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Mutagenicity of the 1-Nitropyrene-DNA Adduct N-(Deoxyguanosin-8-yl)-1-aminopyrene in Escherichia coli Located in a Nonrepetitive CGC Sequence Manny D. Bacolod, Ramji Krishnasamy, and Ashis K. Basu* Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269 Received February 7, 2000
1-Nitropyrene, a common environmental pollutant, forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). Mutational spectra of randomly introduced dGAP in Escherichia coli included many different types of mutations. However, a prior site-specific study in a CGCGAPCG sequence showed only CpG deletions and +1 frame shifts. To further explore the context effects of dGAP in mutagenesis, in this work this adduct was incorporated into a nonrepetitive CGC sequence in single-stranded M13mp7L2 DNA. Upon replication of this construct in repair-competent E. coli, one-base deletions and base substitutions were detected. The -1 frame shifts, whose frequency increased 3-6-fold with SOS (to an average frequency of 1.5%), involved deletion of the adjacent C residues. The base substitutions (∼2.2%) included targeted G-to-T and G-to-C transversions, whose frequencies did not increase with SOS. This suggests that dGAP mutagenesis is highly dependent on the local DNA sequence. 1-Nitropyrene (1-NP),1 a ubiquitous environmental carcinogen (1-8), binds covalently to the C8 position of 2′-deoxyguanosine upon reductive activation to form N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP) (9-11). dGAP induces both base substitution and frame shift mutations, although in bacteria the latter predominate (12-15). Frame shifts induced by dGAP include one-base deletions, one-base insertions, and dinucleotide deletions. To determine the mechanism of mutagenesis by dGAP, we first constructed a single-stranded (ss) M13 genome in which the adduct was placed at the underlined deoxyguanosine of an inserted CGCGCG sequence (16). This DNA sequence was chosen because 1-NP is a very potent mutagen in Salmonella typhimurium frame shift tester strains (such as TA 98) and induces CpG deletion in a repetitive CpG sequence near the reversion site (15). Using this site-specific construct, we found that dGAP induces CpG deletions at a frequency of nearly 2% in repair-competent Escherichia coli strains, which is 20fold greater than that of the control (16). With SOS, the frequency of frame shift mutations is increased. The enhancement in mutation frequency (MF) is due to a +1 frame shift of either C or G residues adjacent to the adduct site. The objective of our current study is to explore the mutagenicity of dGAP in a nonrepetitive CGC DNA sequence. The conformations of dGAP in such a DNA sequence were investigated using an 11-mer, d(CCATCGAPCTACC), by a combination of NMR and molecular mechanics studies (17, 18). These studies showed that the aminopyrene ring of dGAP is intercalated into the DNA helix between two intact Watson-Crick C‚G base * To whom correspondence should be addressed. Telephone: (860) 486-3965. Fax: (860) 486-2981. E-mail: akbasu@ nucleus.chem.uconn.edu. 1 Abbreviations: 1-NP, 1-nitropyrene; AP, 1-aminopyrene; dGAP, N-(deoxyguanosin-8-yl)-1-aminopyrene; ss, single-stranded; ds, doublestranded; MF, mutation frequency.
pairs flanking the adducted site in the duplex 11-mer. The intercalation of the aminopyrene ring results in the displacement of the modified dG ring into the major groove. The glycosidic torsion angle of dGAP is in the syn domain. No hydrogen bond could be detected between the adducted dG and its partner. Thermodynamic studies established that the adducted duplex is most stable when there is no partner opposite dGAP, suggesting a rationale for targeted G deletions (19). With this information on the structure and stability of the adduct on our hands, we carried out an investigation of the effect of dGAP upon replication in E. coli, which is reported here.
