Proc. Nat!. Acad. Sci. USA Vol. 73, No. 11, pp. 3905-3909, November 1976 Biochemistry

5-Bromodeoxyuridine-DNA strand symmetry and the repair of photolytic breaks in Chinese hamster cell chromosomes* (DNA repair/cultured mammalian cells/somatic cell genetics/selection with bromodeoxyuridine-black light) DONALD J. ROUFA Division of Biology, Kansas State University, Manhattan, Kans. 66506 Communicated by Charles D. Michener, August 16,1976

ABSTRACr Experiments described in this report quantitate light-induced nicks (breaks in phosphodiester bonds) in their the black light sensitivities of Chinese hamster lung (CHL) cells DNA. Wild-type cells, which undergo more than one round of containing BrdUrd-DNA of defined composition. Cesium chloride equilibrium gradient centrifugation provides estimates DNA replication during the standard treatment with BrdUrd, both of the percent thymidine replacement by BrdUrd and of incorporate the analog into both strands of their DNA. The the symmetry (unifilar versus bifilar) of BrdUrd incorporation specificity of the selection appears due to CHL cells' inability into the chromosomal DNA duplexes. Radiation damage to to repair the double-stranded scissions that result from irra- BrdUrd-substituted CHL cell DNA and its repair in situ also diation of bifilar BrdUrd-DNA. have been assessed by alkaline sucrose gradient sedimentation. We observe that animal cell sensitivities to visible light (300400 nm wavelength) depend markedly upon the symmetries of MATERIALS AND METHODS BrdUrd-substitution within the cells' DNA. Cells that contain The origin of the CHL V79 cell clone, HT-1, has been described only unifilar BrdUrd-DNA are resistant to black light, whereas before, as have our sources of tissue culture media (Dulbecco's cells that contain bifilar BrdUrd-DNA are extremely photo- sensitive. The former cell populations repair single-stranded modification of Eagle's minimal medium) and reagents (2). nicks (breaks in phosphodiester bonds) in their DNA within 24 Optical grades of cesium chloride and sucrose for analytical hr of irradiation the latter cell populations, however, are not gradient centrifugation, methyl-labeled [3H]- and [14C]thy- able to repair light-induced, double-tranded breaks in their midine were purchased from Schwarz/Mann. [6-3H]BrdUrd DNA. was obtained from New England Nuclear Corp. Nonradioac- tive BrdUrd, thymidine, and hydroxyurea were purchased from We have used 5-bromodeoxyuridine/black light selection to Sigma Chemical Corp. Sarkosyl NL-97 was obtained from ICN isolate temperature-sensitive mutants of Chinese hamster lung Research Products. Bacteriophage T7 [3H]DNA and polyoma (CHL) cells that are unable to replicate DNA at 390 (2). The [3H]DNA form II were the generous gifts of, respectively, selection protocol was modified from the procedure originally Melvin S. Center and Richard A. Consigli of Kansas State developed by Puck and Kao (3), and since has been used by University. several investigators to isolate a variety of temperature-sensitive Tissue Culture Techniques. Procedures for culturing CHL (2, 4, 5) and auxotrophic (6-9) mutants. The rationale for se- cells have been described in detail (2, 11, 12). Cells in monolayer lection is based upon the photosensitivity of 5-bromodeoxyu- culture were treated with 10 LM BrdUrd under the conditions ridine (BrdUrd)-substituted DNA. Cells that synthesize DNA described for each experiment. Where indicated, cells con- at high temperature incorporate the halogenated thymidine taining BrdUrd-substituted DNA were rinsed twice with analog into DNA and sensitize their chromosomes to irradiation phosphate-buffered saline (PBS) and were irradiated with black (2). light according to the method described previously (2). Irra- Several temperature-sensitive, "mutant" CHL cell clones diations werecarried out with the lightsource (two GEF15T8/ now have been examined for their abilities to replicate DNA BLB bulbs in a Blak-Ray reflector, model XX-15) adjusted to at high temperature (2, 9, 10). Many of the temperature-sen- deliver a flux of 8.4 X 105 ergs/cm2-min (140 J/m2-sec). Black sitive clones