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811.Full.Pdf Copyright 0 1986 by the Genetics Society of America DNA SEQUENCE ANALYSIS OF MUTAGENICITY AND SITE SPECIFICITY OF ETHYL METHANESULFONATE IN UVR+ AND UVRB- STRAINS OF ESCHERICHIA COLI PHILIP A. BURNS,' FRANCES L. ALLEN AND BARRY W. GLICKMAN Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, M3J lP3 Canada Manuscript received December 11, 1985 Revised copy accepted April 26, 1986 ABSTRACT EMS-induced mutations within a 180 base pair region of the lacl gene of E. coli were cloned and sequenced. In total, 105 and 79 EMS-induced mutations from a Uvr+ and a UvrB- strain, respectively, were sequenced. The specificity of EMS-induced mutagenesis was very similar in the two strians; G:C + A:T transitions accounted for all but three of the mutants. The overall frequency of induced mutation was fivefold higher in the UvrB- strain compared to the Uvr+ strain. This demonstrates, at the DNA sequence level, that the presumed pre- mutagenic lesion, 06-ethylguanine, is subject to repair by the uvrABC excision repair system of E. coli. An analysis of mutation frequencies with respect to neighboring base sequence, in the two strains, shows that 06-ethylguanine lesions adjacent to A:T base pairs present better targets for the excision repair machin- ery than those not adjacent to A:T base pairs. UTATIONAL spectra produced by mutagens in various repair back- M grounds can provide important information about the role of different premutagenic lesions and repair systems in the mutagenic process. Until re- cently, such studies have involved the characterization of comparatively small numbers of mutants or reversion analyses at relatively few sites. In the system used here, large numbers of E. coli lacZ forward mutations can be readily collected (MILLER1972), cloned by in vitro recombination onto a bacteriophage M 13 vector (SCHAAPER,DANFORTH and GLICKMAN1985) and then sequenced by the chain termination method (SANGERet al. 1980). In this paper, we investigate the mutational spectra produced by EMS in excision-proficient and excision-deficient strains of E. coli. We focused our attention on mutations affecting a 180 base pair (bp) region of the ZacZ gene that codes for the DNA- binding domain of the protein. This permitted the accumulation of data of sufficient density so that site-to-site differences could be deemed significant. EMS is a member of an important class of mutagenic and carcinogenic agents known as alkylating agents. Studies of alkylating agents have revealed a large Abbreviations: EMS, ethyl methanesulfonate; Pgal, phenyl-beta-mgalactopyranoside;Xgal, 5-bromc-4-chloro- 3-indolyl-beta-mgalactoside. i To whom correspondence should be addressed. Genetics 113: 81 1-819 August, 1986. 812 P. A. BURNS, F. L. ALLEN AND B. W. GLICKMAN number of potential sites within DNA at which these compounds can form lesions, including most of the nitrogens and all of the oxygens (SINGERand KUSMIEREK1982). The most important of these lesions with respect to muta- genesis appear to be the alkylated oxygens. Studies have shown that the 06- alkylguanine lesion is able to mispair with thymine and hence give rise to G:C + A:T transitions, both in vitro (ABBOTand SAFFHILL1979) and in vivo (LOE- CHLER, GREENand ESSIGMANN1984). Other studies have demonstrated the miscoding potential of 02-alkylthymine and 04-alkylthymine (HALL and SAFFHILL1983; SINGER, SACIand KUSMIEREK 1983). Most attention with re- spect to mutagenesis has tended to focus on the 06-alkylguanine lesion, as good correlations have been found between the amount of 06-alkylguanine damage and the extent of its repair and mutagenicity (SCHENDELet al. 1978; NEWBOLD et al. 1980; BERANEKet al. 1983). Several pathways exist by which E. coli can repair alkylation damage (LIN- DAHL 1982). At least two distinct repair processes are induced as part of the adaptive response to alkylation treatment (SAMSONand CAIRNS1977). One enzyme induced in response to alkylation damage is 3-methyladenine-DNA glycosylase 11, which is capable of removing potentially lethal N3-methyladen- ine, N7-methyladenine, N3-methylguanine and N7-methylguanine lesions (KAR- RAN, HJELMGRENand LINDAHL1982), and the potentially mutagenic 02-meth- ylthymine lesion (MCCARTHY, KARRANand LINDAHL1984) from the DNA. A second inducible enzyme, 06-alkylguanine-DNA transferase, is able to dealky- late the potentially mutagenic 06-alkylguanine and 04-alkylthymine lesions (MCCARTHY, KARRANand LINDAHL1984). Although methylating agents read- ily induce the adaptive response, ethylating agents are relatively poor inducers, and ethylation damage is less efficiently repaired by the adaptive enzymes (SEDGWICKand LINDAHL1982). In contrast, the uvrABC-dependent excision repair pathway is able to remove 06-ethylguanine, but not 06-methylguanine, from DNA (WARRENand LAWLEY1980; TODD, BROUWERand GLICKMAN 