Enhancement of Postreplication Repair in Chinese Hamster Cells (Alkaline Sucrose Gradients/Ultraviolet/N-Acetoxy-Acetylaminofluorene/DNA) STEVEN M

Proc. Natl. Acad. Sci. USA Vol. 73, No. 7, pp. 2396-2400, July 1976 Biophysics

Enhancement of postreplication repair in Chinese hamster cells (alkaline sucrose gradients/ultraviolet/N-acetoxy-acetylaminofluorene/DNA) STEVEN M. D'AMBROSIO AND R. B. SETLOW Biology Department, Brookhaven National Laboratory, Upton, New York 11973 Contributed by R. B. Setlow, May 14, 1976

ABSTRACT Alkaline sedimentation profiles of pulse-la- hancement of postreplication repair is inhibited by cyclohexi- beled DNA from Chinese hamster cells showed that DNA from cells treated with N-acetoxy-acetylaminofluorene or ultraviolet mide. radiation was made in segments smaller than those from untreated cells. Cells treated with a small dose (2.5 .M) of N-ace- MATERIALS AND METHODS toxy-acetylaminofluorene or (2.5 Jlm-2) 254-nm radiation, sev- Cell Line and Tissue Culture. Chinese hamster cell line V-79 eral hours before a larger dose (7-10 ;&M) of N-acetoxy-acetylaminofluorene or 5.0 Jm-2 of 254-nm radiation, also synthesized was the generous gift of Dr. Richard Bird, Medical Department, small DNA after the second dose. However, the rate at which Brookhaven National Laboratory. Cells were grown in Eagle's this small DNA was joined together into parental size was ap- minimal medium containing 10% fetal bovine serum, 400 preciably greater than in absence of the small dose. This en- ,ug/ml of L-glutamine, 140 units/ml of penicillin, and 140 hancement of postreplication repair (as a result of the initial .ug/ml of streptomycin. small dose) was not observed when cells were incubatedwith Treatment. Approximately 6 X 105 cells were plated in5 ml cycloheximide between the two treatments. Thetresults suggest that N-acetoxy-acetylaminofluorene and ultraviolet-damaged of Eagle's minimal medium in Falcon plastic dishes (60 X 15 DNA from Chinese hamster cells are repaired by similar mm; Becton, Dickson and Co., Oxnard, Calif.), and incubated postreplicative mechanisms that require de novo protein syn- at 38° in a 5% CO2 atmosphere. Parental DNA was labeled by thesis for enhancement. incubation of cells for 15 hr with 0.02 ,Ci of [2-14C]thymidine ([14C]dThd) (50 Ci/mol) per ml. The radioactive medium was DNA synthesized shortly after ultraviolet (UV)-irradiation or replaced with warm nonradioactive medium 30 min prior to chemical treatment of bacteria and mammalian cells is smaller treatment. Cells were treated with various concentrations of than DNA from untreated ones (1-4). Upon further incubation AAAF or irradiated with 254 nm radiation at a fluence rate of of treated cells, the small segments of DNA are joined together 0.36 J-m2 sec-I according to the experimental scheme shown to form large DNA of a size similar to that in untreated cells. in Fig. 1. At the indicated times AAAF in (CH3)2SO was added This type of DNA repair, known as postreplication repair (1), to 5 ml of Eagle's minimal medium to the final concentration is thought to involve the filling in of gaps (1, 5-8) in daughter indicated in the legends of the figures and tables, or the medium DNA left opposite damages in parental DNA by recombina- was removed from the plates and the cells were irradiated. tional exchanges and/or by de novo synthesis of DNA in bac- Control cells