suggesting the involvement of a possible defect in the cellular equilibrium between ligation and ... DNA repair enzymes even though putatively repair- deficient ...
Br. J. Cancer (1984) 49, Suppl. VI, 67-72
The use of recombinant DNA plasmids for the determination of DNA-repair and recombination in cultured mammalian cells R. Cox, W.K. Masson, P.G. Debenham & M.B.T. Webb Division of Cell and Molecular Biology, M.R.C. Radiobiology Unit, Harwell, Didcot, Oxon OXJJ ORD, U.K.
Summary Using the recombinant plasmid pSV2gpt and DNA transfer techniques, cell mediated DNA ligation and recombination of plasmid DNA have been demonstrated in four human cell lines. Data suggesting the involvement of a possible defect in the cellular equilibrium between ligation and exonuclease digestion of double strand DNA scissions in an ataxia-telangiectasia (A-T) cell line is discussed. The same A-T line was grossly proficient in DNA recombination but it will be necessary to distinguish between recombination in coding and non-coding plasmid sequences.
Conventional experimental approaches have yet to provide critical evidence on the link between cellular radiosensitivity and the activity of specific DNA repair enzymes even though putatively repairdeficient cells, such as those from ataxiatelangiectasia human patients have been studied in some detail (Taylor et al., 1975; Cox, 1982; Lehmann, 1982). Experiments utilizing recombinant DNA and gene transfer techniques (e.g. Pellicer et al., 1980; Mulligan & Berg, 1981) may, through their power of resolution, provide new insights into the problem and here we present an outline of some such experiments using a recombinant DNA plasmid to assay DNA repair and recombination in cultured human cells. Our ultimate aim is to introduce, in vitro, inactivating radioproducts into a plasmid encoded, dominant selectable gene and subsequently analyse the fate of such lesions by transferring the damaged DNA into the cells. The capacity of recipient cells to repair the damage and hence express the gene product may then be identified by the formation of viable colonies in a selective medium for which this functioning gene is essential. Since we have yet to construct a suitable plasmid vector for these experiments our initial studies have utilized plasmid pSV2gpt with the damage being introduced in vitro with DNA metabolising enzymes. Plasmid pSV2gpt (Figure 1) was constructed and characterised in the laboratory of Dr. Paul Berg (Mulligan & Berg, 1981) and contains DNA sequences from the bacterial plasmid pBR322, from SV40 virus plus a genomic DNA fragment from Escherichia coli encoding the gpt gene specifying xanthine phosphoribosyl transferase (XPRT, E.C.2.4.2.22). The ampicillin-resistance gene (amp)
allows selective plasmid replication in host bacteria whilst the gpt gene with flanking SV40 promotor and processing sequences allows expression of XPRT activity in recipient mammalian cells. Mammalian cells cannot utilize xanthine as a source of guanosine monophosphate and fail to grow in a selective medium termed XHATM. The appearance of XPRT activity in plasmid transformed mammalian cells allows xanthine utilization and hence selective clonal growth in XHATM medium. In experiments outlined here the ability of SV40 transformed lines of normal, xeroderma pigmentosum (XP) and ataxia-telangiectasia (A-T) cells to rejoin double strand (ds) DNA scissions by conservative (base-pairing dependent) ligation was assessed. DNA ds scissions are introduced in vitro into the gpt region with suitable restriction endonucleases and the linear species generated transferred into the cells. Cell-mediated DNA ligation (rejoining) was then measured, in a semiquantitative way, by the frequency of clonal XPRT+ transformants that arose in that population. Experiments of an essentially similar design were performed to determine the frequency of DNA recombination. In these experiments two DNA fragments from pSV2gpt with nonoverlapping deletions in the gpt region were cotransferred into cells and cell mediated DNA recombination was determined by the frequency at which XPRT+ transformants arose.
