Proc. Natl. Acad. Sci. USA Vol. 76, No. 8, pp. 3927-3931, August 1979 Cell Biology Postreplication repair: Questions of its definition and possible alteration in xeroderma pigmentosum cell strains ( light/DNA replication/pyrimidine dimers) SANG D. PARK* AND JAMES E. CLEAVERt Laboratory of Radiobiology, University of California, San Francisco, California 94143 Communicated by Donald A. Glaser, May 14, 1979

ABSTRACT DNA synthesis in normal cells and in exci- MATERIALS AND METHODS sion-defective and variant xeroderma pigmentosum cells was investigated after irradiation with ultraviolet light. The sizes Cell Growth and Labeling. Primary and simian virus 40 of DNA synthesized during brief pulses of [3Hlthymidine 1-2 (SV40)-transformed human cells were grown in Eagle's mini- hr after irradiation were decreased, the xeroderma pigmento- mal essential medium with 15% fetal calf serum. Cell strains sum variant showing the smallest molecular weight. Once syn- with normal excision repair were GM498 (primary skin fibro- thesized, however, labeled DNA increased in size at the same blasts) and GM637 (SV40-transformed skin fibroblasts). XP cell rate as control in all cell strains, and the rate was relatively in- strains were an excision-defective XP12RO (SV40-transformed sensitive to caffeine. After 2-3 hr, labeled DNA in each cell type reached a maximum size that was less than that in control cells, group A) and an untransformed XP variant, XP4BE. indicating the presence of long-lived blocks to DNA chain Cells were grown in 60-mm petri dishes in [14C]dThd (0.01 growth. This kind of experiment (pulse-chase) has in the past 1uCi/ml, 64 mCi/mmol; 1 Ci = 3.7 X 1010 becquerels) until been used to investigate a repair process believed to be associ- required for use, at which time the medium was replaced with ated with the bypass of damaged sites in parental DNA: nonradioactive medium for 5-20 hr. Cells were then irradiated postreplication repair. We present an alternative model that at an dose rate does not involve a specific postreplication repair mechanism, with 0-10 J of 254-nm UV light per m2 incident but involves normal chain elongation and termination mecha- of 1.3 J/(m2.s), grown for 0-2 hr, labeled for 10 min with nisms in which we conceive that dimers and other damaged [3H]dThd (10 ACi/ml, 50 Ci/mmol), and "chased" in nonra- sites act as all-or-nothing blocks to the progress of replication dioactive medium containing 0.1 mM dThd and 10,gM de- forks. No evidence could be found for any inducible process that oxycytidine. The 3H to 14C ratio remained unchanged (within enhanced the bypass of damaged sites. 20%) during this period, indicating that little labeled DNA was DNA replication in mammalian cells is disturbed in various made during the chase. At various times, trypsinized cell sus- ways by agents that damage DNA. These disturbances include pensions containing 2 X 105 cells per ml or less were prepared a decline and recovery in the rate of DNA synthesis (1-4), a from the irradiated labeled cultures, and 3H and 14C single- decrease in the size of newly synthesized DNA (5, 6), altered strand molecular weights were determined by alkaline sucrose linkage of newly synthesized fragments to higher molecular gradient sedimentation (15). Samples of cell suspension were weight DNA (5, 6), and a decrease in the number of actively also used for determination of the 3H to 14C ratio by precipi- synthesizing replicons (7). Changes in the size of newly syn- tation with 4% perchloric acid, collection on Whatman GF/C thesized DNA have been interpreted in terms of a model, filters, and scintillation counting. "postreplication repair," originally proposed for Labeling Time and Molecular Weight Calculations. Al- (8). According to this model, DNA damage (e.g., pyrimidine though molecular weights in irradiated cells varied with time dimers produced by UV light) interrupts DNA chain growth, after irradiation (Table 1), as observed previously (16-20), in which then resumes beyond the damaged site, leaving a gap the present investigation we concentrated on the fate of labeled opposite the damage that can be filled by recombination or de molecules in irradiated cells during growth after the incorpo- novo synthesis. ration of a brief (10 min) pulse of [3H]dThd. Because DNA We studied the kinetics of DNA synthesis in UV-damaged chain elongation is not decreased in irradiated cells (21), a normal and xeroderma pigmentosum (XP) cells, using both constant labeling time was used to avoid complications that excision-repair-deficient and variant forms of XP.(9, 10), to would occur if the labeling times were varied, thereby pro- investigate the rate at which short fragments of DNA synthe- ducing different lengths of labeled DNA. DNA labeled for 10 sized in irradiated cells increased in size with time after min, shorter than the time taken to synthesize whole replicons pulse-labeling. We assumed that this would allow us to (i) ob- (22), will contain relatively short sections of 3H on their ends, serve the increase in size of DNA chains initially blocked by and the amount of radioactivity per fraction in a sucrose gra- damaged sites, (ii) define the rate of this process, (iii) identify dient will be approximately proportional to the number of defects in various XP cells, and (iv) delineate sites of action of molecules rather than to the total weight of-DNA, as it would caffeine in inhibiting DNA replication. This experimental for uniformly labeled DNA. Therefore, for briefly pulse-labeled approach has been used widely to observe postreplication repair DNA, a calculation from the 3H radioactivity by using the in mammalian cells (5, 6, 10-14). formula defined for weight-average Mrs of uniformly labeled On the basis of our results we present an alternative to the DNA-i.e., Mr = XwiMd/2W6, where wj is the radioactivity prokaryotic postreplication repair model of Rupp and How- in the ith fraction and Mi is the molecular weight of the ith ard-Flanders (8) and explain a wider range of DNA replication fraction will give a value that corresponds approximately to phenomena in UV-damaged mammalian cells. the number average for the DNA molecules in the gradient (23). During a pulse-chase experiment, however, as DNA fragments The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: XP, xeroderma pigmentosum; SV40, simian virus 40. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate * On sabbatical leave from Seoul National University, Seoul, Korea. this fact. t To whom reprint requests should be addressed. 3927 Downloaded by guest on September 27, 2021 3928 Cell Biology: Park and Cleaver Proc. Natl. Acad. Sci. USA 76 (1979)

