rain with high specific activity [3H]thymidine (100 /LCi/ml; 50 C i / mmole). Sufficient amounts of unlabeled thymidine were then added to reduce the specific activity to 98 /~Ci/ml, 10 Ci/mmole. Labeling was terminated 25 min later. After termination of the 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 6-8 months. After developing the slides, areas where the DNA was well spread were examined and the distances between the center of the dense areas of adjacent post-pulse fibers were determined using the corn-

puterized system described earlier (Griffiths and Ling, 1984). Generally, adjacent post-pulse units were similar in size. However, in some cases the lengths were different, probably due to the effect of lesions blocking fork progression. The computerized system (Bioquant II; R and M Biometrics, Nashville) allows for microscopic images to be displayed on a television monitor. Using a digitizing pad and a computer, distances can be measured to an accuracy of around 5%. All slides were coded and scored according to a single-blind experimental design. Statistical analysis involved the Mann-Whitney U test as described previously (Griffiths and Ling, 1984). Results

To determine if exposure to UV results in the use of alternative sites of replicon initiation, a high specific activity-low specific activity labeling protocol described in the previous section was used (Huberman and Riggs, 1968; Hand, 1975; Dahle et al., 1979). The sequential incubation of cells for 25 min in high specific activity [3H]thymidine followed by a 25-min incubation in low specific activity [3H]thymidine results in distinct replication figures after the cells are processed for autoradiographic observation. The replication figure examined in this study was the post-pulse figure. Post-pulse figures, which represent replicons that initiated rephcation during the high specific activity pulse and continued bidirectional replication after the decrease in specific activity, are defined as having a center area of high grain density trailing off to lower grain density on either end. Fig. 2 shows an example of adjacent postpulse figures from AA8 cells. The high and low grain density areas are the result of replication occurring during the high specific activity pulse and the low specific activity pulse, respectively. The site of replicon initiation is presumed to be at the center of the high grain density region. By measuring the distances between the centers of adjacent post-pulse units (inter-origin distances) it is possible to estimate the distances between adjacent sites of replicon initiation within replicon clusters. As reported recently (Griffiths and Ling, 1985), we observed that in wild-type and excision-deft-

42

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40 cient CHO cells there was a small, but significant decrease in the inter-origin distances 2.5 h after exposure to 2.5 or 5.0 J / m 2. In this study we wished to determine how rapidly after exposure to UV this decrease was evident and how long this decrease remained evident in wild-type and excision-deficient CHO cells. Figs. 3 - 6 show the resuits of several experiments with wild-type AA8 cells and the excision-deficient UV-5 cells. In these experiments the inter-origin distances of adjacent post-pulse units were determined for cells labeled either immediately, 3.5 or 5.0 h after exposure to either 5.0 J / m 2 (Figs. 3 and 4) or 10 J / m 2 (Figs. 5 and 6). Immediately after exposure to 5.0 J / m 2 (Figs. 3A and 4A), both the wild-type and the excision-deficient lines exhibited a highly significant ( p < 0.001) decrease in inter-origin distances. At 3.5 h after exposure the excision-deficient line still exhibited a highly significant decrease in inter-origin distances, while the wild-type exhibited only a slight, but still significant ( p < 0.05) decrease. By 5 h after exposure the wild-type ceils exhibited normal inter-origin distances, while the excision-deficient line still exhibited a highly sig-

I

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Fig. 3. Inter-origin distances in asynchronously growing AA8 cells exposed or sham-exposed to 5.0 J / m 2. At various times after exposure (0 h (A); 3.5 (B); 5.0 (C)) [3H]thymidine (100 /~Ci/ml, 50 Ci/mmole was added to the cultures. After 25 min. enough unlabeled thymidine was added to reduce the specific activity to 10 Ci/mmole. After processing for autoradiography, the distances between centers of adjacent post-pulse fibers were measured. The histogram for control cells is represented by the hatched areas and is bounded by the dashed line, while the histogram for treated cells is bounded by the solid line.

nificant ( p < 0.001) decrease. Immediately after exposure to 10 J / m E, the inter-origin distances were decreased even further in both the AA8 line (Fig. 5A) and the UV-5 line (Fig. 6A). The per-