incorporate BrdUrd into their chromosomal DNA light (300-400 nm wavelength) intensities were measured with at all temperatures examined, albeit at rates slightly less than a Blak-Ray longwave meter, model J-221. Cell survival after the rate characteristic of the wild-type clone. We therefore have irradiation was determined by efficiency of colony formation. examined the lethal action of BrdUrd/black light at a molecular Cell concentrations were determined on a Coulter model ZBI level, in order to understand why "slow replicators" survive particle counter. Cells (102, 103, 104, or 105) were inoculated selection. In this report we quantitate the substitution of Chinese into duplicate 60 mm plastic culture dishes (Falcon) and were hamster cell DNA with BrdUrd and correlate defined patterns incubated at 370 in a 10% CO2 atmosphere. Colonies that de- of BrdUrd-substitution with the cells' sensitivities to irradiation. veloped in 7 days were fixed with phosphate-buffered 4% Our data suggest that selective treatment with BrdUrd and formalin, stained with hematoxylin, and scored as described black light enriches cultures with clones that exhibit reduced before (2, 11, 12). Incorporation of ['4C]dThd and [3H]BrdUrd rates of DNA synthesis. Slowly replicating clones incorporate into acid-precipitable DNA was assayed in monolayer cultures BrdUrd into only one strand of their chromosomal DNA, and by procedures described before (2). Rates of thymidine and subsequently survive irradiation by repairing single-stranded, BrdUrd incorporation into DNA were determined under con- ditions where incorporation was linear with time for more than Abbreviations: BrdUrd, 5-bromodeoxyuridine; dThd, thymidine; PBS, phosphate-buffered physiological saline (1); CHL, Chinese hamster 24 hr on a per cell basis. lung; H, a DNA strand heavy because of BrdUrd substitution; L, light Centrifugation Analyses. Chinese hamster cell DNA sub- DNA. stituted with [3H]BrdUrd was analyzed by isopycnic CsCl * This paper is dedicated to the memory of Joseph F. Roufa, M.D., centrifugation according to the procedure of Meyn et al. (13). whose commitments to life inspired all who knew him. Cells growing in monolayer culture were labeled with [14C] 3905 Downloaded by guest on September 30, 2021 3906 Biochemistry: Roufa Proc. Natl. Acad. Sci. USA 73 (1976) dThd (0.25 ACi/ml) and/or [3H]BrdUrd (0.5 ,uCi/ml), as de- scribed for each experiment. Both radioactive substrates were added to cultures at concentrations of 10 AM. Following the labeling period, monolayers were rinsed with PBS and har- vested by treatment with trypsin (12). Cells were collected by centrifugation (700 X g for 10 min) and resuspended in 0.01 M Tris-HCl, 0.15 M NaCl, 0.01 M EDTA, pH 9.5, at a density of approximately 106/ml. The suspensions were adjusted to H-H LL 0.1% Sarkosyl and were incubated at 600 for 30min to dissolve E 11.8 chromatin DNA. Extracts were treated with a Biosonik Sonifier 4 g/CM3 (1 min at 70% maximum power) to reduce DNA to a more homogeneous population of small, double-stranded molecules 1000-3000 nucleotide pairs in length. Preparations were ex- 03 tracted one time with chloroform:isoamyl alcohol (24:1). 0 Samples (0.150 ml) were added to 4.0 ml of CsCl in distilled 8 1 2 3 4 4 water (density = 1.750 g/cm3), overlaid with 0.4 ml of mineral 2- oilt and centrifuged in the SW 50.1 rotor of a Beckman L2-65B ultracentrifuge at 33,000 rpm for 72 hr at 21°. Centrifuge tubes an then~wit 1.7rdfo4h t37 n hi DAwspe were pierced at the bottom and approximately 50 six-drop fractions were collected onto Whatman no. 1 paper filter pads. The pads were dried and assayed in Liquifluor scintillation cocktail (New England Nuclear) for detection of both [3H]- and 8 16 24 32 40 48 [14C]DNA. BOTTOM FRAc~iON TOP The effects of black light upon molecular size of CHL FIG. 1. CsCl gradient equilibrium centrifugation of CHL cell BrdUrd-DNA were assayed by alkaline sucrose gradient cen- DNA. Wild-type CHL cells were labeled for 2 days with in4CodThd and then withi[3HBrdUrd for 2437ehr at and their DNA was pre- trifugation as follows: DNA within monolayer cultures was