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. stranded circle. In a total of four experiments, in which the number of apurinic sites varied from 1 to 5, no mutagenesis was RESULTS detected above background (i.e., <0.5 X 10-6). Table 1 also Inactivation by Depurination. Fig. 1 shows the survival of lists the number of infective centers at each of the levels of single-stranded 4)X174 DNA with increasing time of depuri- depurination. In order to keep this number high (> 106), trans- nation as assayed by transfection. From alkaline sucrose gra- fections for the higher levels of depurination were increased in dients we estimate that approximately one apurinic site is pro- volume while the ratio of DNA to spheroplasts was kept duced every 5 min at pH 5.0 and 70°C. Thus, if Fig. 1 is plotted constant. as survival versus the number of apurinic sites, one obtains a Mutagenesis of Depurinated DNA in SOS-Induced Spher- straight line with a slope of 1, indicating that every apurinic site oplasts. Many lesions in DNA are not mutagenic, or onlyweakly so, under normal conditions but become increasingly mutagenic when the SOS response is induced. This response has been

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Depurination time, min Depurination time, min FIG. 1. Survival of single-stranded 4X174 DNA with increasing time of depurination (pH 5.0, 70°C) and assayed as the surviving frac- FIG. 2. Survival of RF1 DNA as a function of time of depurination tion of infective centers when transfected on E. coli W6 spheroplasts. (pH.4.0, 70°C). Infective center assay was on W6 spheroplasts. Vertical The vertical bars represent the range of duplicate samples. bars represent range of duplicate samples. Downloaded by guest on September 25, 2021 Genetics: Schaaper and Loeb Proc. Nati Acad. Sci. USA 78 (1981) 1775

Table 1. Reversion frequency among progeny phage in to 30-fold above background with DNA containing 3-5 apurinic transfection assay of depurinated single-stranded am3 DNA sites per molecule. Variation in the DNA/spheroplast ratio over Infective Reversion a 20-fold range did not affect the mutagenic response, sug- Apurinic centers,* frequencyt gesting that mutagenesis by depurination is not due to enhanced sites, no. Survival, % no. x 1O6 recombination in SOS-induced cells (unpublished data). o 100 3.5 x 106 1.2 Breakage of Apurinic Sites by Alkali Treatment. In order 0.7 61 2.5 x 106 1.3 to show that the mutagenic response is due to apurinic sites and 2.1 9.5 3.5 X 106 1.0 not to a different type of lesion induced in parallel, we made 3.5 4.5 1.5 x 106 1.4 use ofthefact thatthe stabilityofthe sugar-phosphate backbone at an apurinic (apyrimidinic) site is markedly decreased under * Volume of the assay was varied at a fixed DNA/spheroplast ratio to alkaline conditions (15). Exposure of DNA to pH 13 at 250C for maintain a high number of infective centers. several hours breaks DNA at apurinic sites, whereas nonde- t ± 0.5 x 10-6 (duplicate samples). purinated DNA remains intact. We exposed depurinated and nondepurinated DNA to 0.1 M NaOH at 250C for 2.5 hr and studied most in E. coli (32, 33), but evidence has been accu- verified by neutral and alkaline sucrose gradients that the de- mulating that a similar response may exist in mammalian cells purinated DNA was fragmented in proportion to the number (34, 35). The basic concept is that when the normal progress of of apurinic sites introduced and that the nondepurinated DNA DNA replication is stalled-e.g., by the introduction of lesions was intact (gradients not shown). The predictions, then, are that that constitute a block for the replicating enzyme-a set of new the selective removal of the apurinic sites prior to transfection proteins is induced which allows DNA synthesis to proceed past will abolish the mutagenic response without affecting the sur- the blocking lesion, resulting in increased survival and mark- vival. Both predictions were fulfilled. The survival of the 4X174 edly increased mutagenesis. DNA after depurination was unchanged after alkali treatment Because the one-hit kinetics of inactivation for the single- (Table 2 legend). The strong mutagenic response observed in stranded DNA might be suggestive of a "blocking type" of le- the UV-irradiated spheroplasts was almost completely abol- sion, it seemed pertinent to investigate the mutagenic potential ished after alkali treatment (Table 2). of depurination under conditions of SOS induction. E. coli W6 was irradiated (=80 J/m2) with UV (254 nm) light. After 45 min DISCUSSION of further growth to allow expression of the SOS phenotype, We have observed that heating 4)X174 DNA in acid produces, the cells were converted into spheroplasts. Fig. 3 gives the re- in a time-dependent manner, damage which results in the pro- sults of a representative experiment comparing normal and in- duction of mutations when this DNA is transfected into UV- duced spheroplasts. No difference in survival or burst size of treated E. coli spheroplasts. The most direct explanation for this the depurinated DNA could be detected between the two types observation is the production of apurinic sites as premutagenic of lesions. Exposure of DNA to heat and acid causes depurination spheroplasts (results not shown). However, in contrast to in a quantitative manner, and estimates can be made about the normal spheroplasts, in spheroplasts previously exposed to UV relative frequencies of other products. The 4X174 am3 rever- mutagenesis was easily detectable