were incubated with (CH3)2SO without AAAF. terial and, mammalian cells (4, 6-10). The evidence for the After 5 min of incubation or after UV irradiation, the cells were existence of gaps in mammalian DNA is somewhat equivocal washed twice with 2.5 ml of warm medium. Fifteen to 30 min (5-8). The recombinational pathway in Escherichia coil is later [methyl-3H]dThd (7 Ci/mmol) was added to a final considered to be error-free, whereas the de novo synthesis concentration of 2.4 and 0.6 MACi/ml to treated and untreated pathway, hypothesized to be error-prone (11-13), could lead cells, respectively, and pulsed for 15 or 30 min to label ap- to cell death, mutation, and neoplastic transformation. proximately equal lengths of DNA. Cells were chased with In E. coli the error-prone pathway of postreplication repair unlabeled dThd (4 ,g/ml) for 0-4 hr. has been shown by genetic and biochemical (11-15) analyses Alkaline Sucrose Sedimentation. After an appropriate chase to be induced by UV radiation, and a new protein, associated time, the cells were washed with an EDTA-containing, NaCl with mutation fixation, is made after UV irradiation (14). Re- solution (20) and exposed to 2000 R (0.52 C/kg air) of x-rays to cently Setlow and Grist (16) provided evidence for an enhanced facilitate the unwinding and separation of DNA strands (21). rate of postreplication repair after UV irradiation of fibroblast The cells were suspended in the same solution, and 160,000 cells from xeroderma pigmentosum variants (17). Such individuals in 50 Ml, containing 2000-30 cpm of each isotope, were lysed have the clinical symptoms of the disease, although their cells in 0.2 ml of a 1 M NaOH, 0.01 M EDTA solution layered on top are able to excise UV-induced pyrimidine dimers. Their cells of 5.2 ml of a 5-20% (wt/vol) alkaline sucrose gradient. A 0.2-ml are defective in postreplication repair (18). solution of 60% sucrose (wt/vol) was on the bottom of each Excision repair of DNA in human cells treated with N-ace- gradient. Samples were centrifuged as indicated in the figures toxy-acetylaminofluorene (AAAF) resembles that found after at 200 in a SW 50.1 or SW 60 rotor of a Beckman model L-2 or UV irradiation (19), and both are subject to postreplication L2-65 ultracentrifuge. After centrifugation, a hole was punched repair (4). We compared postreplication repair after UV irra- in the bottom of the tube and 11-drop fractions were collected diation and AAAF treatment and tested the possibility that onto paper strips. The strips were soaked once in 5% trichlo- postreplication repair could be enhanced in Chinese hamster roacetic acid and twice in 95% ethanol, and dried; radioactivity cells by UV and by AAAF. We find an increased rate of was determined in a liquid scintillation counter (22). Spillover postreplication repair of AAAF- or UV-damaged DNA if cells corrections were made (14C to 3H channel: 9%; 3H to 14C are previously treated with AAAF or UV radiation. This en- channel: 5%), and average single-strand molecular weight was computed (23). The molecular weights indicated on some of Abbreviations: UV, ultraviolet; AAAF, N-acetoxy-acetylaminofluorene; the figures were obtained by interpolation and extrapolation dThd, thymidine. from separate experiments on phage DNAs (23). Since the 2396 Biophysics: D'Ambrosio and Setlow Proc. Natl. Acad. Sci. USA 73 (1976) 2397 [14c] dThd 30 30 5-