with 3H-labeled ends link up with one another to larger sizes, the amount of 3H per molecule will increase and the calculated average from the 3H profile (the "apparent Mr") will be in- termediate between true weight and number averages for the DNA. Therefore, in these experiments no one parameter can be used exclusively to describe the complex distribution of molecules with nonuniform distribution of radioactivity. We have consequently displayed complete profiles at several time points and apparent weight-average Mrs at others, and adjusted UV doses for various cell types to obtain DNA sizes that appear to vary over similar ranges, so that errors in the apparent Mr will be similar for each cell type. RESULTS Fraction FIG. 1. Alkaline sucrose gradients from unirradiated normal Decreases in Molecular Weight of DNA Labeled after human fibroblasts (GM637) pulse-labeled with [3H]dThd for 10 min Irradiation. When cells were irradiated with 0-10 J of UV light and analyzed immediately (0), after 0.75 hr of growth (0), or after per m2 and labeled briefly at various times after irradiation, the 2 hr of growth (A). Sedimentation is from right to left. Parental observed Mr decreased and in some cases (XP4BE, GM637) ['4C]dThd DNA peaks coincide with the left peak in the 2-hr-chase began to recover during a 1- to 2-hr period after irradiation profile. (Table 1), as observed previously (16-20). Because of the rapid changes in Mr with time, the relationship between Mr and UV each cell type and about 20-40% of the parental 14C-labeled dose is complex (19, 20). The minimum Mrs observed in the DNA. various cell types at the same dose were lowest in the XP variant Increases in Molecular Weight of DNA during Growth cells (Table 1), as previously observed (10). The control Mrs after Irradiation and Labeling. Growth of the cells after ir- observed in the transformed cells were larger than in the un- radiation and labeling caused the distributions of the 3H-labeled transformed cells, consistent with previous measurements in- DNA to shift to higher Mrs in a qualitatively similar manner dicating that transformation results in an increase in replicon for all cell types and doses (Figs. 1 and 2). The ratios of Mrs of size in human cells (18, 25). 3H-labeled DNA to that of parental 14C-labeled DNA increased Our interest in this study was whether the damage that causes at an approximately linear rate for 2-3 hr that was the same in the observed decrease in Mr can be circumvented during sub- all cell types, and then appeared to approach a plateau, which sequent growth at a rate dependent on the cell type. Therefore, was lower than that of control cells (Fig. 3). The plateau for by using the values in Table 1 as a guide, we chose UV doses so control cells represents the maximum M-rs that can be reliably that the Mrs at the same times after the pulse were similar in observed for single-stranded DNA (26, 27). At times corre- Table 1. DNA molecular weights from human fibroblasts labeled for 10 min with [2H]dThd at 1-2 hr after irradiation with UV light Time after Relative Apparent Dose, irradiation, specific weight-average Cell type J/m2 hr activity* Mr X 10-7t Untransformed Normal (GM498) 0 1.0 3.6 ±0.7 5.2 1 0.85 2.9 + 0.2 10 1 0.78 2.7 ±0.2

