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Mutagenicity of N-Methyl-N'-Nitro-N-Nitrosoguanidine to Escherichia Coli (Xtha-/Exonuclease III/DNA Repair/Alkylation Damage/SOS Response) PATRICIA L

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Mutagenicity of N-Methyl-N'-Nitro-N-Nitrosoguanidine to Escherichia Coli (Xtha-/Exonuclease III/DNA Repair/Alkylation Damage/SOS Response) PATRICIA L Proc. Nati. Acad. Sci. USA Vol. 84, pp. 2891-2895, May 1987 Genetics Loss of an apurinic/apyrimidinic site endonuclease increases the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine to Escherichia coli (xthA-/exonuclease III/DNA repair/alkylation damage/SOS response) PATRICIA L. FOSTER AND ELAINE F. DAVIS Division of Environmental Health, Boston University School of Public Health, and the Hubert H. Humphrey Cancer Research Center, Boston University School of Medicine, Boston, MA 02118 Communicated by Fred Sherman, January 16, 1987 (received for review October 6, 1986) ABSTRACT xthA- Escherichia coli, which are missing a enzyme in spontaneous or induced mutagenesis has been major cellular apurinic/apyrimidinic (AP) endonuclease, are difficult to establish. 5- to 10-fold more sensitive than xthA+ bacteria to mutagenesis Exposure of E. coli to low concentrations of alkylating by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) under con- agents induces the "adaptive response" (20), which includes ditions that induce the "adaptive response." The xthA-- two known DNA repair enzymes-06-methylguanine-DNA dependent mutations are also dependent on SOS mutagenic methyltransferase (21) and 3-methyladenine-DNA glycosyl- processing and consist of both transversion and transition base ase II (TAGII) (22, 23). Two other genes of unknown substitutions. When MNNG-adapted xthA- bacteria are chal- function, alkB (24) and aidB (25), also are induced. O6_ lenged with a high dose of MNNG, more xthA-dependent methyltransferase accurately repairs 06-alkylguanine (21) SOS-dependent mutations are induced, and transversions are and 04-alkylthymine (26), thus virtually eliminating any enhanced relative to transitions. The mutations induced by mutations due to these lesions. TAGII, the product of the challenge are eliminated in xthA- alkA- bacteria, which are alkA gene, removes purines alkylated at the N-3 position (22, also deficient for 3-methyladenine glycosylase II activity. These 23) and pyrimidines alkylated at the 0-2 position (26), leaving data are consistent with the hypothesis that AP sites, at least AP sites. AP sites are also produced after alkylation damage some of which are produced by glycosylase activity, are by the spontaneous loss of alkylated bases, particularly mutagenic intermediates following cellular DNA alkylation. N7-alkylguanine, and by the activities of the constitutive glycosylases formamidopyrimidine-DNA glycosylase and 3- Apurinic and apyrimidinic (AP) sites cause errors in DNA methyladenine glycosylase I (3). Thus, induction of the replication in vitro and are mutagenic when present in adaptive response by alkylating agents leads to the produc- transforming DNA (1, 2). In addition to being a probable tion of AP sites by several different pathways. Based on the cause of spontaneous mutations (3, 4), AP sites have been distribution ofalkyl lesions in DNA (27) and the known repair implicated as a common mutagenic lesion created by a activities, the majority of these AP sites are expected to be number of DNA-damaging agents (reviewed in ref. 1). These due to missing purines. agents include those that make bulky adducts to the DNA, In vitro studies have indicated that AP sites give rise to such as aflatoxin B1 (5), benzo[a]pyrene (6, 7) and p- mutations because DNA polymerases preferentially insert propiolactone (6, 8), as well as simple alkylating agents (6, 9). purines, particularly adenines, opposite such sites during However, the mutagenic role ofAP sites produced in vivo has bypass synthesis (1). Hence, depurination is expected to not been well established. yield a predominance of transversion mutations while The xthA gene ofEscherichia coli encodes exonuclease III, depyrimidination should yield transition mutations. In E. a multifunctional enzyme that is the cell's major AP endo- coli, at least some part of this postulated pathway must be nuclease (10). xthA- bacteria retain only 10-20% of the dependent on SOS mutagenic processing, as mutations are normal cellular AP endonuclease activity (11, 12). xthA- not induced when SOS-deficient cells are transformed with mutants are deficient in the repair of the AP sites created by depurinated DNA (1). the enzymatic removal of misincorporated uracil from DNA Since AP endonuclease activity is the first step in the repair (13). They are also slightly sensitive to killing by methyl of AP sites, we reasoned that if endogenous AP sites are methanesulfonate (11, 12), an alkylating agent that produces mutagenic, the mutagenicity of alkylating agents should be a predominance of N-alkylated DNA bases that give rise to enhanced by the xthA- defect under conditions that (i) maxi- AP sites both spontaneously and enzymatically (3). Other mize the formation of AP sites to saturate compensating phenotypes ofxthA- mutants include increased sensitivity to activities; (ii) minimize