1981). In previous studies of EMS mutagenic specificity, a marked preference for the induction of G:C + A:T transitions has been observed (COULONDREand MILLER1977; PRAKASHand SHERMAN1973). These observations are consistent with a predominant role for the 06-ethylguanine lesion in EMS mutagenesis. However, these studies revealed little about the site-specificity of alkylation- induced mutagenesis. The aim of this study was to investigate the spectrum of mutations induced by EMS in a system that can screen for all classes of mu- tation, and to examine the role of excision repair in the processing of EMS- induced lesions. MATERIALS AND METHODS Strains and media: The strains used in these studies were NR3835: F' lac-pro, A(pro- lac), ara-, thi-, trpE 9777; and NR395 1: NR3835 A(bio-uvrB). Media were as described by MILLER(1972), and COULONDREand MILLER (1977). Treatment: For the EMS treatment, cells were grown to mid-log phase in LB broth, spun down, washed and resuspended in Vogel-Bonner salt solution and then were treated with EMS (Sigma) at 37" for 30 min. After washing, the treated cells were MUTAGENIC SPECIFICITY OF EMS 813 TABLE 1 Classification of EMS-induced ZacZ- mutations in a Uvr' and a UvrB- strain of E. coli No. of occurrences Uvr+ UvrB- Class of mutation 1.5% EMS" 3% EMS' 3% EMSc G:C + A:T transition 46 56 79 A:T + G:C transition 1 1 0 G:C --f T:A transversion 0 1 0 Total 47 58 79 Induced mutation frequencies were as follows: "9 X loe6 at 72% survival; *14 X at 51% survival; '67 X at 2% survival; compared with 0.2 x without treatment. spread on plates containing Pgal (Bachem Fine Chemicals) as the sole carbon source, for the selection of forward lad- mutations, and LB agar and minimal medium plates, for survival. Pgal is a substrate for &galactosidase, but does not induce synthesis of the enzyme. When supplied as a sole carbon source, only cells that synthesize @-galactosidase constitutively (lad- or lacO' mutations) will form colonies. Mutations mapping to the early part of the gene (approximately 180 bp) can be selected using a simple comple- mentation test (MILLER 1972). Cloning and sequencing: The lad- mutations were cloned by in vivo recombination using a specially constructed bacteriophage M 13 strain that carries lacP-lacZ- se- quences. Following infection of Lad- bacteria, recombinant laci--lacZ+ M 13 progeny were selected as blue plaques on an indicator bacterial strain on plates containing Xgal (Research Organics Inc.). Following plaque purification, DNA preparations were made from the recombinant M13 phage carrying the lad- mutations, and these were then sequenced using the dideoxy method of SANCERet al. (1980) and a 15-mer, kindly supplied by S. GILLAM(Vancouver), as a primer. The method is described in detail by SCHAAPER,DANFORTH and GLICKMAN(1 985). RESULTS Within the 180-bp target sequence used in this study, there are 88 known base substitution mutations that will produce a LacI- phenotype (B. W. GLICK- MAN, unpublished results). This total is made up of 25 G:C + A:T and 14 A:T - G:C transitions, plus 11 G:C + C:G, 12 G:C 3 T:A, 15 A:T + T:A and 11 A:T + C:G transversions. The results of sequencing 105 and 79 EMS-induced lacl- mutations isolated in the Uvr+ and UvrB- strains, respectively, are presented in Table 1. All but three of the mutants are G:C 3 A:T transitions. The three exceptions were recovered in the Uvr+ strain and included two A:T + G:C transitions (at positions 117 and 162) and a G:C + T:A transversion (at position 49). The mutation frequencies induced at the 25 potential sites of G:C + A:T transition in the Uvr+ strain after exposure to 3% EMS are shown in Table 2. The distribution of the G:C 3 A:T transitions with respect to the target sequence is shown in Figure 1. The mutations are distributed over 16 sites. The induction frequencies vary up to ninefold from site to site. There are two clear hotspots in the Uvr+ spectrum (positions 56 and 185) that account for 814 P. A. BURNS, F. L. ALLEN AND B. W. GLICKMAN TABLE 2 Induced frequencies of GC -.+ A:T transitions at potential GC + AT mutant sites" in Uvr+ and UvrB- strains following 3% EMS treatment Frequency (X 103 -UvrB- Position Sequence* In Uvr+ In UvrB- Uvr+ 42 CGT 1.0 (4)' 1.7 (2) 1.7 53 TGT 0 0 56 CGC 2.3 (9) 2.5 (3) 1.1 57 TGC 0.3 (1) 5.9 (7) 19.7 75 AGA 0.5 (2) 8.4 (10) 16.8 80 TGA 0.3 (1) 3.4 (4) 11.3 84 GGT 0.8 (3) 5.1 (6) 6.4 90 GGA 0.5 (2) 1.7 (2) 3.4 92 CGG 1.3 (5) 9.3 (1 1) 7.2 93 CGC 1.0 (4) 5.1 (6) 5.1 95 CGT 0 0.8 (1) 104 TGG 0.3 (1) 0.8 (1) 2.7 113 TGG 0 0 120 AGA 0 3.4 (4) 129 CGT 0 0 140 AGT 1.0 (4) 4.2 (5) 4.2 174 GGG 1.6 (6) 0 179 CGG 0 0 185 CGC 2.3 (9) 3.4 (4) 1.5 186 TGC 0.3 (1) 2.5 (3) 8.3 188 TGT 0 2.5 (3) 191 TGT 0.8 (3) 3.4 (4) 4.3 198 CGC 0 0 20 1 GGC 0.3 (1) 0.8 (1) 2.7 206 TGT 0 1.7 (2) Total 14 x 67 X 4.8 AI1 known sites from unpublished spectra and MILLER(1984).
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