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Depurination Causes Mutations in SOS-Induced Cells (Chemical Carcinogenesis/Mutagenesis/Fidelity of DNA Replication/SOS Repair) ROELAND M

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Depurination Causes Mutations in SOS-Induced Cells (Chemical Carcinogenesis/Mutagenesis/Fidelity of DNA Replication/SOS Repair) ROELAND M Proc. Nati. Acad. Sci. USA Vol. 78, No. 3, pp. 1773-1777, March 1981 Genetics Depurination causes mutations in SOS-induced cells (chemical carcinogenesis/mutagenesis/fidelity of DNA replication/SOS repair) ROELAND M. SCHAAPER AND LAWRENCE A. LOEB The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, SM-30, University of Washington, Seattle, Washington 98195 Communicated by Earl P. Benditt, November 17, 1980 ABSTRACT Introduction of apurinic sites into dX174 am3 the apurinic sites. We show that, in accord with previous re- DNA leads to loss of biological activity when measured in a trans- ports, depurination is strongly inactivating but that, under cer- fection assay. For single-stranded DNA, approximately one apu- tain can indeed be rinic site constitutes a lethal hit; for double-stranded (RFI) DNA, conditions, depurination mutagenic. approximately 3.5 hits per strand are lethal. When the reversion frequency of am3 DNA is measured, no increase due to depuri- MATERIALS AND METHODS nation is observed above the background level. However, a large Bacteria and Bacteriophage. E. coli W6, obtained from M. increase in reversion frequency is observed when the same DNA Edgell (University of North Carolina) was used for the prepa- is assayed by using spheroplasts derived from bacteria previously ration of spheroplasts. E. HF4704 HF4714 exposed to UV light. The results suggest that apurinic sites are coli (su-) and (su1l) impediments to a replicating DNA polymerase; however, nucleo- were used to determine titers and reversion frequencies of tides can be incorporated opposite these sites under SOS-induced 4)X174 am3 DNA. The preparation of bacteriophage 4.X174 conditions. We estimate the frequency of mutagenesis per apu- am3 DNA and the isolation of 3H-labeled viral and replicative rinic site to be less than 1 in 1400 in normal spheroplasts and 1 in form I (RFI) DNA of this phage was as described (22). Spher- 100 in SOS-induced spheroplasts. oplasts were prepared as described (22) except that 35% Pentex bovine serum albumin (Miles) was used instead of Povite al- Most chemical carcinogens damage cellular DNA either by a bumin. The efficiency of transfection, measured as infective direct reaction or after metabolic activation (1). It is believed centers, was usually 10-4. that this damage, in the form of adducts to the bases or to the Depurination of DNA. 3H-Labeled single-strand am3 DNA sugar-phosphate backbone, is the initiating event in* tumori- (26 cpm/ng; 0.24 Zg/,.l) in 50 mM Tris'HCl buffer (pH 8.0) genesis. The next step may be the creation of mutations when was diluted with 24 vol of 10 mM sodium citrate/100 mM KC1, the cell tries to process its damaged DNA (2-5). pH 4.80 (250C), to obtain a final pH of 4.95 + 0.05 at 250C. The exact nature of the mutagenic process has not been de- Depurination was achieved by heating the samples for various lineated. Small modifications of bases on DNA may lead to times at 70'C. Under these conditions, an average of one apu- mispairings during DNA replication (6-9). However, many of rinic site was introduced per molecule every 5 min. RFI DNA the more potent carcinogens, such as aflatoxin B1, 2-acetylam- (21.6 cpm of 3H per ng; 4.57 ug/yld) in 10 mM Tris-HCl (pH inofluorene, and benzo[a]pyrene, produce bulky adducts (10) 8.0) was diluted with 24 vol of 10 mM sodium citrate/100 mM and their main initial effect may be to block DNA replication KC1, pH 4.0, and incubated for various times at 70°C. Under (11, 12). In this case, mutagenesis could involve apurinic sites these conditions, approximately 3.5 apurinic sites were intro- as intermediates. Adducts at the N-3 and N-7 positions of the duced per strand every 5 min. Afterdepurination, the DNA was purine bases greatly destabilize the N-glycosylic bond con- rapidly cooled to 0°C and diluted in 50 mM Tris HCl (pH 8.0) necting the base to the sugar-phosphate backbone (13). This prior to transfection. leads to release of the base and produces a chemically stable Quantitation of Apurinic Sites. Hydrolysis of apurinic sites apurinic site (14, 15). We decided to investigate the possible was carried out by incubation of the DNA in 0.1 M NaOH at involvement ofapurinic sites in mutagenesis and carcinogenesis 37°C for 1-2 hr. The average number of breaks per molecule because they are frequent spontaneous lesions (104 per 24 hr was estimated by sedimentation through alkaline sucrose gra- in a mammalian