pared for density gradient analysis as described in Materials and labeled with ['4C]dThd for at least 24 hr followed by Methods. The densities of each gradient fraction (X) were determined [3H]BrdUrd for the lengths of time indicated in each experi- from their refractive indices. [3H(BrdUrd cpm, (0); [14C]dThd cpm, ment. Conditions of labeling were the same as described above. (*. The labeled cells were irradiated with black light (107 ergs/cm2) and either harvested immediately or fed with nonradioactive only one generation. Cellular sensitivity to irradiation therefore medium and returned to a 330 incubator for 24 hr. Cells were appeared to depend upon the amount or the distribution of harvested from the monolayers by treatment with trypsin, and BrdUrd within newly synthesized CHL cell DNA (2). Since were resuspended at a cell density of 106/ml in 0.3 M NaCl, 0.3 pretreatment of CHL cell cultures with aminopterin or fluo- M NaOH, 0.01 M EDTA, 0.1% sodium dodecyl sulfate. After rodeoxyuridine, both known to render animal cells dependent chromatin had dissolved (370, 30 min), DNA in the lysates was upon exogenous thymidine (or BrdUrd), did not enhance the sheared slightly as it was layered above preformed sucrose rate of incorporation of [3H]BrdUrd into DNA (2), the amounts gradients with a large bore (2 mm diameter) plastic pipet of BrdUrd incorporated into CHL DNA under the conditions controlled at a flow rate of 1 ml/min. Linear 5-30% (wt/wt) employed in these experiments appear to be maximal. Thus, sucrose gradients (12.5 ml) were formed above at a 4 ml 40% the amounts of BrdUrd incorporated per unit of DNA repli- sucrose (wt/wt) cushion in SW 27.1 rotor tubes. All sucrose so- cated did not appear to limit the enhancement of photosensi- lutions also contained 0.3 M NaOH, 0.3 M NaCl, and 0.01 M tivity. In addition, several "mutant" CHL temperature-sensitive EDTA. The gradients were centrifuged at 25,000 rpm for 15 clones exhibit marked resistance to BrdUrd treatment at non- hr at 200, and were harvested with an ISCO model 640 gradient permissive temperature, even though their rates of DNA rep- fractionator. Fractions (0.6 ml) were collected, 15 Ag of carrier lication (and thus BrdUrd incorporation) were only slightly less DNA was added to each fraction, and radioactive DNA was than wild type's (unpublished). Thus, large effects upon CHL precipitated by addition of 0.15 ml of 50% trichloroacetic acid. cell sensitivity to BrdUrd/black light irradiation (103-fold) often Precipitates were filtered onto glass fiber discs (Whatman have accompanied very small reductions (<2-fold) in DNA GF/A), washed with 5% trichloroacetic acid, dried, and assayed biosynthetic rates, and suggest that subtle alterations in the in a liquid scintillation counter. All analyses were performed distribution (symmetry) of BrdUrd within newly replicated as double-label experiments with DNA containing both DNA might be responsible for large differences in the cells' [14C]dThd and [3H]BrdUrd. Weight-average molecular weights photosensitivity. of single-stranded DNAs were estimated by comparing the In order to analyze the extent and distribution of BrdUrd radioactive profiles with profiles of single-stranded DNA substitution in various CHL DNAs, we have employed CsCl standards (polyoma DNA II, 1.5 X 106 daltons, and T7 DNA, isopycnic gradient centrifugation. A typical analysis of DNA 1.3 X 107 daltons) by the method of Burgi and Hershey (14). extracted from CHL cells that had been treated first with ['4C]dThd and then with [3H]BrdUrd is illustrated in Fig. 1. RESULTS AND DISCUSSION Three peaks of DNA are evident: a peak of dense [3H]DNA that a of DNA both 3H Photosensitivity of BrdUrd-treated CHL cells bands at 1.78 g/cm3 (H-H), peak containing and 14C labels that bands at 1.75 g/cm3 (H-L), and a peak of When 10,uM BrdUrd is administered for various times to cul- [14C]DNA banding at 1.70 g/cm3 (L-L). The latter peak of tures of CHL cells, the cultures become increasingly more DNA bands at a density identical to that observed for DNA sensitive to irradiation with black light (2). CHL cells were prepared from CHL cells that have not been exposed to BrdUrd 100 fold more sensitive to irradiation after exposure to BrdUrd (not shown). By comparison to the data of others (15), the for two cell generations than after exposure to the analog for densities of BrdUrd-DNAs in this study suggest that >60% of Downloaded by guest on September 30, 2021 Biochemistry: Roufa Proc. Natl. Acad. Sci. USA 73 (1976) 3907 the thymidine in DNA has been replaced by BrdUrd. In addi-

