Repair-deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant, which is noted for its very high sister-chromatid exchange frequencies. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to chlorodeoxyuridine (CldUrd). After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25-30 kilobases (kb) was present. Since the DNA molecules used to produce these transformants were sheared to <50 kb in size, the correcting gene should be small enough to clone in a cosmid vector.

Using drug-resistance markers to select for hybrids after fusion, we have done complementation experiments with ultraviolet light (u.v.)-sensitive mutants and have identified a sixth complementation group, line UV61. Additionally, CHO mutants UV27-1 and MMC-2, isolated in other laboratories, were found to belong to UV group 3, which is represented by line UV24.

To study the behaviour of transfected DNA molecules in repair-deficient cells, we treated plasmid pSN2gpt with either u.v. radiation or cA-diamminedichloroplatinum(II) (cA-DDP) and introduced the damaged DNA into normal CHO cells (AA8) and mutants UV4 and UV5. Unrepaired damage to the plasmid was indicated by loss of colony-forming ability of the transfected cells in selective medium containing mycophenolic acid. With u.v. damage, the differential survival of the cell lines was similar to that seen when whole cells are treated with u.v. However, with cw-DDP damage, mutant UV4 did not exhibit the extreme hypersensitivity (50-fold) that occurs when cells are treated. This result suggests that UV4 cells may be able to repair cross-links in transfected DNA.

In our laboratory we have been characterizing DNA repair mutants of Chinese hamster ovary (CHO) cells as a tool for studying mechanisms of genetic change and mutagenesis (Thompson, 1985; Thompson & Hoy, 1986; Thompson et al. 1986). It is evident that repair mutants of rodent cells provide an important approach for isolating human (and rodent) genes involved in repair pathways. Westerveld et al. (1984) recently cloned a human gene that corrects ultraviolet light (u.v.)-sensitive mutants belonging to UV complementation group 2 described by Thompson et al. (1981). Efforts are under way in our laboratory and others to isolate genes that correct additional mutants that have sufficient hypersensitivity to provide an efficient selection system.

Earlier in our laboratory we identified five complementation groups of u.v. mutants (Thompson et al. 1981; Thompson & Carrano, 1983), all of which involve a defect in the incision step of repair (Thompson et al. 1982a). These mutants, therefore, have properties quite similar to those of xeroderma pigmentosum (XP) cells (Friedberg et al. 1979; Cleaver, 1983). Mutants from each of the groups show a stable phenotype in response to 5-azacytidine treatment, suggesting that they did not arise by gene inactivation associated with methylation (Jeggo & Holiday, unpublished). We have obtained evidence from chromosome mapping studies that the five complementation groups represent at least four different human genes. In hybrid cells, the mutation of line UV20 (group 2) was corrected by a gene on human chromosome 19 (Thompson et al. 1985; Rubin et al. 1985). Mutant UV5 (group 1) also appears to be corrected by human chromosome 19, but the mutants of groups 3, 4 and 5 show correction by chromosomes 2, 16 and 13, respectively (L. Thompson, M. Siciliano & A. Carrano, unpublished data). Correction by different chromosomes indicates that different human genes are involved. It is conceivable that the two complementation groups corrected by chromosome 19 are due to interallelic complementation of a single gene. However, this possibility seems unlikely because the mutants in groups 1 and 2 consistently show very different phenotypes in terms of their sensitivity to DNA cross-linking agents (Hoy et al. 1985). The fact that two of the u.v. repair genes are linked on chromosome 19 could have functional significance, especially if they are close together. The isolation of the UV5-complementing gene is needed to give the appropriate DNA probes to determine the linkage. Gene transfer experiments in progress indicate that the human gene that corrects UV5 is functional in purified DNA and small enough to clone in a cosmid vector (unpublished data).

We are also proceeding to clone a human gene that corrects the EM9 mutant line, which is noted for its greatly elevated frequency of sister-chromatid exchange (SCE), defective repair of strand breaks, and reduced rate of DNA maturation when bromodeoxyuridine (BrdUrd) is in the template strand (Thompson et al. 19826; Dillehay et al. 1983). The SCEs in EM9 cells result primarily from the BrdUrd incorporation that is used in the standard SCE protocol (Pinkel et al. 1985). All the enzymes involved in DNA metabolism that have been examined in EM9 cells were found to be normal (for references, see Thompson et al. 1985).

In hybrid cells, the EM9 defect is corrected by human chromosome 19 (Siciliano et al. 1986). DNA isolated from one of these resistant hybrids was also shown to correct EM9 in the DNA transfection procedure (Thompson et al. 1985). The data presented here on recent DNA transformants give our progress toward isolating the transferred human gene. We hope to use this gene to determine what gene product is defective in EM9 cells and to use this product to study the mechanism of SCE formation.

In addition, we present the results of recent efforts to identify new UV complementation groups of CHO cells. A sixth group has been found with line UV61. We also describe the behaviour of mutants UV5 (group 1) and UV4 (group 2) in response to transfected plasmid DNA that has been damaged with u.v. or cis- diamminedichloroplatinum(II) (cis-DDP).