cell) (14) and carcinogen treatment may en- dients (5-20% sucrose in 0.3 M NaOH/0.8 M NaCVl1 mM hance depurination as much as 100- to 1000fold (16, 17). EDTA) in a SW 50.1 rotor. For single-stranded DNA, sedi- Depurination has generally been considered to be nonmu- mentation was at 35,000 rpm for 15 hr and the number of sites tagenic (18, 19). In contrast, it has been shown (20, 21) that was estimated from the ratio of linear to circular molecules in depurination of homopolymer templates like poly[d(A-T)] or the range of 0 to 2 apurinic sites. For double-stranded DNA, poly[d(G-C)] leads to increased mismncorporation of noncom- sedimentation was at 42,000 rpmfor 17 hr, and number average plementary nucleotides when copied by purified DNA molecular weight was obtained by the graphical procedure of polymerases. Lehmann and Ormerod (23) using DNA containing 5 to 20 In this report, we describe the mutagenic consequences of apurinic sites. The Z1, Z4, and Z10 Hae III restriction fragments depurination in vivo. We have depurinated bacteriophage served as markers (25.1, 11.2, and 1.35% of the +X genome 4X174 am3 DNA in vitro and transfected this DNA into Esch- length, respectively). Neutral sucrose gradients (5-20%) were erichia coli spheroplasts. Because the DNA of the amber mu,, run for 4.5 hr at 45,000 rpm in SW 50.1 rotors. tant am3 was used, the reversion of this amber phage to wild Transfections. In transfection assays, the depurinated DNA type could be used as a measure of the mutagenic potential of was diluted in at least 10 vol of 50-mM Tris-HCl (pH 8.1). The final DNA concentration was chosen. not to exceed 0.2 ,ug/mn1 The publication costs of this article were defrayed in part bypage charge because at greater concentrations the linearity between the payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: RFI, replicative form I. 1773 Downloaded by guest on September 25, 2021 1774 Genetics: Schaaper and Loeb Proc. Nad Acad. Sci. USA 78 (1981) amount of input DNA and the resulting number of infective on the DNA probably constitutes a lethal hit. Double-stranded centers is no longer observed. Within each experiment the ratio DNA is much more resistant to inactivation (Fig. 2). We esti- of DNA to spheroplasts was always constant. An equal volume mate that about 3.5 apurinic sites per strand constitute a lethal of spheroplasts was added to the DNA solution, and after 12 min hit. The resistance of double-stranded DNA is in accord with at 370C a small sample was taken to determine the number of studies on bacteriophage T7 (25) and simian virus 40 (26) in surviving infective centers by plating on HF4714 (su'). To the which 7-8 and 4-5 apurinic sites were required for one lethal remainder, 1 vol of PAM (22) medium was added and incubation hit, respectively. These results presumably reflect the repair was continued for an additional 1.5 hr. After freezing at -70GC, capacity of the cell for apurinic sites on double-stranded DNA thawing, and the addition of a few drops of chloroform, the re- by either excision (27) or an insertase type of repair (28, 29). version frequency of the progeny phage was determined by The difference between survival of single- and double-stranded using HF4704 and HF4714 as indicator bacteria. The details of DNA is similar to that reported for UV-treated DNA (30). all transfection and plating procedures have been described (22, Mutagenesis Due to Depurination. Previous studies (22, 24) 24). have shown that single-stranded 4X174 am3 can revert to wild SOS-Induction. E. coli W6 was grown to OD&% of 0.60. The type in three ways. The TAG codon at positions 586-588 on the cells were harvested by centrifugation, resuspended in 0.1 M 4~Xchromosome map (31) converts to aTGG (tryptophan), TTG NaCl/0.01 M MgSO4 at 1/10th of the original volume, and ir- (leucine), or TCG (serine) codon. The fact that in all cases the radiated in thin layers in plastic Petri dishes with UV light (80 substitution occurs opposite the adenine of the amber codon J/m2) produced by a G15T8 General Electric bulb (254 nm). allows one to study the mutagenic consequences of depurina- After irradiation, cells were resuspended in the original volume tion. The amr3 phage has the additional advantage of a low back- of fresh M-9 medium and incubated for 45 min at 370C in sub- ground reversion frequency, 10-6, when measured as progeny dued light. After this period, the cells were harvested and pro- phage (24). Table 1 shows a representative experiment mea- cessed into spheroplasts. Every experiment was performed suring the reversion frequency of the progeny phage as a func- with fresh spheroplasts after 2-3 hr of incubation on ice to de- tion of the number of apurinic sites introduced per (X single- velop competence.
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