SEDIMENT 0 ± AAAF ± AAF2 a ±UV, ±UV2 IN ALKALI U- FIG. 1. Experimental procedure for treating cells with AAAF or 0 UV radiation. Cells were incubated with [14C]dThd for 15 hr, to label w parental DNA, and the medium was changed. After 30 min in non- 4 radioactive growth medium, the cells were incubated with or without AAAF1 for 5 min or irradiated with 254-nm radiation (UV1), and 2-12 z hr later with or without AAAF2 or UV2. The cells, after 15-30 min in 0 fresh medium, were pulsed with [3H]dThd for 15-30 min and chased a-w with unlabeled dThd for various periods of time. 10 15 20 25 5 10 15 20 25 FRACTION sedimentation constant of large DNA decreases with centrifugal FIG. 2. Alkaline sucrose gradient profiles (sedimentation to the force (24-27), the apparent molecular weight of parental size left) of DNA from cells treated with AAAF or UV radiation. (a) Cells DNA depends on the particular centrifugation conditions used, were treated with 0 (0); 7.0 (A); or 10 (X) gM AAAF. They were as seen in a comparison of Figs. 2a and b. However, the amount pulse-labeled with [3H]dThd for 30 min, chased for 135 min, and of daughter DNA under the parental peak is insensitive to ex- centrifuged at 25,000 rpm for 120 min in a SW 50.1 rotor. (b) Cells conditions. were irradiated with 0 (0); 5.0 (); or 7.5 (A) J.m-2 ofUV radiation. perimental Treated and untreated cells were pulse-labeled for 30 min with Chromatography. Approximately 6 X 105 cells were grown [3H]dThd, chased for 60 min, and centrifuged at 30,000 rpm for 130 in 1 ml of Eagle's minimal medium at 38' in 35 X 10 mm min in a SW 50.1 rotor. The lower apparent molecular weight ofpa- Falcon plastic dishes. The DNA was labeled by incubation of rental DNA, compared to (a), arises because of the use of a higher cells for 15 hr in 0.3 MCi of [8-14C]deoxyguanosine (5 Ci/mol) centrifugal force (see Materials and Methods). Parental DNA (o) per ml. After the final treatment with AAAF, the cells were labeled with [14C]dThd is shown from cells given 10 1sM AAAF and removed from the plates with an EDTA-containing solution 10 J-M2 of UV radiation. and resuspended in 1 ml of 1 M NaOH for 18 hr at 340 to hy- drolyze RNA (28). The DNA was precipitated with 350 Ml of time to 4 hr (data not shown) increased the size of DNA ob- 65% trichloroacetic acid, washed twice with 95% ethanol, and tained from treated cells to that from parental DNA. hydrolyzed with-formic acid (29). The hydrolysate was spotted The size of parental DNA from treated cells was similar to on Whatman no. 1 chromatography paper and chromato- that from untreated cells. The values of apparent average sin- graphed with butanol-water (86:14) as the solvent. After the gle-strand molecular weight were 3.4, 4.3, and 3.2 X 108 for solvent front reached the bottom of the paper, about 18 hr, the cells treated with 0, 7, and 10,uM AAAF, respectively, and 2.1, chromatogram was dried and cut into 2 X 2 cm strips. The ra- 2.2, and 1.8 X 108 for cells irradiated with 0, 5.0, and 7.5 J.m-2 dioactivity of each strip was determined in a toluene-based of 254-nm radiation. scintillation fluid for 10 min in a BeckmAn scintillation counter. For determination of the number of thymine-containing dimers Enhancement of postreplication repair after UV irradiation, the DNA was labeled with [3H]dThd, hydrolyzed, and chromatographed in two dimensions (29). If cells were treated with 2.5 zM AAAF or 2.5 J m-2 of 254-nm Chemicals. AAAF was the generous gift of Dr. J. A. Miller radiation and 2 hr later with 5.0 AAAF or 5.0 J.m-2 UV ra- (McArdle Laboratory for Cancer Research, Madison, Wisc.). diation according to the experimental scheme shown in Fig. 1, (CH3)2SO was purchased from Fisher Chemical Co. Cyclo- the pulse-labeled DNA was of higher molecular weight than heximide was obtained from Sigma Chemical Co. [14C]dThd DNA from cells treated with 5.0uM AAAF or 5.0 Jm-2 alone and [methyl-3H~dThd were products of New England Nuclear (Fig. 3). Similar enhancements of postreplication repair were (Boston, Mass.). [8- 4C]Deoxyguanosine in 30% ethanol was observed after treating cells with higher concentrations of purchased from Schwarz/Mann (Orangeburg, N.Y.). Eagle's AAAF1 and AAAF2 (Fig. 4), or UV1 and UV2 (R. B. Setlow, minimal medium, fetal bovine serum, L-glutamine, and peni- unpublished observations), longer periods between the two cillin-streptomycin were obtained from Grand Island Biological treatments (up to 12 hr), as well as longer (3 hr) and shorter (1 Co. (Grand Island, N.Y.). hr) chase times (unpublished observations). The various treatments we have used inhibit the rate of DNA synthesis per culture by 30-70%, and hence could change the RESULTS distribution of the lengths of growing replicons. Such changes Postreplication repair would come about if only the initiation of new replicons were inhibited, as is observed shortly after x-irradiation (30). How- The alkaline sucrose gradient profiles of pulse-labeled DNA ever, UV radiation and presumably AAAF, primarily inhibit chased for 2.2 hr, from cells treated with 0, 7, or 10MgM AAAF, the average rate of chain elongation and not initiation, as judged are shown in Fig. 2a. Similar data are shown in Fig. 2b for by the fact that net [3H]dThd incorporation does not decline pulse-labeled DNA, chased for 1 hr, from cells irradiated with with time after irradiation and cells continue to enter the S 0, 5.0, and 7.5 Jm-2 of 254-nm radiation. Less of pulse-labeled phase (31). Moreover, as indicated in Fig. 4, the initial treatment DNA from cells treated with AAAF or UV radiation is of the alone inhibits, and does not enhance, the appearance of pulse- same size as pulse-labeled DNA from untreated cells. For larger labeled DNA of parental size. doses of AAAF or UV radiation, the sedimentation constant of Treatment of cells with AAAF1 or UV1 radiation could result the pulse-chased DNA is appreciably lower, and hence its in membrane alterations or changes in cell size that could di- single-strand molecular weight is smaller. Increasing the chase minish the accessibility of cellular DNA to AAAF2 or UV2 ra- 2398 Biophysics: D'Ambrosio and Setlow Proc. Natl. Acad. Sci. USA 73 (1976)