Culture conditions

Stock cell lines were grown in suspension as described earlier (Thompson et al. 1980, 1982a,b) in a-MEM medium supplemented with 10 % foetal bovine serum (K. C. Biological, Lenexa, KA). Medium contained 100 units ml −1 of penicillin and 100 μgml −1 of streptomycin sulphate. Stocks were renewed every 3 months from material in liquid nitrogen and periodically tested for mycoplasma. The mutant lines were checked about once a month for sensitivity. Plasticware for monolayer culture and colony formation was from Corning.

Cell fusion complementation tests

For fusion, 2 ×101 cells (1:1 ratio of each type) were inoculated into a 6cm dish and allowed several hours for attachment. Cells were exposed for 60s to 2ml of a solution containing 47% polyethylene glycol 1000 (Baker Chemical Co.) and 10% dimethyl sulphoxide (DMSO) in medium. The cells were rinsed twice with 10% DMSO in medium and incubated in normal medium for ≈24 h for hybrid cell formation. Cells were then trypsinized, counted, and plated at 2 ×101 cells per 10 cm dish. After 4h dishes were rinsed with phosphate-buffered saline and irradiated with far u.v. as described by Thompson et al. (1980). For colony formation, cells were then incubated 10 days in normal medium or medium containing 1 mM-ouabain (Sigma) plus hypoxanthine (74 μM), amethopterin (550nM), and thymidine (41 μM), HAT medium. Colonies around the periphery of the dish, where shielding occurs during irradiation, were not counted.

Transfection of EM9 with sheared DNA

DNA from the secondary transformant 9TT3 (Thompson et al. 1986) was sheared so that almost all the molecules were below 50 kb. This was done by passing the DNA four times through a 30 gauge needle. Sheared genomic DNA was combined with pSV2gpt DNA in a 1:1 ratio (w/w). Calcium phosphate precipitates were prepared as described by Corsaro & Pearson (1981). To each dish of 2 ×101 EM9 cells containing 10 ml of medium, 40 μg of DNA in 1ml was added. Sixty dishes were exposed to DNA for 20 h. Dishes were rinsed with 10ml of medium and given 30 ml of fresh medium for a 48 h incubation for expression. Each dish was trypsinized and replated into three replicates with 30 ml of medium containing both MAXTA supplements and CldUrd (chlorodeoxyuridine) as described by Thompson et al. (1985) for selection of gpi-positive, repairproficient cells. On days 4 and 8, 15 ml of fresh medium was added, and only the CldUrd selection was continued. Eleven to 15 days after plating, five colonies were isolated and grown to mass culture under CldUrd selection.

Transfer of pSV2gpt DNA treated with u.v. radiation or cis-DDP

For exposure to u.v. radiation, pSV2gpi DNA was diluted into a solution containing 0 ·25 M- CaCU, 1 mM-Tris - HCl (pH 8), and 10mM-NaCl; 0 ·5 ml was irradiated in a 35 mm plastic dish. A 3 μg sample of precipitated DNA (see above) was added in a volume of 0 ·5 ml to each 10 cm dish containing 2 ×101 cells. A lower volume of precipitate was used here, compared to the case of genomic DNA, to reduce toxicity. The precipitate was left on the cells for 16 –20 h. Then the dishes were rinsed, given fresh medium, and incubated for 24 h for expression of gpt function. Cells were trypsinized and replated at 1 ×101 to 2 ×101 cells per 10 cm dish in MAXTA-supplemented medium (Thompson et al. 1985) containing 2-5 times the standard concentration of glutamine. Three replicate dishes were plated for each dose point, and duplicate untreated controls were used in each experiment. Plating efficiency was determined at each dose by plating 300 cells into three replicate dishes, and these values (0 ·7 –0 ·95) were used to calculate the frequency of MAXTA- resistant colonies per viable cell plated. MAXTA selection dishes were incubated 12 –14 days, and plating efficiency dishes were incubated 7 –9 days.

For exposure to cis-DDP, the DNA in 0·4ml of 10 mM-Tris. HCl buffer (pH8) was combined with cis-DDP (100 μgml−1 in 10 mM-Tris. HCl) and incubated at 37°C for 1 h. The DNA in 0·3 M- sodium acetate (pH 5) was precipitated with ethanol at −70°C, then redissolved in 10 mM- Tris -HCl, 10mM-EDTA (pH 7·4) for calcium phosphate precipitation. A 2μg sample of DNA in 1 ml was added to each 10 cm dish containing 4×101 cells in 10 ml of medium, and the cells were incubated at 37·C for 4h. The medium was aspirated gently, and 2·5 ml of 20% glycerol in medium (v/v) at room temperature was added to the side of the dish. After 1 min, the glycerol was removed, and the dishes were rinsed three times with serum-free medium and given 20 ml of growth medium. After 24 h incubation the cells were plated for MAXTA selection as described above for u.v.-treated DNA.