tion, >93% of the DNA contained by the CHL cells analyzed CO was rendered dense by BrdUrd substitution (H-H or H-L) during the 24 hr of treatment with the analog, in agreement with the CHL cell generation time of 14 hr at 370 as measured -J by cell counts (unpublished). Since prolonged exposures (48 and o B 72 hr) to [3H]BrdUrd did not reduce the fraction of (L-L) DNA 0 extracted from CHL cultures similar to the one analyzed in Fig. 1, it appeared that the unsubstituted DNA (L-L) was contained by cells which lost viability during or after prelabeling with ["4C]dThd. To confirm this interpretation ['4C]dThd-labeled cultures were exposed to medium that contained BrdUrd and [3H]deoxycytidine. DNA was prepared from the culture and U-° 0.01 was analyzed by isopycnic CsCl centrifugation as in Fig. 1 (not 0 shown). The [3H]deoxycytidine-labeled DNA banded only with the H-H and H-L duplex DNAs, demonstrating that the [14C]dThd-labeled fraction of L-L DNA failed to incorporate both BrdUrd and deoxycytidine, and thus did not replicate after the 14C prelabeling. All of the DNA contained within repli- cating cells thus appeared uniformly substituted with BrdUrd, 0 DISTRIBUTION \ of the genome, at a res- 1- EXPT.HitsH-L L-L and no detectable regions replicating < ~~~A0 0 100 olution of DNA segments of 1000-3000 nucleotide pairs in a: 8 4.8 15.2\ U_ I 0 25 714\ length as in these experiments, avoided BrdUrd substitution \ during the standard exposure period. D 67 28 5 i D We have examined the symmetries of BrdUrd substitution >0.001 , | J within DNA prepared from cultures of CHL cells treated with 25 50 75 100 the analog according to several labeling protocols and have ergs/cm' X lo-' compared the cells' sensitivities to irradiation to the distribution FIG. 2. Sensitivity of CHL cells to BrdUrd/black light: depen- of BrdUrd within their duplex DNAs. These experiments are dence upon symmetry of BrdUrd substitution. Cultures of wild-type summarized in Fig. 2. The distributions of bifilar and unifilar CHL cells were not exposed to BrdUrd (A), or were treated with 10 BrdUrd-DNA in each cell population were analyzed by CsCl jiM MM~r for 14 hr at 37° (C), 10 1AM BrdUrd for 24 hr at 37° (D), gradient centrifugation (see Fig. 1), and are summarized in the or 10,gM BrdUrd for 6 hr at 33° following reversal of a "double thy- insert to Fig. 2. Curves A, C, and D illustrate the effect of ex- midine block" (B). The cultures were irradiated with the doses of cell cultures to BrdUrd for increasing black light indicated and survivors were determined by quantitative posing randomly growing efficiencies of colony formation. The distributions, expressed as %, lengths of time. As indicated in the insert, DNA extracted from of BrdUrd in the DNA of each population of cells were determined cultures treated with [3H]BrdUrd as in A, C, and D contained by CsCl equilibrium gradients on parallel cultures labeled with increasing proportions of bifilar (H-H) BrdUrd-DNA. In ex- I14C]dThd and [3H]BrdUrd as described in Materials and Meth- periment B synchronous cells, accumulated at the Gl-S phase ods. boundary by a "double thymidine block" (16), were treated with BrdUrd for 6 hr at 33'. Since the S phase of CHL cells at basis for these differences and to analyze the consequences of 330 is 7 hr (unpublished), no cells within population B were able black light irradiation on the integrity of duplex BrdUrd-DNA to replicate a chromosome twice during the 6 hr exposure to from Chinese hamster cells, we have analyzed the molecular BrdUrd. Thus, we expected and observed no bifilar BrdUrd- size of DNAs extracted from irradiated cultures by alkaline DNA in the sample prepared from the