Materials and Methods Materials. Caution: 1-NP and its derivatives are carcinogenic to rodents and should be handled carefully. 1-NP, 1-aminopyrene, and m-chloroperoxybenzoic acid were obtained from Aldrich Chemical Co. (Milwaukee, WI). [γ-32P]ATP was from Du Pont New England Nuclear (Boston, MA). T4 polynucleotide kinase and DNA ligase were obtained from New England Biolabs (Beverly, MA). E. coli strains GW5100 (JM103, P1-) and DL7 (AB1157, lac∆U169, uvr+) that carry a chromosomal lac deletion have been described previously (14). Methods. Oligodeoxynucleotides were synthesized on an Applied Biosystems, Inc., model 380B DNA synthesizer, using the phosphoramidite method. HPLC separations were performed using reverse-phase columns (Phenomenex C-18, 5 µm particle size, 4.6 mm × 250 mm). The 11-mer containing dGAP was synthesized as described previously (19, 20). All M13 minipreps were carried out using Qiagen Spin M13 Kits purchased from Qiagen Inc. (Valencia, CA). DNA sequencing reactions were performed using Big Dye Terminator Cycle Sequencing Ready Reactions from PE Applied Biosystems (Foster City, CA). The sequencing runs were carried out in an ABI Prism 377XL DNA Sequencer at the University of Connecticut Biotechnology Center (Storrs, CT). (1) Construction of Site-Specifically Modified M13 Genomes. Scheme 1 shows the steps for construction of sitespecifically modified ssM13 genomes and the subsequent assay
10.1021/tx000023r CCC: $19.00 © 2000 American Chemical Society Published on Web 05/25/2000
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Scheme 1. Construction Scheme of M13mp7L2 Containing a Site-Specific dGAP and Subsequent Steps for Mutational Analysis
for mutagenesis. Bacteriophage M13mp7L2 DNA (400 µg) was digested with EcoRI (3200 units) for 2 h at 25 °C in 1 mL of 100 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 50 mM NaCl. Agarose gel electrophoresis indicated no visible band for the remaining circular DNA. An equimolar ratio of a scaffold 50mer was annealed to the linear ssDNA at a concentration of 100 ng/mL by heating at 75 °C for 15 min followed by slow cooling to room temperature over a period of 3-4 h. A 10-fold molar excess of the modified or unmodified 5′-phosphorylated 11-mer was ligated into the gap of this annealed DNA in the presence of 800 units of T4 DNA ligase in 40 mM Tris-HCl buffer (pH 7.8), 8 mM MgCl2, 16 mM dithiothreitol, and 1 mM ATP at 16 °C for 48 h. After ethanol precipitation, an additional round of EcoRI (5 units/µg of DNA) digestion was carried out for 4 h to linearize any uncut or religated DNA. To remove the 50-mer scaffold from the M13 DNA, each DNA solution was heated at 100 °C for 45 s and rapidly cooled to 0 °C. Prior to heating, a 10-fold molar excess of an “anti-scaffold” 50-mer that contained the DNA sequence complementary to the scaffold oligomer was added to prevent the scaffold from reannealing on the M13 DNA (21). To monitor whether removal of the scaffold was quantitative, an aliquot of gapped circular M13 DNA was subjected to the same steps of denaturation and analyzed by agarose gel electrophoresis. The ligation efficiency was determined by comparing the slower running circular DNA band with the faster running linear DNA with the aid of a Kodak Digital Science Electrophoresis Documentation and Analysis System 120 and 1D Image Analysis Software. (2) SOS Induction and Transformation in E. coli. Repaircompetent E. coli (DL7) cells were grown in 100 mL cultures in Luria broth to a density of 1 × 108 cells/mL and then harvested by centrifugation at 5000g for 15 min at 0 °C. The cells were resuspended in an equal volume of ice-cold deionized water and recentrifuged at 5000g for 30 min. This procedure was repeated except the cells were resuspended in 50 mL of ice-cold deionized water. The bacterial pellet was resuspended in 1 mL of glycerol/ water (10% v/v) and kept on ice until further use. To induce SOS, the following additional steps were introduced after the first centrifugation. The cells were resuspended in 50 mL of 10 mM MgSO4 and treated with UV light (254 nm) (20 J/m2) in 25 mL aliquots in 150 mm × 50 mm plastic Petri dishes. The