Molecular hybridization

DNAs were digested with restriction enzymes, separated by electrophoresis, and transferred from agarose gels to nitrocellulose filters by the method of Southern (1975). Prehybridization of filters was done at 65°C with 25ml of a solution containing 5×SSC (1×SSC is 150mM-NaCl, 15mM-sodium citrate), 50μgml−1 of denatured salmon sperm DNA, and 0·1% each of Ficoll, bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), and sodium dodecyl sulphate (SDS). Hybridization was carried out for 18 h in 20 ml of a solution consisting of the prehybridization ingredients plus 10 % dextran sulphate and the probe DNA, which contained 4·6× 101ctsmin−1 in ≈ 300 ng of the Alu family BLUR-8 sequence (Deininger et al. 1981) isolated as a Bam HI fragment. The probe was labelled with [α- 1P]dCTP by nick translation. Filter washes were done as follows: first, in 2×SSC + 0·1 % each of Ficol, BSA, PVP and SDS (once for 5 min, three times for 15 min each); second, in 1×SSC + 0·1 % each of SDS, Ficoll, BSA and PVP (four times for 30min each); third, in 1×SSC at room temperature. The filter was exposed to Kodak X-OMAT AR5 film with an intensifying screen (Cronex Lightning-Plus, DuPont) for ≈3 days.

Progress toward isolating a human DNA repair gene that corrects the EM9 mutation

Earlier, we introduced into EM9 cells human DNA sequences that normalized the SCE level and restored resistance to mutagens (Thompson et al. 1985). It is interesting to note that, unlike SCEs, chromosomal aberrations induced by BrdUrd remained slightly elevated in the primary transformants (Thompson et al. 1985). This result suggested that the transfected human gene was unable to correct fully all aspects of the biochemical defect in EM9.

Our strategy has been to identify the correcting human gene in the background of EM9 hamster DNA on the basis of the linkage of the gene to the human Alu family repetitive sequences. These repetitive sequences are associated with most genes (Schmid & Jelinek, 1982). The initial transfection was done with high molecular weight DNA (>160 kb) isolated from hybrid cells, which were derived by fusing EM9 cells with normal human lymphocytes. A 3·8 kilobase (kb) human EcoRI restriction fragment, which was present in 6/6 primary transformants (Thompson et al. 1985), looked promising as being part of the repair gene, and this sequence was isolated from a cosmid library of DNA from one of the primary transformants (see below). However, this fragment was later found to be absent in 3/6 secondary transformants (Thompson et al. 1986). To determine whether the repair gene was small enough to clone in a cosmid vector, a DNA transfer was performed using sheared DNA. DNA from the secondary transformant 9TT3 was sheared such that most of the molecules were 25–50 kb in size. This DNA was coprecipitated with pSV2gpt DNA and used to treat 1·2×101 EM9 cells. Five independent colonies were obtained that were able to grow in medium containing both MAXTA and CldUrd, implying that cotransfer of functional gpt and repair genes had occurred.

During the course of these experiments the resolution of the human restriction fragments on Southern blots was improved by using the 300 base-pair (bp) Alu family sequence (BLUR-8) as the probe and by optimizing the hybridization conditions. (The analysis of the primary clones was done using total human DNA, and the presence of large human restriction fragments could not be discerned in the high molecular weight region of the blots.)

The DNAs of the five tertiary clones were analysed for the presence of human sequences (Fig. 1). The prominent feature of these FcoRI-digested DNAs was the presence of a band corresponding to a human fragment of 25–30 kb in each of the transformants. The restriction fragment at 3·8kb in the lane of 9TT3 donor DNA was not present in any of the tertiary clones. This result implies that the 3·8 kb fragment was a sequence flanking the complementing gene and was lost when the DNA was sheared. No bands were visible in the control lane of CHO DNA (from mutant UV135). The important large common fragment has similar intensity in each of the five independent transformants, suggesting that it is present as a single copy in each cell line. Lane 1 of Fig. 1, which illustrates the detection of human repetitive sequences, contains the recombinant cosmid pH9T12-l present at a level equivalent to five copies per cell. This cosmid clone contains an insert of about 40 kb of human DNA, which includes the 3·8 kb fragment seen in the primary transformants.

Fig. 1.

Autoradiogram of a Southern transfer of DNA and hybridization to detect human sequences in tertiary transformants of mutant EM9. DNA from the secondary transformant 9TT3 was sheared to <50 kb and used to produce the five transformants designated 9TTT1, 9TTT2, 9TTT3, 9TTT4 and 9TTT5. DNAs were digested with.EcoRI, subjected to electrophoresis in a 0·8% agarose gel, transferred to nitrocellulose, and probed with nick-translated BLUR-8 Alu family sequence. The common fragment at ≈25 kb in the five transformants probably contains repair gene sequences. The lanes containing UV135 DNA are negative controls (see the text), and the lane containing DNA from the cosmid pH9T12-l is a positive control. The positioning of the molecular weight (in kb) markers (lambda phage DNA intact and digested with Hind III) takes into account the curvature of the gel. See Materials and Methods for hybridization conditions.