with depurinated <4X174 am3 sion system is highly specific in that it measures only single-base DNA. The enhancement of mutagenesis was proportional to substitutions at position 587 in the 4OX chromosome-i.e., at the number of apurinic sites introduced. In three different ex- the adenine in the TAG-amber codon. Changes allowed are the periments, this enhancement of mutagenesis varied from 10- A--G transition and the A-RT transversion (22, 24). It seems reasonable that this adenine will be the likely target ofany treat- ment that causes reversion. However, the possibility that dam- age at sites other than position 587-for instance, at the pre- ceding guanine at position 588-is responsible for reversion occurring at 587 cannot be completely eliminated. Assuming that depurination is a predominantly random event (and we know of no evidence to the contrary), this adenine will be re- moved in proportion to the average number of apurinic sites Table 2. Reversion frequency among progeny phage after transfection of depurinated and alkali-treated single-stranded am3 DNA on normal and induced spheroplasts Frequency x 106 Apursnic Normal spheroplasts Induced spheroplasts no.* No alkali Alkali No alkali Alkali 0 1.2 1.2 1.2 1.5 2.7 1.3 1.4 7.6 1.7 4.5 1.5 1.3 20 2.3 After depurination, DNA samples were incubated with an equal vol- Apurinic sites, no. ume of 0.2 M NaOH for 2.5 hr at room temperature, neutralized, and transfected as normal. Control DNA samples were incubated at room FIG. 3. Reversion frequency among am3 progeny phage as a func- temperature without alkali addition. Results are means of duplicate tion of the number of apurinic sites introduced. Single-stranded am3 samples. Duplicate samples varied by about 20%. DNA was depurinated and transfected. Spheroplasts were W6, either * Survivals were 100%, 7.0%, and 1.4% before alkali treatment and normal or SOS-induced. The vertical bars represent the range of du- 97%, 5.5%, and 1.25% after alkali treatment (based on pretreatment plicate transfections. titer). Downloaded by guest on September 25, 2021 1776 Genetics: Schaaper and Loeb Proc. Nad Acad. Sci. USA 78 (1981) introduced per molecule. When the survival of these molecules (Fig. 1). However, alternative explanations such as hydrolysis is measured, a strong inactivation is seen (Fig. 1) to the extent of apurinic sites in spheroplasts cannot be ruled out. that approximately one apurinic site constitutes a lethal hit on This finding of mutagenesis at apurinic sites could be of im- a single-strand molecule; however, no mutagenesis is observed portance for mechanisms of mutagenesis and carcinogenesis in under these conditions (Table 1). In contrast, when the spher- general. Apurinic sites are a major lesion both spontaneously oplasts are previously exposed to UV, a clear mutagenic re- and under conditions of treatment with chemical carcinogens. sponse occurs (Fig. 3). This response is proportional to the num- The point that may have been overlooked in previous assess- ber of apurinic sites introduced. ments of their role in mutagenesis is their interaction with in- It is unlikely that any other lesion introduced by the same ducible error-prone repair systems like the E. coli SOS system. treatment is responsible for the observed mutagenesis. The Those previous studies denying a role for apurinic sites in mu- other known major reaction is cytosine deamination. We cal- tagenesis were mainly based on alkylation of bacteriophage T4 culate from the data of Lindahl et al. (14, 36) and the pH de- and indirect induction of apurinic sites by release of alkylated pendence of cytidine deamination (37) that, at pH 5, depuri- bases by heat treatment (18). Inactivation but no change in nation is favored over deamination, the rate constants being mutation frequency was observed. However, mutagenesis due 10-6 and 2 x 10-9 s-', respectively. Thus, only 1 uracil is in- to alkylation itself interfered, and no interaction with the host troduced per 1000 apurinic sites. Therefore, at the depurination SOS system could have been measured. The latter argument levels utilized, cytosine deamination will have no detectable also applies to the heat mutagenesis experiments described by effect on the survival of the molecules. Most importantly, since Baltz et aL (40) and Bingham et aL (41). the TAG codon does not contain a cytosine, there will be no We suggest a role for apurinic sites in mutagenesis by chem- effect on the reversion frequency. ical carcinogens based on the following observations. Bulky car- Deamination of adenine, converting it to hypoxanthine, is cinogens terminate DNA synthesis at the site of modification mutagenic because hypoxanthine pairs with cytosine upon rep- of DNA as shown by Moore and Strauss (11) and Hsu et aL (12). lication, thus causing the A- G transition and reverting am3 Blockage of DNA replication leads to SOS-induction in bacteria to wild type. Adenine deamination occurs at only 2% of the rate (33), and evidence is accumulating that similar events take place of cytosine deamination (27) and we calculate that, upon intro- in eukaryotic cells (34, 35). Modification of bases in DNA also duction of 1-5 apurinic sites per molecule the frequency of leads to enhanced rates of depurination (13), as exemplified by adenine deamination at position 587 will be 0.2-1.0 x 10-7, the potent mutagen and carcinogen aflatoxin B1 for which the which is too low to have any effect on the reversion frequency. major adduct is to the N-7 position of guanine (42, 43). Finally, Moreover, this type of mutagenesis is an example of so-called our study shows that, under SOS-induced conditions, apurinic direct mutagenesis and therefore should be independent of the sites can be a major source of mutations. Alternatively, they may induction of the E. coli SOS system. As far as we know, no other themselves function as inducing signals for an error-prone DNA heat- or acid-induced adenine modifications have been de- repair pathway. scribed. If they exist, they must be assumed to be rare. Based on the concept of an apurinic site as a functional block- Finally, as shown in Table 2, alkali treatment of the depu- ing lesion, we can use the mutagenesis data in Tables 1 and 2 rinated DNA before transfection effectively abolishes the mu- to estimate the mutagenic potential of apurinic sites under nor- tagenic effect. Alkali treatment constitutes a convenient way of mal and induced removing all molecules with one or more apurinic sites from the conditions. At any average number of apurinic total population of surviving molecules. The prediction then is sites introduced per molecule, the surviving fraction will be that, if the apurinic sites are indeed the cause of the mutations, equal to the fraction of the molecules that escapes depurination alkali treatment should abolish the mutagenic response which and the revertants will arise from the fraction that has only one is what was observed. apurinic site. This is because bypass of the lesion must be con- In these initial studies, we have observed mutagenesis after sidered rare and multiple bypasses should therefore be negli- exposure of E. coli to UV underconditions known to induce SOS gible. The fractions f(0) and f(l) with zero and one site per mol- repair. The simplest explanation is that the error-prone repair ecule are given by the Poisson distribution. They are respectively system induced in the bacterium persists in the spheroplasts e- and re-r; r is the average number of hits introduced. If we and is responsible for the enhanced mutagenesis. The need for assume that depurination hits purines at random, the chance this SOS system suggests that apurinic sites may be one of those of the adenine in the am3 codon being removed should be ap- lesions, like pyrimidine dimers (38), that constitute a block for proximately 1/2500 per hit introduced because the 4X genome DNA replication. The stringent fidelity requirements of the consists of about 5000 nucleotides. The increase in reversion normal replicating complexes may not allow the stable incor- frequency R due to depurination should then be given by: poration of a nucleotide opposite the lesion. It has been pos- (chance of position 587 being hit) times (fraction of the mole- tulated that, under SOS conditions, these stringent require- cules that contain one hit) times (chance P that an apurinic site ments become relaxed and copying occurs with possibly random at 587 will indeed yield a mutation) divided by (the total number insertion opposite lesions (32, 33). However, because no defi- of surviving molecules). The result is: nite evidence has been presented in support of this hypothesis, 1 Pf(1) Pr other mechanisms of mutagenesis cannot be ruled out. In ex- AR = ~.= periments with purified E. coli DNA polymerase I and homo- 2500 f(0) 2500 polymer templates, apurinic sites slowed the rate of polymer- In the case of normal spheroplasts, one can see from Table 1 ization but by no means were an absolute block (21). As a matter that at 4% survival, r = 3.5 and AR is < 10-6. This means that of fact, misincorporation opposite apurinic sites was the mech- P is less than 1 in 1400. For the induced situation of Table 2, anism proposed in that study. The replicative enzyme in E. coli, AR =20 x 10-6, and it follows that P is about 1 in 100. If one the DNA polymerase III holoenzyme, is a much more faithful assumes that base insertion opposite an apurinic site is random, enzyme than is DNA polymerase I (39), and it well might fail then the bypass chance of an apurinic site should be 1.3 times to copy apurinic sites under normal conditions but could do so this value. If other mechanisms, such as hydrolysis of apurinic under conditions with relaxed fidelity requirements. Compat- sites, contribute to the observed inactivation, then the muta- ible with the idea of blockage is the one-hit inactivation kinetics genic response results from a smaller number of apurinic sites Downloaded by guest on September 25, 2021 Genetics: Schaaper and Loeb Proc. NatL Acad. Sci. USA 78 (1981) 1777

and our calculated frequencies are an underestimate. Stark et 18. Lawley, P. D. & Martin, N. C. (1975) Biochem. J. 145, 8591. al. (44) showed that aflatoxin B1 treatment of Salmonella ty- 19. Drake, J. W. & Baltz, R. H. (1976)Annu. Rev. Biochem. 45, 11-37. phimurium caused mutations to 8-azaguanine resistance at fre- 20. Shearman, C. W. & Loeb, L. A. (1977) Nature (London) 270, 537-538. quencies of about 1 mutation per 27-37 aflatoxin adducts, and 21. Shearman, C. W. & Loeb, L. A. (1979)J. Mot Biol 128, 197-218. thus values of this order of magnitude may be quite significant. 22. Kunkel, T. A. & Loeb, L. A. (1979)J. Biol Chem. 254,5718-5725. 23. Lehmann, A. R. & Ormerod, M. G. (1970) Biochim. Biophys.Acta We thank Drs. Barry Glickman, Clyde Shearman, and Helen Zakour 204, 128-143. for their generous counsel and assistance. This study was supported by 24. Kunkel, T. A. & Loeb, L. A. (1980)J. 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