Table 1. Amounts of AAAF-modified guanine (a) (b) and thymine-containing dimers > 12 Percentage of total radioactivity* 0 X 8-6 AAAF- Thymine- modified containing o 9 Treatmentt guaninet dimers AAAF2 0.10 z 5 01 02 1035202 AAAF1 + AAAF2 0.17 UV2 0.38 UV1 + UV2 0.42 FRACTION * The percentages of AAAF-modified [8-14C]guanine and [3H]thy- FIG. 3. Sedimentation profiles of DNA from cells treated as mine-containing dimers were determined using paper chroma- outlined in Fig. 1. (a) Cells were treated with (0) or without (@) 2.5 tography (see Materials and Methods). MM AAAF1 and again, 2 hr later, with 5.0 MM AAAF2. They were t See Fig. 1. Cells were treated with or without 2.5MM AAAF or 2.5 pulse-labeled for 30 min with [3H~dThd, chased for 120 min, and J.m-2 of UV radiation, followed 2 hr later with 140 MM AAAF or centrifuged at 25,000 rpm for 120 m3 in a SW 50.1 rotor. Parental 99J-m-2 ofUV radiation. DNA (o), labeled with [14C]dmhd, is shown from cells given AAAFL $ AAAF-modified [8-'4C]guanine migrated to an RF of0.7. plus AAAF2 and is similar to that from cells given AAA2 alone. (b) Cells were irradiated (0) or not irradiated (-)with 2.5 J-m~2 of UV radiation, and after 2 hr, with 5.0 J-m~2. They were puLsed-labeled fortuitous enhancement of postreplication repair rate is also for 30 min, chased for 60 mm and centrifuged for 160 min at 25,000 unlikely. rpm in a SW 60 rotor. Parental DNA (o) is shown from cells given The number of alkali-, acid-, and heat-stable AAAF-modified UV1 plus UV2 irradiation. guanine and pyrimidine dimers (see ref. 29) and AAAF-modified guanine not lost by depurination, calculated from the data diation. To check for possible changes in uptake of AAAF2 re- in Table 1, was about 80 per 108 daltons of DNA for AAAF- sulting from AAAF1 treatment, we treated cells with a very modified guanine and 250 per 108 daltons for thymine-con- large dose of AAAF2 (140 ,M), and the fraction of modified taining dimers for cells given 140 AM AAAF and 99 J m-2 of guanine was measured. Table 1 shows that the two treatments, UV radiation. if anything, result in more modified guanine than AAAF2 treatment alone. Table 1 also shows that the same number of Effect of cycloheximide on postreplication repair thymine-containing dimers are produced after irradiation of Incubation of cells with cycloheximide (5 ,ug/ml) until 30 mi cells with 99 J.m2 whether or not cells were first irradiated before the pulse chase had no effect on the size of parental DNA with UV1. Therefore, the possibiity that membrane or cell size (Fig. 5). Protein synthesis, determined by [3H]leucine uptake changes caused by AAAF1 or UV1 radiation could lead to the into acid-precipitable material, was inhibited moretthan 90%