Fig. 1.

Autoradiogram of a Southern transfer of DNA and hybridization to detect human sequences in tertiary transformants of mutant EM9. DNA from the secondary transformant 9TT3 was sheared to <50 kb and used to produce the five transformants designated 9TTT1, 9TTT2, 9TTT3, 9TTT4 and 9TTT5. DNAs were digested with.EcoRI, subjected to electrophoresis in a 0·8% agarose gel, transferred to nitrocellulose, and probed with nick-translated BLUR-8 Alu family sequence. The common fragment at ≈25 kb in the five transformants probably contains repair gene sequences. The lanes containing UV135 DNA are negative controls (see the text), and the lane containing DNA from the cosmid pH9T12-l is a positive control. The positioning of the molecular weight (in kb) markers (lambda phage DNA intact and digested with Hind III) takes into account the curvature of the gel. See Materials and Methods for hybridization conditions.

As an additional control, two of the lanes in Fig. 1 contained UV135 DNA to which was added the equivalent of 1× or 10× copies per cell of the pSV2gpt DNA. Because of the cotransfer procedure used, all the transformants contain pSV2gpt. Since the 300 bp BLUR-8 sequence was isolated from pBR322, which has homology with pSV2gpt, we wanted to ensure that the BLUR-8 probe was not contaminated with vector sequences. No bands were evident in these two control lanes. Because these transformants were made from DNA <50 kb, the human fragment at ≈30 kb probably contains at least a part of the correcting gene. The gene may extend beyond this fragment since none of the transformants recovered show evidence of breakage of this fragment. The gene should be small enough to clone in a cosmid vector, which can accept inserts up to about 45 kb. Efforts toward reaching this objective are under way.

Line UV61 represents a sixth complementation group for u.v. sensitivity

Preliminary tests on the mutant line UV61 (previously designated 6-56-37 by Busch (1980)) were performed by the rapid complementation procedure described earlier (Thompson et al. 1981). These results suggested that this mutant might complement the five existing groups (Thompson et al. 1981; Thompson & Carrano, 1983) but were equivocal because the u.v. sensitivity of UV61 is less than that of the other mutants. As shown in Fig. 2, the u.v. fluence required to cause a given level of killing of UV5 cells is only about 60% as much as that required to give the same killing of UV61. The D37 values for UV5, UV61 and parental AA8 cells are 2·2, 3·8 and 10·6 J m−2, respectively. It is of interest to note that these D37 values for UV5 and AA8 are somewhat higher than our previously published values (Thompson et al. 1980, 1981). We attribute these differences to changes in culture conditions, possibly the use of dialysed serum in earlier experiments.

Fig. 2.

u.v. survival curves of parental AA8 and mutants UV5 and UV61. Cells were irradiated at a density of 2×101 cells per 10cm dish, trypsinized, and plated at varying densities for colony formation. Error bars are standard errors of the means for values from two or three experiments. Average plating efficiencies for UV5, UV61 and AA8 were 0·86, 0·88 and 0·90, respectively. Symbols: (▵) AA8; (○) UV61; (▫5) UV5.

Fig. 2.

u.v. survival curves of parental AA8 and mutants UV5 and UV61. Cells were irradiated at a density of 2×101 cells per 10cm dish, trypsinized, and plated at varying densities for colony formation. Error bars are standard errors of the means for values from two or three experiments. Average plating efficiencies for UV5, UV61 and AA8 were 0·86, 0·88 and 0·90, respectively. Symbols: (▵) AA8; (○) UV61; (▫5) UV5.

Drug resistance markers were used to make the complementation tests more efficient. This approach is illustrated by the data in Table 1 for the mutants UV4 and UV41, which have been assigned to complementation groups 2 and 4, respectively (Thompson et al. 1981). The use of one line having both thioguanine and ouabain resistance (shown by the designation TOR) allows one to select for hybrids without having a selectable marker on each of the fusion partners (Baker et al. 1974). By selecting for hybrids after u.v. treatment, the background of colonies from surviving parental cells is eliminated. As shown in the last line of Table 1, when UV4 was fused with UV41-TOR, the frequency of hybrids was 0·0012 in the absence of u.v. irradiation. After exposure to 6Jm−2, about 50% of these hybrids survived, indicating that they were relatively resistant although not as resistant as AA8 cells. (This incomplete complementation was shown previously by survival curves of individual hybrid clones (Thompson et al. 1981).) When UV41 was crossed with its derivative line UV41-TOR, no u.v.-resistant hybrids were formed, which was the expected result. Also, in the three self-cross controls, there were no detectable u.v.- resistant colonies, again as expected.

Table 1.