XP variant (XP4BE) 0 1.0 2.9 i 0.1 2.8 1 0.60 1.75 + 0.05 2 0.51 1.70 + 0.20 5.2 1 0.29 1.04 5.2 2 0.22 1.68 Transformed Normal (GM637) 0 1.0 6.3 + 0.5 5.2 1 0.44 2.7 + 0.6 5.2 2 0.37 4.5 + 0.3

Excision-defective 0 1.0 4.7 + 0.2 (XP12RO) 0.5 1 0.65 4.2 2 0.55 4.0 1.4 1 0.79 3.4 2 0.62 3.3 5.2 1 0.40 1.6 2 0.28 1.2 * Specific activities were determined from the 3H-to-14C ratio in DNA and normalized to a value of 1.0 for each control. t See Materials and Methods. Data are shown as mean. SEM is shown when three or more separate determinations were made. With our dosimetry a dose of 5 J/m2 produces dimers spaced by a length of approximately 107 daltons, number average, of single-stranded DNA (24). Downloaded by guest on September 27, 2021 Cell Biology: Park and Cleaver Proc. Natl. Acad. Sci. USA 76 (1979) 3929

..4 lg l- E C4~~~ ~ ~

s K, , 1, 5 0. 4-0~~~~~~~~~~~~I1.o> 3

0.5-

1 2 3 4 5 1 2 3 4 5 6 51 152025 5 10152025 5 10152025 Fraction Time after irradiation, hr