60H

I- 501-

0 40H c- 0 w 30h

z w 20H w 0.

10-

CONTROL 3.5 MM AAAF, 3.5 MM AAAF, 0 AAAF, 0 UV1 2.5 Jm2UV1 2.5Jm2UV1 0 UV, 0 AAAF2 1 0MM AAAF2 1 0pM AAAF2 0 UV2 0 UV2 5.0 Jm-2UV2 5.0 Jm-2UV2 FIG. 4. The percentage of pulse-labeled DNA sedimenting as high-molecular-weight DNA. Cells were given the treatments shown, separated by 2 hr, as outlined in Fig. 1. Cells treated with AAAF were pulsed-labeled for 30 min and chased for 135 min before sedimentation. Cells given no UV2 irradiation were pulse-labeled for 15 min; those given UV2, for 30 min, and chased for 1 hr before sedimentation. High-molecular-weight DNA was taken as the cumulative fractions from the bottom of gradients that included 60% of parental 14C radioactivity (fractions 1-11 in Figs. 2a and 3a, 1-14 in Figs. 2b and 3b). Similar patterns are obtained for cumulative fraction that included 30-70% of parental 14C radioactivity. Biophysics: D'Ambrosio and Setlow Proc. Natl. Acad, Scd. USA 73 (1976) 2399

W F - ~~~~~(a). b Do - 2k 0 0 10. 48- 0

0 5 9OI 02 O I 02 2 4- 4 IO 52 5 5 OI 02 4 w a.) FIG. 6.UIn AFehneeto earo AF n w 5 10 152025 510 152025 a. FRACTION 5 10 1520 25 5 10 1520 25 FIG. 5. Effect of cycloheximide on the enhancement of postre- FRACTION plication repair in cells treated with AAAF. (a) Cells were treated with 2.5MuM AAAFI, and incubated for 2.5 hr with (-) or without (0) 5 Mg FIG. 6. UV and AAAF enhancement of repair of AAAF- and ofcycloheximide per mL Cells were washed twice with Eagle's minimal UV-damaged DNA. (a) Cells were treated (0) or not (0) with 2.5 medium, pulse-labeled with [3H]dThd for 30 min, and chased with J-M-2 of 254-nm radiation, and after 2 hr treated with 10MuM AAAF. unlabeled dThd for 3.5 hr before sedimentation. 14C-Labeled parental They were pulsed with [3H]dThd for 30 min, chased for 135 min, and DNA (o) is shown from cells given AAAF1 and cycloheximide. (b) sedimented at 25,000 rpm for 120 min. (b) Cells were treated (0) or Cells were treated with 2.5 ,M AAAF1, immediately incubated for not (0) with 2.5 MM AAAF and after 2 hr exposed to 10 J.m-2 Of 2.5 hr with (-) or without (0) 5.0Mug/ml of cycloheximide, and after 254-nm UV radiation. They were pulsed with [3H]dThd for 30 min, removal of cycloheximide, treated with 5.0 gM AAAF2. They were chased for 120 min, and sedimented at 30,000 rpm for 130 min. 14C- pulse-labeled for 30 min, chased with unlabeled dThd for 3.5 hr, and Labeled parental DNA (o) is shown from cells treated with both UV sedimented. Parental DNA (o), labeled with [14C]dThd, is shown radiation and AAAF. from cells given AAAF1 plus AAAF2. in the presence of pulse-labeled DNA was considerably smaller than DNA from cycloheximide,.and although cycloheximide cells to was removed from the cells 30 min prior to a [3H]dThd pulse, given two treatments but incubated in the medium the rate of DNA synthesis was inhibited 50%. This inhibition containing the essential amino acid (S. M. D'Ambrosio, un- of DNA synthesis seems to be due to a decrease in both the rate published observation). These results suggest that de novo of protein synthesis is required for enhancement of postreplication chain growth and the initiation of new DNA chains as a direct of AAAF- and result of the inhibition of protein synthesis (31, 