Illustration of complementation tests using drug resistance markers with mutants from groups 2 and 4

Illustration of complementation tests using drug resistance markers with mutants from groups 2 and 4
Illustration of complementation tests using drug resistance markers with mutants from groups 2 and 4

Similarly, UV61 was fused with mutants from each of the five complementation groups that had the ‘TOR’ phenotype, as shown in Table 2. The lines used were derivatives of UV5, UV20, UV24, UV41 and UV135, which belong to groups 1 through 5, respectively. These mutants were all isolated from the AA8 parental line. Mutant UV27-1, which belongs to group 3 (see below), has a different origin. In each cross of UV61 with a TOR line, a high frequency of u.v.-resistant hybrids was formed. These frequencies were in the same range as that seen with the complementing pair of mutants shown in Table 1. In each of the self-crosses in Table 2, no colonies were seen. These results indicate that UV61 complements each of the first five groups.

Table 2.

Line UV61 complements mutants from UVgroups 1 through 5

Line UV61 complements mutants from UVgroups 1 through 5
Line UV61 complements mutants from UVgroups 1 through 5

We showed above that the u.v. sensitivity of UV61 differs from that of the other mutants shown in Table 2. It is interesting to note that the biochemical defect in UV61 also appears to differ. Groups 1 through 5 have essentially no incision after u.v. treatment (Thompson et al. 1982a), but UV61 has an intermediate level of unscheduled DNA synthesis (D. Bootsma, personal communication), suggesting partial incision activity.

Mutant UV27-1 belongs to complementation group 3

Clone ‘27-1’ was isolated by Wood & Burki (1982). (We added the ‘UV’ prefix to indicate that it belongs to the collection of u.v.-sensitive lines.) On the basis of the data in Table 3, we have assigned UV27-1 to group 3, which contains UV24. In each cross with the TOR lines several hundred colonies were obtained, except with UV24-TOR. (We previously reported (International Conference on Mechanisms of Antimutagenesis and Anticarcinogenesis, Lawrence, KA, Oct. 6–10, 1985) that UV24 complemented UV27-1, but this result was in error due to mislabelling of a frozen stock.) Another mutant line MMC-2, which was isolated by Robson et al. (1985), was also found to belong to group 3 (results not shown).

Table 3.

UV27-1 belongs to complementation group 3

UV27-1 belongs to complementation group 3
UV27-1 belongs to complementation group 3

Survival of plasmid pSV2gpt in normal and u.v.-sensitive lines

The plasmid pSV2gpt provides a convenient system for evaluating the repair of damaged DNA molecules introduced by the calcium phosphate precipitation procedure. In MAXTA selection medium, which includes mycophenolic acid, the bacterial gpt gene serves as a dominant selectable marker (Mulligan & Berg, 1981). Loss of gpt function can be determined by measuring the survival of transfected cells in MAXTA medium. One advantage of this approach to studying repair is that the damage is localized to the target DNA sequence.

In the present study we were interested in the question of whether the u.v. repair mutants would exhibit a repair-deficient phenotype when the plasmid was damaged with mutagens and transfected into the cells. Cells that can repair damage that would otherwise inactivate the plasmid should express a functional gpt gene and form colonies in selective medium. Thus, frequencies of transformation were determined as a function of the dose of DNA-damaging agent to which the plasmid was exposed.

As seen in Fig. 3, when the plasmid was treated with u.v. radiation, it was three to four times more resistant when transfected in the normal CHO cells (AA8) compared with the repair-deficient lines UV5 and UV4 (groups 1 and 2, respectively). These two mutants, which have similar survival responses to u.v. (Thompson et al. 1980; Busch et al. 1980), also showed the same response with irradiated plasmid. The differential survival between the normal and mutant cells was slightly less than that obtained with irradiated cells. Perhaps the repair of transfected plasmid DNA is less efficient than genomic DNA. At low u.v. fluence we observed a twofold enhancement of the frequency of transformation to the gpt+ phenotype in normal CHO cells, but the u.v.-sensitive lines showed no evidence of this effect. Overall, our results with irradiated plasmid differ in several respects from those seen with normal and XP human cell lines. The normal human cells showed higher levels of enhancement of transformation by u.v., and the same effect was seen with XP cells (Spivak et al. 1984; van Duin et al. 1985). In both studies the XP cells showed transformation frequencies that were as high as those obtained with normal cells, suggesting that in the plasmid transformation assay the XP (group A) cells could repair u.v. damage. However, with damaged viral DNA a repair defect has been seen in XP cells. Using a plaque assay for host cell reactivation of adenovirus 2, Day (1974) consistently found XP lines to have a more sensitive response to u.v.- irradiated virus than did normal human cells.

Fig. 3.

Relative frequency of MAXTA-resistant colonies of AA8, UV4 and UV5 cells transfected with u.v.-irradiated pSV2gpt DNA. The absolute frequencies of MAXTA- resistant colonies in the absence of u.v. damage were5×10−5 to 18×10−5, 3×10−5, and 8×10−5 to 18×10−5 for AA8, UV4 and UV5, respectively. Error bars show standard errors of the mean for repeat experiments of AA8 and UV5. For the AA8 data an exponential best fit was done for the points between 400 and 2600 J m−2 and the remainder of the curve was drawn by eye. For UV5, a linear best fit with a line going through a survival of 1 - 0 at zero dose was performed. Symbols: (○) AA8; (▴) UV4; (▫) UV5.