FIG. 2. Alkaline sucrose gradients from irradiated normal FIG. 3. Relative molecular weights (ratios of apparent weight (GM637), excision-defective (XP12RO), and variant (XP4BE) cells averages for 3H pulse-labeled DNA compared to "4C-labeled parental grown 0-1 hr after irradiation before labeling for mm with DNA in each gradient) of normal, excision-defective, and XP variant [3H~dThd and then grown for an additional 0-3.5 hr before analysis. cells labeled 0-2 hr after exposure to UV and grown for various times. (Left) GM637 cells labeled for mmn immediately after exposure to (Top Left) Normal SV40-transformed GM637 cells after exposure 5.2 J of UV light per m2 and grown for 0 hr (top), 0.75 hr (middle), 2 to 5.2 J/m2. (Top Right) Excision-defective XP12RO SV40-trans- hr (bottom, *), or 3.5 hr (bottom, 0). (Center) Excision-defective formed cells after exposure to 2.8 J/m2. (Bottom Left) Normal GM498 XP12RO cells labeled hr after exposure to 2.8 J of UV light per m2 cells after exposure to 13 J/m2. (Bottom Right) Variant XP4BE cells and grown for 0 hr (top), hr (middle), or 2 hr (bottom). (Right) XP after exposure to 2.8 J/m2. 0, Control cells grown after a 10-min variant cells (XP4BE) labeled hr after exposure to 2.8 J of UV light [3H]dThd pulse at zero time and harvested at various times thereafter; per m2 and grown for 0 hr (top), 0.5 hr (middle), or 1.5 hr (bottom). v, cells labeled immediately after irradiation and grown for various All data have been drawn as actual counts in each gradient and not times; *, cells labeled 1 hr after irradiation and grown for various expressed as percentage of radioactivity or corrected for differences times; *, cells labeled 2-2.5 hr after irradiation and grown for various in cell numbers applied to the gradient; sedimentation is from right times; open symbols, cells grown in 1-2mM caffeine exclusively after to left. Arrow indicates the peak position of parental "'C-labeled DNA the labeling period. Each chase sequence was taken from one or two (100-120 S) in each gradient. In every case "'C-labeled DNA had a centrifugation studies; the Mrs are slightly different from those in unimodal profile similar to the control 2-hr chase shown in Fig. 1. Table 1 which was assembled from a larger number of pooled deter- minations. sponding to the period of the plateaus, the profiles were bimodal for GM637, but such fine structure in profiles was less clear for the other cell types. One component of the bimodal peak for function of the rate of postreplication repair. Inspection of Fig. GM637 was similar in size to parental DNA and the other was 3 indicates that, by this definition, the rate of a supposed smaller (Figs. 1 and 2). When3H-3abeledthe initial Mr of DNA postreplication repair process is only slightly lower than the rate was low, the plateau was also low for each cell type, as though at which Mrs increase in control cells and is independent of cell some DNA chains whose replication was blocked during the type-i.e., does not appear to be defective in the XP variant. labeling period remained blocked for long periods. Comparison of our results with others indicates that the rate of The presence of caffeine during the postlabeling period had postreplication repair, so defined, was also the same in normal, only slight effects on the initial rate of increase in Mrs and excision-defective, and XP variant cells (10, 11) and was un- mainly affected the rate at which the final plateau was reached affected by dose fractionation (13). But because of the few in irradiated cells. These effects were similar in control and determinations made at widely spaced time intervals and the irradiated cultures of each cell type. omission of unirradiated controls, the significance of the data was overlooked (10-13). Our results also indicate that there is DISCUSSION no radiation-induced system that increases the rate of postre- Our results indicate that there is a cell-type-dependent decrease plication repair at the longer times after irradiation. of the sizes in which DNA is synthesized during the first few Pulse-chase experiments may therefore not define a post- hours after irradiation (Table 1). If this represents interruption replication repair process as described by the Rupp and How- of nascent DNA strands by UV-damaged sites, then more in- ard-Flanders model (8). The differences in DNA sizes seen at terruptions occur (with consequent lower Mrs) in the XP variant a single time point after a chase are actually a reflection of the strain than in normal or excision-defective XP strains. The fact that the initial sizes of the labeled pieces are different, not minimum Mr in the XP variant corresponds approximately to that the rates of increase during the chase are different. We interruptions at almost every dimer (ref. 24; Table 1). Conse- have similarly observed that caffeine decreases the sizes in quently, the chain elongation seen during subsequent growth which DNA molecules are synthesized after irradiation and must involve completion of daughter DNA strands larger than eliminates recovery of DNA synthesis (19, 20) but has little the spacing of pyrimidine dimers in parental DNA. effect on the rate at which they increase during the chase (Fig. It has been assumed previously that chasing pulse-labeled 3). cells for various times allows investigation of the way replication To explain these results we propose an alternative model, has bypassed dimers, leaving gaps filled in by a postreplication because that of Rupp and Howard-Flanders (8) may be inap- repair mechanism (5, 6, 10-12). The rate at which Mrs increase propriate for mammalian cells (28). Our model is based on re- after irradiation and labeling (Fig. 3) should therefore be a cent observations made by DNA fiber autoradiography of Downloaded by guest on September 27, 2021 3930 Cell Biology: Park and Cleaver Proc. Natl. Acad. Sci. USA 76 (1979) nuclear DNA (29, 30) and a study of mitochondrial DNA (31), abnormal termini for replicon synthesis, which is completed which suggest that dimers can be long-lived blocks to replication by chain growth from adjacent unblocked forks (Fig. 4). Slow forks and (Fig. 4) is based on the following premises: leakage past blocks or excision of damaged sites at blocked forks (i) DNA replication proceeds bidirectionally from origins may also become important at high doses when there are many that function coordinately in clusters, initiate at similar times, damaged sites per replicon. and terminate at sites between origins that may not be deter- The unique feature of our model is that dimers are not by- mined by a specific base sequence (22, 32, 33). passed during replication, leaving a gap to be filled in re- (ii) Damaged sites block replication forks on an all-or- troactively (8), but merely block replication and become rep- nothing basis [we can then define a probability of blocking lication termini. Not all damaged sites block fork progression "P(block)"], but DNA chain growth at unblocked forks pro- [i.e., P(block) < 1], and those that do not are assumed to cause ceeds at normal rates in irradiated cells (28). Caffeine may in- no interruptions in the daughter strands. The decreased Mr soon crease P(block), thereby reducing initial Mrs (19,20), but have after labeling is caused by blockage of some forks, but the no effect on continued chain growth. normal rate of increase during the chase (Fig. 3) is from normal (iii) P(block) is close to 1 immediately after irradiation (i.e., elongation of unblocked forks and linkage of adjacent replicons forks are blocked at most damaged sites) and decreases to 0 with by normal termination mechanisms. time as cells increase in their ability to replicate without in- Previous experiments in which a pulse of [3H]dThd was terruption by damaged sites (16-20); the decline in Mr in the followed by a chase in BrdUrd and photolysis with 313-nm light first hr after irradiation is also complicated by end-labeling of in order to measure the size of purported gaps opposite dimers replicons made just before irradiation becomes blocked. (6, 34) may actually have estimated the size of the termination (iv) Blocked forks are relieved predominantly by acting as regions containing damaged sites. Our model predicts that if