43 50-

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Fig. 4. Inter-origin distances of asynchronously dividing UV-5 cells exposed or sham-exposed to 5.0 J / m 2. Conditions were similar to those for Fig. 3 with A representing inter-origin distances immediately after exposure, B representing interorigin distances 3.5 h after exposure and C representing interorigin distances 5.0 h after exposure or sham exposure. TABLE 1 E F F E C T OF UV L I G H T ON I N T E R - O R I G I N DISTANCES IN CHO CELLS

Cell line

Time after exposure

Decrease (%) in mean inter-origin distances

(h)

5.0 J / m 2

10.0 J / m 2

AA8

0 3.5 5.0

22.1 6.5 Nd a

41.5 11.2 Nd

UV-5

0 3.5 5.0

25.1 29.9 30.1

34.1 31.0 35.4

Nd, no decrease.

0

25

Inter-Origin

50

75

Distance

100 (pm)

Fig. 5. Inter-origin distances of asynchronously dividing AA8 cells exposed or sham-exposed to 10 J / m 2. Conditions were similar to those for Fig. 3 with A representing inter-origin distances immediately after exposure, B representing interorigin distances 3.5 h after exposure and C representing interorigin distances 5.0 h after exposure or sham exposure.

centages of decrease in inter-origin distances are tabulated in Table 1. At 3.5 and 5.0 h after exposure to 10.0 J/rn 2 the extent of activation of alternative sites in the UV-5 line was similar to that observed immediately after exposure. Although the AA8 line still exhibited a highly significant (p < 0.001) decrease in inter-origin distances 3.5 h after exposure (Fig. 5B), the extent of activation was less than that observed immediately after exposure. As was the case for exposure to 5.0

44 50-

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Fig. 6, Inter-origin distances of asynchronously dividing UV-5 cells exposed or sham-exposed to 10.0 J / m 2. Conditions were similar to those for Fig. 3 with A representing inter-origin distances immediately after exposure, B representing interorigin distances 3.5 h after exposure and C representing interorigin distances 5.0 h after exposure or sham exposure.

J / m 2, the AA8 cells exhibited normal inter-origin distances by 5.0 h after exposure to 10 J / m 2 (Fig. 5C). Discussion It has been known for some time that mammalian cells do not use all potential sites of replicon initiation. Using DNA fiber autoradiography,

Taylor and Hozier (1976) showed that along the mammalian DNA there are potential sites of replicon initiation every 4 /~m. In addition, several laboratories have shown that agents such as caffeine induce cells to use additional or alternative sites of replicon initiation (Lehmann, 1972; Griffiths et al., 1978; Tatsumi and Strauss, 1979). The results reported here show clearly that exposure of CHO cells to UV light results in a temporary decrease in inter-origin distances. There are 2 possible mechanisms to explain this decrease in inter-origin distances: (1) exposure to UV activates alternative sites of replicon initiation; or (2) exposure to UV selectively inactivates replicon clusters with long inter-origin distances. UV appears to inhibit the activation of a certain fraction of replicon clusters (Kaufmann and Cleaver, 1981; Painter, 1985) and if for some reason these cluster had long (> 30 #m) inter-origin distances, the average inter-origin distance in irradiated cells would decrease. This decrease would not be due to the activation of alternative sites but would be the result of scoring the non-inactivated replicon cluster which would contain replicons with shorter than average inter-origin distances. Although it is possible that this second mechanism :plays some role in the shift in inter-origin distances, it is also clear that this cannot be the only factor involved. We base this conclusion on the fact that exposure to UV results in the appearance of inter-origin distances much shorter than those normally observed in unirradiated ceils (e.g. Figs. 4B and C, 5A). For example, in Fig. 4C there is about a 30% decrease in inter-origin distances, yet almost 10% of the distances are less than 15/~m in irradiated cells, a distance rarely observed in unirradiated cells. In addition, approximately 40% of the distances are between 15 and 20/~m, while less that 5% of the distances are this short in unirradiated cells. Such a large increase in segments of this length cannot be accounted for by the rather modest degree of inhibition of initiation of replicon clusters. [Since exposure of CHO cells to fluences around 5.0 J / m 2 inhibits thymidine incorporation by less than 30% 3.5 h after exposure (Griffiths and Ling, 1984), the number of inactivated clusters must also be less than 30%.] We (Dahle et al., 1980; Griffiths and Ling, 1985) and others (Cleaver, 1978; Painter, 1985)