Fig. 3.

Relative frequency of MAXTA-resistant colonies of AA8, UV4 and UV5 cells transfected with u.v.-irradiated pSV2gpt DNA. The absolute frequencies of MAXTA- resistant colonies in the absence of u.v. damage were5×10−5 to 18×10−5, 3×10−5, and 8×10−5 to 18×10−5 for AA8, UV4 and UV5, respectively. Error bars show standard errors of the mean for repeat experiments of AA8 and UV5. For the AA8 data an exponential best fit was done for the points between 400 and 2600 J m−2 and the remainder of the curve was drawn by eye. For UV5, a linear best fit with a line going through a survival of 1 - 0 at zero dose was performed. Symbols: (○) AA8; (▴) UV4; (▫) UV5.

In a second set of transfection experiments we used the compound cA-diammine- dichloroplatinum(II) (cis-DDP), which, like u.v., produces bulky adducts but also produces DNA cross-links. This compound has been shown to produce DNA intrastrand cross-links as well as low, but potentially toxic, levels of interstrand cross-links (Plooy et al. 1985) in CHO cells. Under our conditions of treatment of the plasmid a very high percentage of the molecules should have interstrand cross-links (Poll et al. 1984). The pattern of responses with the three cell lines was similar to that seen with u.v. except that the mutants showed less difference compared with AA8 (Fig. 4). UV5 cells had a response that was about twice as sensitive as that of AA8 cells, and UV4 cells had a threefold more sensitive response. This result for UV5 is similar to the differential sensitivity seen with cA-DDP-treated cells in a cytotoxicity assay (Hoy et al. 1985). However, the behaviour of UV4 contrasts sharply with our finding that UV4 cells were 50 times more sensitive than AA8 in the cytotoxicity assay (Hoy et al. 1985). Since UV4 cells were also very sensitive to many other agents known to produce DNA interstrand cross-links, the results obtained with cis-DDP- treated plasmid were unexpected.

Fig. 4.

Relative frequency of MAXTA-resistant colonies of AA8, UV4 and UV5 cells transfected with pSV2gpf DNA treated with CTS-DDP. The absolute frequencies of MAXTA-resistant colonies in the absence of ds-DDP were 19×10−5 to 23×10−5, 14 × 10−5, and 13×10−5 to 20×10−5 for AA8, UV4 and UV5, respectively. Error bars show standard errors of the mean for repeat experiments. For each cell line an exponential best fit was obtained with a line going through a survival of 1·0 at zero dose. Plasmid DNA was exposed to cis-DDP for 1 h at 37°C. Symbols: (▫) AA8; (▵) UV4; (○) UV5.

Fig. 4.

Relative frequency of MAXTA-resistant colonies of AA8, UV4 and UV5 cells transfected with pSV2gpf DNA treated with CTS-DDP. The absolute frequencies of MAXTA-resistant colonies in the absence of ds-DDP were 19×10−5 to 23×10−5, 14 × 10−5, and 13×10−5 to 20×10−5 for AA8, UV4 and UV5, respectively. Error bars show standard errors of the mean for repeat experiments. For each cell line an exponential best fit was obtained with a line going through a survival of 1·0 at zero dose. Plasmid DNA was exposed to cis-DDP for 1 h at 37°C. Symbols: (▫) AA8; (▵) UV4; (○) UV5.

These results suggest that cross-links might not be nearly as toxic to UV4 when introduced in the plasmid as when present in the genomic DNA molecules. One interpretation is that in UV4 cells cross-links in naked DNA molecules can be repaired rapidly and efficiently compared with DNA in nucleosomes. For example, evidence has been presented that many of the mutations in the u.v. excision repair pathway in human cells act at the level of chromatin rather than unprotected DNA (Mortelmans et al. 1976; Kano & Fujiwara, 1983). In addition, the critical unhooking event for cross-links may have much faster kinetics than the removal of pyrimidine dimers (Reid & Walker, 1969) and occur before integration of the plasmid DNA into the genome can occur. Our results with UV4 are analogous to those seen with Fanconi’s anaemia (FA) cells, which are characteristically very hypersensitive to killing by cross-linking agents (Ishida & Buchwald, 1982). Poll et al. (1984) found FA cells to be very sensitive to killing by cis-DDP, but in a host cell reactivation assay in which simian virus 40 (SV40) DNA was treated with cis-DDP, the FA cells showed a normal response. Alternatively, the unexpected results with UV4 might be due to a different spectrum of lesions formed in vitro versus in vivo. Clearly much remains to be learned about the role of chromatin and other factors in the nucleotide excision repair process in both rodent and human cells.

This work was performed under the auspices of the US Department of Energy by the Lawrence Livermore National Laboratory under contract no. W-7405-ENG-48.