32). The sedi- repair UV-damaged DNA. mentation profiles of untreated pulse-labeled cells previously Effect of UV-irradiation and AAAF on enhancement of incubated with or without cycloheximide were identical (data repair of AAAF- and UV-damaged DNA not shown), and indicate that the normal rate of strand joining and was not Pulse-labeled DNA from cells treated with 10,M AAAF re- elongation significantly altered by cycloheximide. mained near If cycloheximide was added after an or the top of the alkaline sucrose gradients after a immediately AAAF1 135-min chase (Fig. 6a). However, when cells were exposed to UV1 treatment of 2.5 MuM or 2.5 J-m-2, most of the newly syn- 2.5 UV irradiation thesized DNA was considerably smaller than DNA from cells Jm-2 of and 2 hr later treated with 10,gM given similar treatments but no cycloheximide (Fig. 5b, Table AAAF, a significant portion of the pulse-labeled DNA moved 2) and was about the same as alone in the absence of further down the gradient (Fig. 6a). Similarly, the pulse-labeled AAAF2 DNA from cells treated with 2.5 ,M AAAF 2 hr prior to a 10 cycloheximide. On the other hand, cycloheximide had only a m-2 small effect if AAAF2 was omitted (Fig. 5a, Table 2). Further, J UV irradiation moved further down the gradient than if cells were incubated in Eagle's minimal medium lacking cells exposed only to 10 J.m-2 of UV radiation (Fig. 6b). L-methionine between AAAF1 and AAAF2 treatments, the DISCUSSION Table 2. Effect of cycloheximide on the Treatment of Chinese hamster cells with AAAF or UV radiation enhancement of postreplication repair by UV radiation significantly enhances the subsequent rate of postreplicative repair of AAAF-damaged or UV-irradiated DNA. We have also % of pulse- observed a similar enhancement of postreplicative repair in cells labeled DNA of xeroderma pigmentosum variant sedimenting as (16). high-molecular- Studies on excision repair of parental DNA in human cells Treatment* weight DNAt [Chinese hamster cells have very low levels of excision repair for UV damage (33, 34)] indicated that 7MuM AAAF made re- No UVI, + cycloheximide, no UV2 21 pairable damage equivalent to 5 Jm-2 of UV radiation (19). UV1, - cycloheximide, UV2 16 Other studies (35) and ours (unpublished) indicate that 10,uM UV1, + cycloheximide, UV2 12 AAAF and 10 J m-2 of UV radiation give similar postreplication No UV,, + cycloheximide, UV2 10 repair patterns. The approximate equivalence in dosimetry * from the measure of excision repair and postreplication repair Cells were irradiated with or without 2.5 J.m-2 (UV1), incubated is satisfying. The enhancement of postreplicative repair for 4 hr with or without 3 Mg/ml of cycloheximide, and then ex- of posed or not exposed to 5.0 J.m-2 (UV2). After 30 min in Eagle's AAAF- and UV-damaged DNA by both AAAF and UV irra- minimal medium without cycloheximide, the UV-irradiated and diation is consistent with the current hypothesis (19) that DNA nonirradiated cells were pulse-labeled with [3H]dThd for 30 and damages from these two agents (presumably alkylated purines 15 min, respectively (no chase). and pyrimidine dimers) are repaired by the same and/or t See Fig. 4. overlapping mechanisms. 2400 Biophysics: D'Ambrosio and Setlow Proc. Natl. Acad. Sci. USA 73 (1976)

