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

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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 un- treated 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-acetyl- aminofluorene 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.
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