Baker
,
R. M.
,
Brunette
,
D. M.
,
Mankovitz
,
R.
,
Thompson
,
L. H.
,
Whitmore
,
G. F.
,
Siminovitch
,
L.
&
Till
,
J. E.
(
1974
).
Ouabain-resistant mutants of mouse and hamster cells in culture
.
Cell
1
,
9
21
.
Busch
,
D. B.
(
1980
).
Large scale isolation of DNA repair mutants of Chinese hamster ovary cells. Ph.D. dissertation, University of California, Berkeley
.
Busch
,
D. B.
,
Cleaver
,
J. E.
&
Glaser
,
D. A.
(
1980
).
Large scale isolation of UV-sensitive clones of CHO cells
.
Somat. Cell Genet.
6
,
407
418
.
Cleaver
,
J. E.
(
1983
). Xeroderma pigmentosum. In
The Metabolic Basis of Inherited Disease
(ed.
J. B.
Stanbury
,
J. B.
Wyngaarden
,
D. S.
Fredrickson
, J. L. Goldstein & M. S. Brown), 5
th
edn
, pp.
1227
1248
.
New York
:
McGraw-Hill
.
Corsaro
,
C. M.
&
Pearson
,
M. L.
(
1981
).
Enhancing the efficiency of DNA-mediated gene transfer in mammalian cells
.
Somat. Cell Genet.
7
,
603
616
.
Day
,
R. S.
(
1974
).
Cellular reactivation of ultraviolet-irradiated human adenovirus 2 in normal and xeroderma pigmentosum fibroblasts
.
Photochem. Photobiol.
19
,
9
13
.
Deininger
,
P. L.
,
Jolly
,
D. J.
,
Rubin
,
C. M.
,
Friedmann
,
T.
&
Schmid
,
C. W.
(
1981
).
Base sequence studies of 300 nucleotide renatured repeated human DNA clones
.
J. molec. Biol.
151
,
17
33
.
Dillehay
,
L. E.
,
Thompson
,
L. H.
,
Minkler
,
J. L.
&
Carrano
,
A. V.
(
1983
).
The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells
.
Mutat. Res.
109
,
283
296
.
Friedberg
,
E. C.
,
Ehmann
,
U. K.
&
Williams
,
J. J.
(
1979
).
Human diseases associated with defective DNA repair
.
Adv. Radiat. Biol.
8
,
85
174
.
Hoy
,
C. A.
,
Thompson
,
L. H.
&
Salazar
,
E. P.
(
1985
).
Defective cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents
.
Cancer Res.
45
,
1737
1743
.
Ishida
,
R.
&
Buchwald
,
M.
(
1982
).
Susceptibility of Fanconi’s anemia lymphoblasts to DNA- cross-linking and alkylating agents
.
Cancer Res.
42
,
4000
4006
.
Kano
,
Y.
&
Fujiwara
,
Y.
(
1983
).
Defective thymine dimer excision from xeroderma pigmentosum chromatin and its characteristic catalysis by cell-free extracts
.
Carcinogenesis
4
,
1419
1424
.
Mortelmans
,
K.
,
Friedberg
,
E. C.
,
Slor
,
H.
,
Thomas
,
G.
&
Cleaver
,
J. E.
(
1976
).
Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells
.
Proc. natn. Acad. ‘Sci. U.S.A.
73
,
2757
2761
.
Mulligan
,
R. C.
&
Berg
,
P.
(
1981
).
Selection for animal cells that express the Escherichia coligene coding for xanthine-guanine phosphoribosyltransferase
.
Proc. natn. Acad. Sci. U.S A.
78
,
2072
2076
.
Pinkel
,
D.
,
Thompson
,
L. H.
,
Gray
,
J. W.
&
Vanderlaan
,
M.
(
1985
).
Measurement of sister chromatid exchanges at very low bromodeoxyuridine substitution levels using a monoclonal antibody in Chinese hamster ovary cells
.
Cancer Res.
45
,
5795
5798
.
Plooy
,
A. C. M.
,
Fichtinger-Schepman
,
A. M. J.
,
Schutte
,
H. H.
, van
Dijk
,
M.
&
Lohman
,
P. H. M.
(
1985
).
The quantitative detection of various platinum-DNA-adducts in Chinese hamster ovary cells treated with cisplatin; application of immunochemical techniques
.
Carcinogenesis
6
,
561
566
.
Poll
,
E. H. A.
,
Abrahams
,
P. J.
,
Arwert
,
F.
&
Eriksson
,
A. W.
(
1984
).
Host-cell reactivation of cis-diamminedichloroplatinum(II)-treated SV40 DNA in normal human, Fanconi anaemia and xeroderma pigmentosum fibroblasts
.
Mutat. Res.
132
,
181
187
.
Reid
,
B. D.
&
Walker
,
I. G.
(
1969
).
The response of mammalian cells to alkylating agents. II. On the mechanism of the removal of sulfur-mustard-induced cross-links
.
Biochim. biophys. Acta
179
,
179
188
.
Robson
,
C. N.
,
Harris
,
A. L.
&