Damaged Chain site Terminus growth=/'I_

Gapped Blocked synthesis \\ synthesis Origin Replicon 0 0 0 ---- 0-0 0 wTt ' s - Is -7 double strand DamagedDamagedsitesite ~~~DNA Abnormal I nitiation Gap filling terminus and a termination a b Termination 3H of replicons of # replicons ft T TT T _ T < Blocked I -~~~~~replication Chain elongation (unlabeled chase) Chain Chain growth Terminus growth Terminus at damaged site 4

Normal chain growth Termination (both forks) Termination unimpeded by one damaged site

Blocked _ / fork Normal c chain growth ( (one fork only) Long-lived gap d Blocked fork Blocked Na fork ______

Long-lived gaps in daughter strands FIG. 4. Interpretative model for events during DNA replication in UV-damaged cells. (a) Events for two approaching replication forks from adjacent origins according to the Rupp and Howard-Flanders model (8) in which dimers are bypassed, the gap is filled in retroactively, and replicons are terminated at the normal site. Question mark indicates the ambiguity in the model about the presence of gaps opposite the undamaged parental strand. (b) Events for two approaching replication forks from adjacent origins according to the model presented in this report. A blocked fork becomes an abnormal termination site. (c) Events for two approaching forks from adjacent origins when both become blocked. (d) Summary of our model for a stretch of DNA with several origins. Replication is initiated during the [3H]dThd pulse (shaded areas indicate 3H-labeled DNA), and replicons are completed during the subsequent chase in unlabeled medium. Only damaged sites that actually cause blocks are indicated for simplicity. In this model the possibility that blocking is an all-or-nothing event with a probability less than 1 is entertained to accommodate the recovery of DNA replication (16-20). Downloaded by guest on September 27, 2021 Cell Biology: Park and Cleaver Proc. Nati. Acad. Sci. USA 76 (1979) 3931