cells in population B. In sucrose gradient sedimentation. Initially we attempted to an- the absence of bifilar BrdUrd-DNA, CHL cells were only alyze very high-molecular-weight CHL DNAs by Iysing cells modestly sensitive to black light (17% survived 107 ergs/cm2 after layering them on the sucrose gradients. This procedure irradiation). When CHL cells contained 25% bifilar BrdUrd- allowed us to visualize single-stranded DNA up to 109 daltons, DNA (Curve C), they were 23 times more sensitive to black but resulted in heterogeneous and nonreproducible sedimen- light (0.75% survival). When 67% of the DNA contained tation profiles. Although we observed as much as 30% of the BrdUrd in bifilar distribution (Curve D), the cells were 1700 CHL DNA sedimenting with an apparent molecular weight times more sensitive to black light (0.01% survival). This ex- >5 X 108, 70% of the DNA in these preparations tailed into periment indicates that the sensitivities of mammalian cells lower molecular weight regions of the gradients (3 to 5 X 107). exposed to black light irradiation are affected markedly by the For this reason we expose CHL DNA to mild shear as it is symmetry of the BrdUrd-substitution, since cells in each pop- layered on the sucrose gradients (see Materials and Methods). ulation (B, C, and D) contained only slightly different molar Chinese hamster DNA prepared for alkaline sucrose gradients amounts of BrdUrd in their DNA; i.e., the molar ratios of as described in Materials and Methods sediments to the dense BrdUrd in the DNAs prepared from populations B, C, and D sucrose cushion as a population of molecules with single-strand can be calculated from data in the insert to Fig. 2 to be 2:3:4, molecular weights of 3 to 10 X 107 (Fig. 3). respectively. To examine the extent of photolytic damage to CHL cell of irradiated CHL BrdUrd- BrdUrd-DNA, cultures were prelabeled for 24 hr with Fragmentation and repair [14C~dThd and transferred into medium that contained 10 uM DNA [3H]BrdUrd for 14 hr (one generation) at 37°. Under these The data described above suggested that there are substantial conditions 70-75% of the DNA within the cultures was substi- differences between the cells' tolerances to irradiation of unifilar tuted with BrdUrd in unifilar symmetry, as judged by CsCl and bifilar BrdUrd-chromosomes. To investigate the molecular gradient analysis (see insert to Fig. 2). The cultures were irra- Downloaded by guest on September 30, 2021 3908 Biochemistry: Roufa Proc. Natl. Acad. Sci. USA 73 (1976)

-z

w 25 C),)3: 0 cr 20 0

15 r- I 0e 'aa -ia ' 10 x -J E C,)H a C.2 z 0RHS 5 z 0 1x 0 -c 0 I z 0 0 0 z w -c C- 0 0 3 0 -ox 0 r6 t 4 8 12 16 20 24 T 16 TOP FRACTION BOTTOM FIG. 3. Alkaline sucrose gradient analysis of CHL cell DNA. 12 Chinese hamster cells were labeled with ['4C]dThd and harvested for sucrose gradient analysis according to the procedure described in Materials and Methods. Radioactive cell suspensions (0.5 ml) (5 X 8 105 cells) and 50,000 cpm each of T7 [3HJDNA and polyoma (PY) [3H]DNA II were layered on the gradient and centrifuged as described in Materials and Methods. The gradients were harvested and frac- 4 tions (0.6 ml) were assayed for acid-precipitable [3H]- and ["4C]DNAs. 4 8 12 16 20 24 28 4 8 12 16 20 24 28 Molecular weights were estimated from the T7 and polyoma DNA TOP FRACTION BOTTOM standards by the method of Burgi and Hershey (14). The broken line FIG. 4. Black-light-induced fragmentation of CHL cell DNAs indicates the position of the 40% (wt/wt) sucrose cushion. (0), containing uni- and bifilar BrdUrd. Wild-type cells, prelabeled for [14C]DNA; (0), [3H]DNAs. 48 hr with [14C]dThd, were transferred into medium containing 10 gM [3H]BrdUrd for 14 hr (A and B) or for 48 hr at 370 (C and D). Under these conditions, cultures in A and B contained 70-75% unifilar diated and DNA was prepared for alkaline sucrose gradient BrdUrd-DNA and in C and D contained 85-90% bifilar BrdUrd-DNA. analysis. As illustrated in Fig. 4A, ['4C]dThd-labeled strands All cultures were exposed to 107 ergs/cm2 of black light irradiation of DNA sedimented to the dense sucrose cushion, identical to