Since enhanced postreplicative repair is not observed if cells 12. Witkin, E. M. & George, D. L. (1973) Genetics (Suppl.) 73, are incubated with cycloheximide or in Eagle's minimal me- 91-108. Repair dium lacking L-methionine after a normally inducing dose of 13. Radman, M. (1975) in Molecular Mechanisms for of DNA, eds. Hanawalt, P. C. & Setlow, R. B. (Plenum Press, New AAAF or UV radiation, we conclude that de novo protein York), pp. 355-368. synthesis is required for induction of enhancement. 14. Sedgwick, S. G. (1975) Nature 255,349-350. We conclude from our data that there are at least two 15. Sedgwick, S. G. (1975) J. Bacteriol. 123, 154-161. postreplication repair pathways in Chinese hamster cells. The 16. Setlow, R. B. & Grist, E. (1976) Biophys. J. 16, 183a. first is constitutive and slowly fills in the presumptive gaps left 17. Cleaver, J. E. & Bootsma, D. (1975) Annu. Rev. Genet. 9,19-38. opposite the AAAF or UV lesion in parental DNA. It is resistant 18. Lehmann, A. R., Kirk-Bell, S., Arlett, C. F., Paterson, M. C., to cycloheximide. The second is enhanced by AAAF treatment Lohman, P. H. M., DeWeerd-Kastelen, E. A. & Bootsma, D. and UV irradiation of cells and increases significantly the rate (1975) Proc. Nati. Acad. Sci. USA 72,219-223. repair of AAAF-or UV-damaged DNA above 19. Regan, J. D. & Setlow, R. B. (1973) in Chemical Mutagens of postreplication A. to constitutive pathway. The enhanced Principles and Methods for their Detection, ed. Hollaender, that due primarily the (Plenum Press, New York), Vol. 3, pp. 151-170. pathway is not observed in cells incubated with cycloheximide. 20. Setlow, R. B., Regan, J. D., German, J. & Carrier, W. L. (1969) Since an induced postreplication repair pathway seems to be Proc. Natl. Acad. Sci. USA 64,1035-1041. responsible for increased mutagenesis in E. coli (36), and since 21. Elkind, M. M. & Kamper, C. (1970) Biophys. J. 10, 237-245. an enhancement of repair is observed in cells of xeroderma 22. Carrier, W. L. & Setlow, R. B. (1971) Anal. Biochem. 43,427- pigmentosum variants after UV irradiation (16) and AAAF 432. treatment (S. M. D'Ambrosio, unpublished observation), we 23. Regan, J. D. & Setlow, R. B. (1974) Cancer Res. 34, 3318- hypothesize that the enhanced repair pathway may be re- 3325. sponsible for chemical and UV carcinogenesis in mammals. 24. Hutchinson, F. (1975) in Molecular Mechanisms for Repair of DNA, eds. Hanawalt, P. C. & Setlow, R. B. (Plenum Press, New York), pp. 703-707. We are grateful to E. Grist for her assistance. This research was 25. Elkind, M. M. (1971) Blophys. J. 11, 502-520. carried out at Brookhaven National Laboratory under the auspices of 26. Lett, J. T. (1975) in Molecular Mechanisms for Repair ofDNA, the U.S. Energy Research and Development Administration. eds. Hanawalt, P. C. & Setlow, R. B. (Plenum Press, New York), pp. 655-664. 1. Rupp, W. D. & Howard-Flanders, P. (1968) J. Mol. Biol. 31, 27. Palcic, B. & Skarsgard, L. D. (1972) Int. J. Radiat. Biol. 21, 291-304. 417-433. 2. Setlow, R. B. & Setlow, J. K. (1972) Annu. Rev. Biophys. Bioeng. 28. Korn, E. D. (1957) in Methods in Enzymology, eds. Colowick, 1,293-346. S. & Kaplan, N. (Academic Press, New York), Vol. 4, pp. 615- 3. Strauss, B. S. (1974) Life Sci. 15, 1685-1694. 642. 4. Lehmann, A. R. (1974) Life Scs. 15,2005-2016. 29. Carrier, W. L. & Setlow, R. B. (1971) in Methods in Enzymology, 5. Iyer, V. N. & Rupp, W. D. (1971) Biochfm. Biophys. Acta 228, eds. Grossman, L. & Moldave, K. (Academic Press, New York), 117-126. Vol. 21, pp. 230-237. 6. Meneghini, R. & Hanawalt, P. C. (1975) in Molecular Mecha- 30. Painter, R. B. & Young, B. R. (1975) Radiat. Res. 64,648-656. nisms for Repair ofDNA, eds. Hanawalt, P. C. & Setlow, R. B. 31. Housman, D. & Huberman, J. A. (1975) J. Mol. Biol. 94,173-181. (Plenum Press, New York), pp. 639-642. 32. Gautschi, J. R. & Kern, R. M. (1973) Exp. Cell Res. 80,15-26. 7. Buhl, S. & Regan, J. (1973) Nature 246,484. 33. Trosko, J. E., Chu, E. & Carrier, W. (1965) Radiat. Res. 24, 8. Higgins, N. P., Kato, K. & Strauss, B. (1976) J. Mol. Biol. 101, 667-672. 417-425. 34. Hart, R. W. & Setlow, R. B. (1974) Proc. Natl. Acad. Sci. USA 9. Rupp, W. D., Wilde, C. E., Reno, D. L. & Howard-Flanders, P. 71,2169-2173. (1971) J. Mol. Biol. 61, 25-44. 35. Goodman, J. I., Trosko, J. E. & Yager, J. D. (1976) Chem. Biol. 10. Ganesan, A. K. (1974) J. Mol. Biol. 87, 103-119. Interact. 12, 171-182. 11. Witkin, E. M. (1969) Annu. Rev. Genet. 3,525-552. 36. Sedgwick, S. G. (1975) Proc. Natl. Acad. Sd. USA 72,2753-2757.