45

have proposed that exposure to UV results in the activation of alternative sites of replicon initiation. In these models it was proposed that this activation would not be evident until DNA polymerases were blocked by UV-induced DNA lesions. The results reported here suggest that this activation may occur almost immediately after exposure. Since the high specific activity pulse lasted 25 min and since activation was observed when the high/low activity labeling protocol was initiated immediately after exposure, it is possible to conclude that this activation occurs within the first 25 min following UV. It will be necessary to use shorter high specific activity pulse intervals to determine if there is any delay in this activation. The fact that this activation appears to be so rapid suggests that some process other than, or in addition to, DNA chain blockage may be involved in this activation. The fact that exposure to ionizing radiation, which strongly inhibitis the activation of replicon clusters, does not result in the activation of alternative sites of replicon initiation (Griffiths et al., 1978) suggests that inhibition of initiation of replicon clusters is not involved in this process. Since exposure to UV results in the production of a large number of bulky DNA lesions and exposure to X-rays does not (Cerutti, 1975), it appears possible that the distortion in the DNA resulting from the presence of bulky DNA lesions, such as pyrimidine dimers, may be partially responsible for the activation of alternative sites of replicon initiation. Work with other mutagens/carcinogens that either do or do not produce bulky DNA lesions should help determine the role such lesions play in this activation. For the wild-type AA8 cells, this activation of alternative sites of replicon initiation is evident immediately (Fig. 3A) and 2.5 h (Griffiths and Ling, 1985) after exposure. By 3.5 h after exposure to 5.0 or 10.0 J / m 2 (Figs. 3B and 5B) there appears to be only minor activation and by 5.0 h (Figs. 3C and 5C) there is no evidence for activation. For the excision-deficient UV-5 cells, activation is evident at all times tested. We have previously shown that the UV-5 cells also show no recovery in the rate of thymidine incorporation even after exposure to low (0.8 J / m 2) fluences of UV light while the AA8 line exhibits fairly rapid

recovery at least for fluences of 6.5 J / m 2 and lower (Griffiths and Ling, 1984). In addition, the UV-5 line exhibited prolonged blockage of DNA chain growth, while the AA8 line recovered normal rates of DNA chain growth within a few hours (Griffiths and Ling, 1985; Meechan et al., 1986). In fact, there appears to be a correlation between the loss of the activation of alternative sites of initiation and the ability of cells to replicate normal length fragments. For example, at 0 and 2.5 h after exposure of cells to 10.0 J / m 2 there is a strong inhibition in thymidine incorporation, a strong blockage of DNA chain growth and a strong activation of alternative sites. At 3.5 h after exposure to 10 J / m 2 there is still a depression in thymidine incorporation, but cells exhibit only a slight blockage in DNA chain growth (resuits not shown) and only a slight activation of alternate sites of replicon initiation (Fig. 5B). Although thymidine incorporation is still depressed 5.0 h after exposure, there is no evidence of alternative sites being activated (Fig. 5C) and DNA chain growth is no longer blocked (results not shown). Thus, it is tempting to postulate that the removal or alteration of the blocking lesions in the AA8 cells not only results in normal rates of DNA chain growth, but also in the deactivation of alternative sites. The UV-5 cells which lack the ability to remove or alter these lesions thus exhibit a prolonged activation of their alternative sites. Work with other cell lines, other fluences, and other times should help determine if this correlation is valid. Conclusion

Both wild-type and excision-deficient cells exhibited an activation of alternative sites of replicon initiation for the first few hours after exposure to UV. Excision-deficient cells exhibited this activation at later times (e.g. 5.0 h) after exposure, while the wild-type cells did not. Since the activation of alternative sites of initiation does not remove DNA lesions, but allows the cells to replicate DNA which contains mutagenic and carcinogenic lesions, this process may result in increased mutagenesis or carcinogenesis. Specifically, when alternative initiation sites are activated (e.g. i a in Fig. 1), lesions that had been blocks are no longer

46

blocks because they will now be encountered by the lagging strand which is thought to be capable of replicating past lesions (Meneghini et al., 1981; Painter, 1985). Thus, since pyrimidine dimers are thought to be non-instructive (Friedberg, 1985) this would increase the frequency of mis-incorporation. This would occur either under conditions where the lagging strand insects bases directly across from lesions or where a gap is left and then later filled in with the lesion still present. Further work with other cell lines such as human cells and with other mutagens/carcinogens should determine the relative role this recovery process plays in mutagenesis and carcinogenesis.