and either analyzed by alkaline sucrose gradient centrifugation im- nontreated DNA (Fig. 3). In contrast, [3H]BrdUrd-substituted mediately (A and C) or after 24 hr continued incubation at 330 (B and strands sedimented more slowly than the 14C-strands, indicating D). (0), % total [14C]dThd cpm; (0), % total [3H]BrdUrd cpm. light-induced scissions of only the BrdUrd-containing DNA strands. When BrdUrd was omitted from cultures or when DNA strand had occurred, we would not have observed a sig- cultures were not irradiated, both DNA strands (the '4C- and nificant reduction in the weight average distribution of that 3H-strands) cosedimented to the dense sucrose cushion (not DNA in the alkaline gradients. If as many as 1000 nicks had shown). Irradiation of CHL cells containing unifilar BrdUrd- been generated in each chromosome, the mean molecular DNA therefore "nicks" the BrdUrd-strand of DNA, a result weight of the BrdUrd-DNA would have been reduced to 0.9 consistent with the data of several other laboratories (reviewed X 107 or slightly smaller than the T7 DNA standard (1.3 X in ref. 17). 107). Inasmuch as CHL cells contain a genome of approximately Since Chinese hamster cell DNA contains 26% thymidine 3.6 X 1011 daltons of DNA (unpublished data), and since the (15), the average single-stranded chromosomal DNA molecule clone used in these studies contains 19 chromosomes per cell, (of 9.5 X 109 daltons, see above) contains approximately 7.5 X maximum chromosome-size, single-stranded DNA in this cell 106 residues of thymidine. Assuming uniform and random re- line is, on the average, 9.5 X 109 daltons. This assumes maxi- placement of 60% of the thymidine residues with BrdUrd (Fig. mum DNA size when each chromosome possesses a single du- 1), the average DNA strand is substituted with only 4.5 X 106 plex DNA molecule (18). Since the mild shear used in our residues of BrdUrd. Irradiation induced 100-1000 nicks per preparation reduces the single-stranded molecular weight average DNA strand, and thus must have involved fewer than (weight-average) of CHL DNA to 9 X 107 daltons (data not 0.02% of the BrdUrd residues contained by each chromosomal shown), the average molecule of chromosomal DNA is broken DNA molecule. approximately 100 times during extraction. After irradiation Wild-type cells survive high doses of black light after unifilar of [3H]BrdUrd-substituted cells, [3H]DNA fragments were BrdUrd substitution (Fig. 2). Since it appears from the experi- reduced further in size (Fig. 4A), suggesting that each chro- ment summarized in Fig. 4A that 100-1000 nicks are generated mosomal BrdUrd-DNA strand experienced at least 100 addi- within the BrdUrd-containing strand of each chromosome by tional nicks due to the irradiation. If fewer nicks per BrdUrd- irradiation, it is likely that nicked DNA is edited by cellular Downloaded by guest on September 30, 2021 Biochemistry: Roufa Proc. Nati. Acad. Sci. USA 73 (1976) 3909 repair mechanisms shortly after irradiation. We therefore have molecular basis for black light sensitivity, yielding a selective analyzed irradiated uni- and bifilar CHL BrdUrd-DNAs im- kill factor of 104 after BrdUrd treatment (Fig. 2D), appears to mediately after black light treatment (Fig. 4A and C) and after be mammalian cells' inability to repair irradiated bifilar a "repair" period of 24 hr in continued tissue culture (Fig. 4B BrdUrd-DNA (Fig. 4). In contrast, irradiated unifilar and D). As described above, irradiation of unifilar BrdUrd- BrdUrd-DNA is repaired within 24 hr (Fig. 4B), and is only DNA resulted in the nicking of the [3H]BrdUrd-strand only moderately selective in tissue culture (Fig. 2B). (Fig. 4A). After 24 hr of continued culture, however, the 3H- labeled strand was repaired sufficiently to cosediment with the The author wishes to thank Drs. R. A. Consigli and M. S. Center for [14C]dThd-labeled strand (Fig. 4B). Since 83% of the cells with their critical reading of this manuscript and for their suggestions during only unifilar BrdUrd-DNA did not survive 1o0 ergs/cm2 irra- the course of these experiments. He also acknowledges the technical diation (Fig. 