Materials and methods Cells and cell culture media Permanent human cell lines transformed with SV40 C) The Macmillan Press Ltd., 1984
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R. COX et al. V-.. II
Pvu
Hir
Kpn I
Figure 1 Diagrammatic representation of recombinant plasmid pSV2gpt. - DNA sequences from plasmid pBR322; ) DNA sequences from SV40 simian virus; -'.' DNA sequences from E. coli (encoding the gpt gene). Positions of sites for major restriction endonucleases and amp (ampicillin resistance) gene and plasmid replication origin (ori) are indicated.
iHI
Bgl 11
Figure 2 Diagrammatic representation of pSV2gpt fragments used in DNA recombination experiments. DNA sequence, restriction endonuclease and gene notation as in Figure 1.
pSV2gpt TRANSFER, DNA LIGATION/RECOMBINATION
virus were used in all experiments. The diploid strains from which these derived were: MRC5 and W138 (normal), XP12R0 (classical X.P.) and AT5B1 (classical A-T). Cells were maintained at 370 in 95% air: 5% CO2 in Eagles MEM plus 10% foetal calf serum (MEMFCS) supplemented with antibiotics and a 1 to 5 split was used for subculture. Cell monolayers were dissociated with 0.1% EDTA. trypsin plus 0.4 mg ml1 Determination of clonal viability was performed in MEMFCS. XPRT+ transformants were selected in XHATM medium (MEMFCS with xanthine, 10 ug ml- 1; hypoxanthine, 100 ,M; aminopterin, 0.4pM; thymidine, 15MM; and mycophenolic acid, 25igml-1) at a density of 2.105 cells per 9cm diameter dish. Clonal viability was scored after 14 days incubation, XPRT+ transformants after 21 days. Plasmid DNA Plasmids pSV2gpt and pBR322 were maintained in E. coli HB1I1 and plasmid DNA extracted and purified using conventional methods (Clewell & Helinski, 1970). Plasmid DNA was characterised by restriction endonuclease digestion and electrophoresis in 0.8% agarose slab gels containing ethidium bromide. DNA concentrations were determined spectrophotometrically. Enzymes and DNA digestions Restriction endonucleases EcoRl, Hind III, Bgl II, Kpn I and Xor II were obtained variously from Biolabs, BRL, CBL and BCL. Enzyme incubation conditions were those recommended by the supplier except that bovine serum albumin was omitted. A 10 fold excess of enzyme was used in all digestions and any evidence of incomplete digestion of DNA prompted a second round of digestion. Single strand specific nuclease SI was obtained from Sigma and BCL. Incubation conditions were similar to those of the supplier except that for Kpn I cut plasmid incubation periods were reduced to avoid
over-digestion. Isolation of DNA fragments Following sequential restrictions endonuclease digestion, two DNA fragments were generated from pSV2gpt DNA. Fragment A contained sequences between the Xor II and Kpn I sites of Figure 1, fragment B, the sequences between EcoRl and Hind III. These fragments were separated by preparative agarose gel electrophoresis, eluted from the gel and
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freed from ethidium bromide by extraction with H20 saturated butanol. DNA-transfer Transfer of plasmid DNA to cells was performed using the calcium phosphate-DNA co-precipitation method of Frost & Williams (1978). Monolayer cells (-2.106 cells per 80sqcm flask) were exposed to 2ml of the DNA precipitate for 20min. Fresh MEMFCS (25 ml) was added and the cells incubated at 37° for 24 h. A 3 min pulse with 15% glycerol in isotonic HEPES saline pH 7.2 was followed by a 4 day incubation period in fresh medium to allow expression of the gpt gene. At this time clonal viability and frequency of XPRT+ transformants was determined. The frequency of XPRT+ transformants was calculated per viable cell at the time of selection.
Results
Cloning efficiencies The cloning efficiencies of MRC5 and AT5B1 (following DNA transfer and expression) were generally between 30 and 60%. The XPl2RO and W138 lines gave cloning efficiencies of 10%. Plasmid DNA transfer frequencies The frequencies of XPRT+ transformants varied between the cell lines and also varied (approximately linearly) with DNA concentration. No XPRT+ clones were seen in mock transfers or transfers with pBR322 DNA. Maximum transfer was obtained with intact pSV2gpt plasmid at a concentration of 20 Mg ml- 1 and under the conditions specified, XPRT+ frequencies varied between 10-4 and 10-3 with frequencies in the four lines usually increasing in the order of W138