Acknowledgments We thank Dr. L. Thompson for supplying us with the CHO cells. We also thank Dr. P.J. Meechan for his comments and suggestions concerning this work. The work was supported by U.S.P.H.S. grant CA 32579 awarded by the National Cancer Institute.

References Ahmed, F.E., and R.B. Setlow (1977) DNA repair in V-79 cells treated with combinations of ultraviolet light and Nacetoxy-2-acetylaminofluorene, Cancer Res., 37, 3414-3419. Berger, C.A., and H.J. Edenberg (1986) Pyrimidine dimers block simian virus 40 replication forks, Mol. Cell. Biol., 6 3443-3450. Cerutti, P.A. (1975) Repairable damage in DNA: overview, in: P.C. Hanawalt and R.B. Setlow (Eds.), Molecular Mechanisms for Repair of DNA, Part A, Plenum, New York, pp. 3-12. Cleaver, J.E. (1978) DNA repair and its coupling to DNA replication in eukaryotic cells, Biochim. Biophys. Acta, 516, 489-516. Dahle, D.B., T.D. Griffiths and J.G. Carpenter (1979) Inhibition of deoxyribonucleic acid synthesis and replicon elongation in mammalian cells exposed to methyl methane sulfate, Radiat. Res., 78, 542-549. 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. Edenberg, H.J. (1976) Inhibition of DNA replication by ultraviolet light, Biophys. J., 16 849-860.

Friedberg, E.C. (1985) DNA Repair, Freeman, New York, pp. 405-408. 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., J.G Carpenter and D.B. Dahle (1978) DNA synthesis and cell survival following X-irradiation of mammalian cells treated with caffeine or adenine, Int. J. Radiat. Biol., 33,493-505. Hand, R. (1975) DNA replication in mammalian cells, Altered patterns of initiation during inhibition of protein synthesis, J. Cell Biol., 67, 761-773. Huberman, J.A., and A.D. Riggs (1968) On the mechanism of DNA replication in mammalian chromosomes, J. Mol. Biol., 32, 327-341. Kaufmann, W.K., and J.E. Cleaver (1981) Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts, J. Mol. Biol., 140, 171-187. Kaufmann, W.K., J.E. Cleaver and R.B. Painter (1980) Ultraviolet radiation inhibits replicon initiation in S phase human cells, Biochim. Biophys. Acta, 608, 191-198. Lehmann, A.R. (1972) Effect of caffeine on DNA synthesis in mammalian cells, Biophys. J., 12, 1316-1325. Makino, F., and S. Okada (1975) Effects of ionizing radiations on DNA replication in cultured mammalian cells, Radiat. Res., 62, 37-51. Meechan, P.J., J.G. Carpenter and T.D. Griffiths (1986) Recovery of subchromosomal DNA synthesis in synchronous V-79 Chinese hamster cells after ultraviolet light exposure, Photochem. Photobiol., 43,149-156. 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. Painter, R.B. (1985) Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light, Mutation Res., 145, 63-69. Painter, R.B., and B.R. Young (1975) X-Rays induced inhibition of DNA synthesis in Chinese hamster ovary, human HeLa and mouse L cells, Radiat. Res., 64, 648-656. Park, S.D., and J.E. Cleaver (1979) Post-replication repair: definition and possible alterations in xeroderma pigmentosum cell strains, Proc. Natl. Acad. Sci. (U.S.A.), 76, 3927-3931. Tatsumi, K., and B.S. Strauss (1979) Accumulation of DNA growing points in caffeine-treated human lymphoblastoid cells, J. Mol. Biol., 135, 435-449. Taylor, J.H., and J.L. Hozier (1976) Evidence for a four micron replication unit in CHO cells, Chromosoma, 57, 341-350. Watanabe, I. (1974) Radiation effects on DNA chain growth in mammalian cells, Radiat. Res., 58, 541-556.