2B), repair of the unifilar BrdUrd-DNA either is aid of Ms Harriet Meinicke and Mr. Jack Mollenkamp, without whose not complete or is not accurate in the majority of cells treat- skillful assistance these experiments could not have been undertaken, ed. and Mrs. Susan Hughes for her aid in preparing this manuscript. This work was supported by Grants NP-135A from the American Cancer When bifilar BrdUrd-DNA was irradiated, damage to DNA Society and GM23013 from the U.S. Public Health Service. This is was more extensive than was observed after irradiation of un- Contribution no. 1280-A, Division of Biology, Kansas Agriculture ifilar BrdUrd-DNA (Fig 4C). DNA fragments of T7 DNA size Experiment Station. (1.3 X i07 daltons) are produced by irradiating CHL cells that contain bifilar BrdUrd-DNA. Those fragments contained both 1. Dulbecco, R. & Vogt, M. (1954) J. Exp. Med. 99,167-199. [3H]BrdUrd and [14C]dThd, since 30% of the 14C label cosedi- 2. Roufa, D. J. & Reed, S. J. (1975) Genetics 80, 549-566. mented with the 3H in fractions 14-21 (Fig. 4G). During pro- 3. Puck, T. T. & Kao, F.-T. (1967) Proc. Natl. Acad. Sci. USA 58, longed exposure to [3H]BrdUrd (48 hr at 370) (i) a substantial 1227-1234. amount of ['4C]dThd-DNA had been repaired (non-semicon- 4. Naha, P. M. (1970) Nature 228, 166-168. servatively) with patches of BrdUrd-containing polynucleotide, 5. Scheffler, I. E. & Buttin, G. (1973) J. Cell. Physiol. 81, 199- or (ii) irradiation of bifilar BrdUrd-DNA resulted in double- 216. 6. Taylor, M. W., Souhrada, M. & McCall, J. (1971) Science 172, stranded breaks in the duplex DNA, or (id) irradiation induced 162-163. sister strand exchanges between [3H]- and [14C]DNAs. By 7. Chang, T.-Y. & Vagelos, P. R. (1976) Proc. Natl. Acad. Sci. USA whatever mechanism, radiation damage not only was more 73,24-28. extensive to bifilar BrdUrd-DNA than to unifilar BrdUrd-DNA, 8. Chu, E. H. Y., Sun, N. C. & Chang, C. C. (1972) Proc. Natl. Acad. but also, as shown in Fig. 4D, it was not repaired by CHL cells Sci. USA 69,3459-3463. during the 24 hr following black light treatment. Bifilar 9. Haralson, M. A. & Roufa, D. J. (1975) J. Biol. Chem. 250, BrdUrd-DNA within irradiated cells was degraded further to 8618-8623. fragments of 0.3 to.0.5 X 106 daltons during the 24 hr that fol- 10. Roufa, D. J. & Haralson, M. A. (1975) in DNA Replication and lowed irradiation. These fragments also appeared to involve Its Regulation, eds. Goulian, M. & Hanawalt, P. (W. A. Benja- DNA derived from both the 3H- min, New York), pp. 702-712. and 14C-labeled strands of the 11. Roufa, D. J., Sadow, B. N. & Caskey, C. T. (1973) Genetics 75, CHL DNA duplex. 515-530. The experiments described in this report demonstrate that 12. Gillin, F. D.,'Roufa, D. J., Beaudet, A. L. & Caskey, C. T. (1972) treatment of mammalian somatic cells with BrdUrd/black light Genetics 72,239-252. enriches the cell cultures for clones that do not synthesize bifilar 13. Meyn, R. E., Hewitt, R. R. & Humphrey, R. M. (1975) in Methods BrdUrd-DNA during the treatment. The procedure has been in Cell Biology (Academic Press, New York), Vol. IX, pp. used to obtain in tissue culture a variety of auxotrophic mutants 103-113. (3, 6, 7, 19), nutritionally limited mutants (8), and tempera- 14. Burgi, E. & Hershey, A. D. (1963) Biophys. J. 3,309-321. ture-sensitive mutants possessing several conditional defects 15. Bick, M. D. & Davidson, R. L. (1974) Proc. Natl. Acad. Sci. USA (2, 4, 5). Several CHL clones which we have isolated attained 71,2082-2086. 16. Puck, T. T. (1964) Cold Spring Harbor Symp. Quant. Biol. 29, resistance to BrdUrd/black light by reducing their rates of DNA 167-176. replication only 20-60% (unpublished). Slight reductions in 17. Hutchinson, F. (1973) Rev. Biophys. 6,201-246. rates of DNA synthesis were sufficient to insure that no mutant 18. Kavenoff, R., Klotz, R. C. & Zimm, B. H. (1973) Cold Spring cells replicated a chromosome twice in BrdUrd-a condition Harbor Symp. Quant. Biol. 28, 1-8. necessary for the generation of bifilar BrdUrd-DNA. The 19. Kao, F.-T. & Puck, T. T. (1967) Genetics 55,513-524. Downloaded by guest on September 30, 2021