Hickson
,
I. D.
(
1985
).
Isolation and characterization of Chinese hamster ovary cell lines sensitive to mitomycin C and bleomycin
.
Cancer Res.
45
,
5304
5309
.
Rubin
,
J. S.
,
Prideaux
,
V. R.
,
Willard
,
H. F.
,
Dulhanty
,
A. M.
,
Whitmore
,
G. F.
&
Bernstein
,
A.
(
1985
).
Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene
.
Molec. cell. Biol.
5
,
398
405
.
Schmid
,
C. W.
&
Jelinek
,
W. R.
(
1982
).
The Alu family of dispersed repetitive sequences
.
Science
216
,
1065
1070
.
Siciliano
,
M. J.
,
Carrano
,
A. V.
&
Thompson
,
L. H.
(
1986
).
Assignment of a human DNA repair gene associated with sister chromatid exchange to chromosome 19
.
Mutat. Res.
174
,
303
308
.
Southern
,
E. M.
(
1975
).
Detection of specific sequences among DNA fragments separated by gel electrophoresis
.
J. molec. Biol.
98
,
503
517
.
Spivak
,
G.
,
Ganesan
,
A. K.
&
Hanawalt
,
P. C.
(
1984
).
Enhanced transformation of human cells by UV-irradiated pSV2 plasmids
.
Molec. cell. Biol.
4
,
1169
1171
.
Thompson
,
L. H.
(
1985
).
DNA repair mutants, in Molecular Cell Genetics
(ed.
M.
Gottesman
), pp.
641
667
.
New York
:
John Wiley & Sons
.
Thompson
,
L. H.
,
Brookman
,
K. W.
,
Dillehay
,
L. E.
,
Carrano
,
A. V.
,
Mazrimas
,
J. A.
,
Mooney
,
C. L.
&
Minkler
,
J. L.
(
1982b
).
A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister chromatid exchange
.
Mutat. Res.
95
,
427
440
.
Thompson
,
L. H.
,
Brookman
,
K. W.
,
Dillehay
,
L. E.
,
Mooney
,
C. L.
&
Carrano
,
A. V.
(
1982rz
).
Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions
.
Somat. Cell Genet.
8
,
759
773
.
Thompson
,
L. H.
,
Brookman
,
K. W.
,
Salazar
,
E. P.
,
Fuscoe
,
J. C.
&
Weber
,
C. A.
(
1986
). DNA repair genes of mammalian cells. In
Antimutagenesis and Anticarcinogenesis: Mechanisms
(ed.
D. M.
Shankel
,
P. E.
Hartman
,
T.
Kada
, &
A.
Hollaender
), pp.
349
358
.
New York
:
Plenum Press
.
Thompson
,
L. H.
,
Busch
,
D. B.
,
Brookman
,
K.
,
Mooney
,
C. L.
&
Glaser
,
D. A.
(
1981
).
Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells
.
Proc, natn. Acad. Sci. U.SA.
78
,
3734
3737
.
Thompson
,
L. H.
&
Carrano
,
A. V.
(
1983
). Analysis of mammalian cell mutagenesis and DNA repair using in vitro selected CHO cell mutants. In
Cellular Responses to DNA Damage, UCLA Symposia on Molecular and Cellular Biology, New Series, vol. 11
(ed. E. C. Friedberg &
B. A.
Bridges
), pp.
125
143
.
New York
:
Alan R. Liss
.
Thompson
,
L. H.
&
Hoy
,
C. A.
(
1986
). Using repair-deficient Chinese hamster ovary cells to study mutagenesis. In
Chemical Mutagens
(ed.
F. J.
de Serres
), pp.
285
325
.
New York
:
Plenum Press
.
Thompson
,
L. H.
,
Mooney
,
C.L.
,
Burkhart-Schultz
,
K.
,
Carrano
,
A. V.
&
Siciliano
,
M. J.
(
1985
).
Correction of a nucleotide-excision-repair mutation by human chromosome 19 in hamster-human hybrid cells
.
Somat. Cell molec. Genet.
11
,
87
92
.
Thompson
,
L. H.
,
Rubin
,
J. S.
,
Cleaver
,
J. E.
,
Whitmore
,
G. F.
&
Brookman
,
K.
(
1980
).
A screening method for isolating DNA repair-deficient mutants of CHO cells
.
Somat. Cell Genet.
6
,
391
405
.
van Duin
,
M.
,
Westerveld
,
A.
&
Hoeijmakers
,
J. H. J.
(
1985
).
UV stimulation of DNA- mediated transformation of human cells
.
Molec. cell. Biol.
5
,
734
741
.
Westerveld
,
A.
,
Hoeijmakers
,
J. H. J.
,
van Duin
,
M.
,
de Wit
,
J.
,
Odijk
,
H.
,
Pastink
,
A.
,
Wood
,
R. D.
&
Bootsma
,
D.
(
1984
).
Molecular cloning of a human DNA repair gene
.
Nature, Land.
310
,
425
429
.
Wood
,
R. D.
&
Burki
,
H. J.
(
1982
).
Repair capability and the cellular age response for killing and mutation induction after UV
.
Mutat. Res.
95
,
505
514
.