two forks approaching from adjacent origins are blocked there 8. Rupp, W. D. & Howard-Flanders, P. (1967) J. Mol. Biol. 31, will be long-lived interruptions in the daughter strands, which 291-304. E. & Bootsma, D. Annu. Rev. Genet. 9, 19- is what we observed at the longer chase times (Figs. 3 and 4) and 9. Cleaver, J. (1975) in Rupp and How- 38. which would not have been expected the 10. Lehmann, A. R., Kirk-Bell, S., Arlett, C. F., Paterson, M. C., ard-Flanders model (8). Our model also provides an explanation Lohman, P. H. M., de Weerd-Kastelein, E. A. & Bootsma, D. of observations made with enzyme digestion that are inexpli- (1975) Proc. Nati. Acad. Sci. USA 72,219-223. cable in the model of Rupp and Howard-Flanders (8). Digestion 11. Lehmann, A. R., Kirk-Bell, S., Arlett, C. F., Harcourt, S. A., de of DNA made in irradiated cells with the UV-specific T4 en- Weerd-Kastelein, E. A., Keijzer, W. & Hall-Smith, P. (1977) donuclease V was expected to produce double-strand breaks Cancer Res. 37, 904-910. because gaps in newly synthesized DNA were believed to be 12. Bowden, G. T., Giesselbach, B. & Fusenig, N. E. (1978) Cancer opposite dimers; however, none were found (35, 36). In the Res. 38,2709-2718. model we present there are no gaps opposite dimers but only 13. d'Ambrosio, S. M. & Setlow, R. B. (1977) Proc. Natl. Acad. Sci. blocked forks at dimer sites. USA 73, 2396-2400. Our interpretation is especially relevant to the XP variant. 14. Painter, R. B. (1978) Nature (London) 275,243-245. XP variant cells 15. Cleaver, J. E., Thomas, G. H., Trosko, J. E. & Lett, J. T. (1972) The large differences between normal and Exp. Cell Res. 74, 67-80. detected by Lehmann et al. (10, 11) were obtained with the use 16. Buhl, S. N., Setlow, R. B. & Regan, J. D. (1973) Biophys. J. 13, of long labeling times in the presence of caffeine and, although 1265-1275. only one (11) or two (10) chase times were used, the major effect 17. Lehmann, A. R. & Kirk-Bell, S. (1972) Eur. J. Biochem. 31, was expressed in the size of the labeled DNA made, not on its 438-445. subsequent rate of elongation. Therefore, although the variant 18. Cleaver, J. E., Williams, J., Kapp, L. N. & Park, S. D. (1978) in has a unique response to UV damage, this does not involve a DNA Repair Mechanisms, eds. Hanawalt, P. C., Friedberg, E. defect in bypassing of dimers during a postlabeling chase, but C. & Fox, C. F. (Academic, New York), pp. 85-93. rather an increased probability that replication forks will be 19. Cleaver, J. E., Thomas, G. H. & Park, S. D. (1979) Biochim. blocked by damaged sites, as we have reported (19). Biophys. Acta, in press. In conclusion, we believe that pulse-chase experiments do 20. Park, S. D. & Cleaver, J. E. (1979) Nucleic Acids Res. 6, 1151- not clearly identify the action of a unique repair process in- 1159. 21. L. F. & Painter, R. B. (1976) Biophys. J. 16,883-889. in damaged sites, but rather can be inter- Povirk, volved bypassing 22. Edenberg, H. J. & Huberman, J. A. (1975) Annu. Rev. Genet. preted equally satisfactorily in terms of the blocking of DNA 9,245-284. replication forks and termination by normal mechanisms at the 23. Lehmann, A. R. & Ormerod, M. H. (1970) Biochim. Biophys. damaged sites. The search for molecular mechanisms involved Acta 204, 120-143. in DNA replication may, according to our interpretation, be 24. Williams, J. I. & Cleaver, J. E. (1979) Biochim. Biophys. Acta, more profitably focused on determining the termination sites in press. between replication origins in damaged cells, the factors that 25. Kapp, L., Park, S. D. & Cleaver, J. E. (1979) Exp. Cell Res., in regulate the probability that damaged sites will block fork press. progression, and the cause of the increased probability in the 26. Lett, J. T., Klucis, E. S. & Sun, C. (1970) Biophys. J. i0, 277- XP variant. 292. 27. Cleaver, J. E. (1974a) Radiat. Res. 57,207-227. We are grateful to Mr. G. H. Thomas and Mr. W. C. Charles for 28. Cleaver, J. E. (1978) Biochim. Biophys. Acta 516, 489-516. technical assistance and to Drs. R. B. Painter, S. Wolff, A. B. Blumen- 29. Doniger, J. (1978) J. Mol. Biol. 120, 433-446. thal, A. R. Lehmann, and E. C. Friedberg for numerous discussions 30. Edenberg, H. J. (1976) Biophys. J. 16,849-860. about the significance of these data. This work was supported by the 31. Edenberg, H. (1978) in DNA Repair Mechanisms, eds. Hanawalt, U.S. Department of Energy. P. C., Friedberg, E. C. & Fox, C. F. (Academic, New York), pp. 1. Cleaver, J. E. (1965) Biochim. Biophys. Acta 108, 42-52. 489-492. 2. Domon, M. & Rauth, A. M. (1968) Radiat. Res. 35,350-368. 32. Tapper, D. P. & DePamphilis, M. L. (1978) J. Mol. Biol. 120, 3. Rude, J. M. & Friedberg, E. C. (1977) Mutat. Res. 42, 433- 401-422. 442. 33. Lai, C. J. & Nathans, D. (1975) J. Mol. Biol. 97, 113-118. 4. Painter, R. B. (1977) Nature (London) 265,650-651. 34. Buhl, S. N., Setlow, R. B. & Regan, J. D. (1973) Int. J. Radiat. Biol. 5. Cleaver, J. E. & Thomas, G. H. (1969) Biochem. Biophys. Res. 22,417-424. Commun. 36, 203-208. 35. Meneghini, R. & Hanawalt, P. (1976) Biochim. Biophys. Acta 6. Lehmann, A. R. (1972) J. Mol. Biol. 66,319-337. 425,428-437. 7. Painter, R. B. & Young, B. R. (1976) Biochim. Blophys. Acta 418, 36. Clarkson, J. M. & Hewitt, R. R. (1976) Biophys. J. 16, 1155- 146-153. 1164. Downloaded by guest on September 27, 2021