the mutagenicity ofotherlesions such as the oxidizing agent H202 (14), slight sensitivity to near UV 06-alkylguanine; and (iii) allow for SOS mutagenic processing. light (15), and partially defective expression of heat shock These conditions were achieved by exposing E. coli to a low proteins (16). Since the xthA gene product is also a duplex- concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MN- specific DNA 3'-5' exonuclease (10), can remove a variety of NG), thus inducing both the adaptive response (20) and the SOS blocking groups from 3' termini in duplex DNA (17, 18), has response (9). When both of these responses are active, xthA- RNase H activity (10), and can recognize and nick near urea bacteria are more mutable by MNNG than xthA+ bacteria. residues in DNA (19), it is not known what contribution, if any, its AP endonuclease activity may make to these other MATERIALS AND METHODS phenotypes. Despite the several phenotypes of xthA- bac- Bacterial Strains. All genetic manipulations were as de- teria and the many activities of exonuclease III, a role for this scribed (28). The E. coli K-12 strains used for most of the The publication costs of this article were defrayed in part by page charge Abbreviations: AP, apurinic/apyrimidinic; MNNG, N-methyl-N'- payment. This article must therefore be hereby marked "advertisement" nitro-N-nitrosoguanidine; TAGII, 3-methyladenine-DNA glycosyl- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ase II. Downloaded by guest on September 25, 2021 2891 2892 Genetics: Foster and Davis Proc. Natl. Acad. Sci. USA 84 (1987) experiments reported here are derivatives of PF260 [F- ara with MNNG-exposed cells to give the number of MNNG- A(gpt-lac-pro) thi hisS supD60]. hisS is a deletion ofthe entire induced mutations per plate. This difference was then divided histidine gene cluster (29). One set of experiments used by the number of cells plated. In all cases except xthA+ and PF352 and PF353 (9), which are hisG- hisD' derivatives of umuC::TnS/F' hisG428 strains (see Table 1), the induced AB1157 (F- argE3 his4 leuB6 proA2 thr-J ara-14 galK2 mutations per plate were greater than twice the spontaneous lacYl mtl-i xyl-S thi-J rpsL31 supE44 tsx-33; see ref. 29) and background; for all experiments with xthA- alkA+ MS23 (AB1157 his' alkA-; see ref. 30), respectively. To umuDC+/F' hisG46 strains, the induced mutations per plate ensure that the strains were isogenic, PF353 and an xthA- were 20-40 times the spontaneous background. derivative of it were transduced to alkA' and tetracycline Analysis of Mutations. Revertants of hisG46 were single resistance with a Plvir lysate of NK5526 (alkA' hisG::TnJO; colony purified and infected with M13hol67.18 (34). To select obtained from B. Bachmann, Yale University). Methyl meth- for recombinant phage, a His- recipient (PF665 = P9OC anesulfonate resistant transductants were then made hisG- recAl3 hisG- hisD'/F::Tn3) was infected with selection for hisD+ as described (9). Results with these strains did not the His' phenotype. Single-stranded DNA was prepared and differ from those with PF352 and PF353. sequenced by standard dideoxy methods with a synthetic xthA- derivatives were constructed by transducing cells oligonucleotide priming close to the hisG46 region. In a few with a Plvir lysate of BW9115 [A(xth-pncA) pheSl ; see ref. cases, M13 crosses were repeated selecting for histidinol 11; obtained from B. Weiss, The Johns Hopkins University], utilization in WB351 (34) to ensure that spontaneous muta- selecting for resistance to both 1-2 mM 6-aminonicotinamide tions were not being generated during the phage crosses; no and fluorophenylalanine (ref. 40; the effective concentrations differences in the sequences were found. True revertants of varied among strains), and screening for methyl methanesul- hisG428 were identified by their ability to transfer the His' fonate sensitivity. umuC: :TnS derivatives were constructed phenotype by mating. To determine the percentage of true by transduction with a Plvir lysate ofGW2100 (obtained from revertants of hisG428 (Table 1), 291 revertants from MNNG- G. Walker, Massachusetts Institute of Technology) and treated cultures and 100 revertants from nontreated cultures screening kanamycin-resistant transductants for lack of UV of the xthA- umuDC+ strain and 50 revertants from both mutability to rifampicin resistance. treated and untreated cultures of each of the other strains F'4 episomes with either the hisG46 or hisG428 mutations were analyzed. The different classes of hisG428 revertants in were mated into recipient E. coli strains using appropriate our strains could not be identified using thiazole alanine selection. The F'400 episome carrying the his gene cluster of sensitivity as described for S. typhimurium (32). Salmonella typhimurium was obtained from R. Brent (Mas- To calculate the MNNG-induced frequency of mutational sachusetts General Hospital) as hisO1242, hisG46, events, the fraction of the total analyzed represented by a gnd: :TnJO, rfb+. A hisG428 construction was made by given class of mutation was multiplied by the total number of transducing a S. typhimurium strain carrying the episome as colonies on the plate from which the revertants were taken.
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