cultures were incubated in Luria broth at 37 °C for 40 min so SOS functions could be maximally expressed. Following SOS induction, these cells were centrifuged, deionized, and resuspended in glycerol/water in a manner similar to that described above except all manipulations were carried out in subdued light. For each transformation, 60 µL of the cell suspension was mixed with 60 ng of M13 construct and transferred to the bottom of an ice-cold Bio-Rad Gene-Pulser cuvette (0.1 cm electrode gap). Electroporation of cells was carried out in a Bio-Rad GenePulser apparatus at 25 µF and 1.8 kV with the pulse controller set at 200 Ω. Immediately after electroporation, 1 mL of SOC medium (22) was added and the mixture was transferred to a 1.5 mL microcentrifuge tube. Following a 1 h recovery at 37 °C, the cells were centrifuged at 15000g for 5 min to isolate the phage-containing supernatant. (3) Analysis of Transformants by Oligonucleotide Hybridization. Progeny phage that produced clear plaques in the presence of IPTG and X-gal were analyzed by a differential oligonucleotide hybridization technique as reported previously (21). The M13 progeny were plated overnight in 15 mm × 50 mm Petri dishes at a density of 80-100 plaques per plate. The plaques were transferred to Magnagraph nylon transfer membranes (Osmonics Inc.). The filters were sequentially washed with a denaturing buffer (0.5 M NaOH and 1.5 M NaCl) for 30 s and a neutralizing solution [0.5 M Tris-HCl (pH 8.0) and 1.5 M NaCl] for 5 min, followed by a rinse with 2× SSC (1.5 M NaCl and 0.2 M sodium citrate) for 5 min. The liberated DNA was cross-linked to the filter with UV light (1200 × 100 µJ/cm2). The membranes were left in a hybridization vial containing 20 mL of prehybridization solution [6× SSC and 5× Denhardt’s (22)] and incubated for 3 h at 50 °C inside a hybridization incubator (Robbins Scientific). After the prehybridization, 0.15 µg of 32Plabeled 17-mer probe (5′-ATTGGTAGCGATGGCAC) was added along with 20 mL of hybridization solution (6× SSC, 5× Denhardt’s, 100 µg/mL yeast RNA, 0.05% sodium pyrophosphate, and 0.1% SDS). After incubation for 12-15 h at 50 °C, the membranes were washed three times with 20 mL of wash solution (6× SSC, 0.05% sodium pyrophosphate, and 0.1% SDS) first at 40 °C for 20 min, then at 45 °C for 20 min, and finally at 50 °C for 30 min. Exposure of the autoradiographic film to
Mutagenicity of the 1-Nitropyrene-DNA Adduct
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Table 1. One-Base Deletions at the 5′-CGC-3′ Sequencea dGAP
dG expt
plaques screened
1
2295
2
1076
total
3371
1
1667
2
1176
total
2843
5′-C 0 (0)b 0 (0)
0 (0) 0 (0)
G 0 (0) 0 (0)
0 (0) 0 (0)
3′-C 0 (0) 2 (0.19)
0 (0) 0 (0)
others 0 (0) 0 (0)
0 (0) 0 (0)
plaques screened
total (%)
Without SOS 0 1938 0.19
718
0.10
2656
With SOS 0
706
0
630
0
1336
5′-C
G
3′-C
others
total (%)
2 (0.10)b 0 (0)
0 (0) 0 (0)
3 (0.15) 3 (0.42)
0 (0) 1c (0.14)
0.25 0.56 0.41
4 (0.57) 0 (0)
0 (0) 0 (0)
9 (1.3) 7 (1.1)
0 (0) 0 (0)
1.9 1.1 1.5
a
Phenotypically detectable mutants confimed by DNA sequencing. Several mutants from both control and dGAP-containing constructs involved a single-base deletion at the ligation sites. These are likely to be the result of genetic engineering manipulations and were subtracted from our calculations. b Deletion frequency of each expressed as a percentage is shown in parentheses. c Deletion of 5′-CGAPCT3′. Table 2. Base Substitutions Detected at or Near the dG or dGAP Sitea dGAP
dG expt
plaques screened
1
231
2
129
total
360
1
325
2
180
total
GfG
GfT
GfC
GfA
231 (100)b 129 (100)
0 (0) 0 (0)
0 (0) 0 (0)
0 (0) 0 (0)
325 (100) 180 (100)
0 (0) 0 (0)
505
0 (0) 0 (0)
0 (0) 0 (0)
total % base substitution
plaques screened
Without SOS 0 180 0
158
0 With SOS 0
338 439
0
234
0
673
GfG
GfT
GfC
GfA
174 (96.7)b 156 (98.7)
3 (1.7) 1 (0.63)
3 (1.7) 1 (0.63)
0 (0) 0 (0)
total % base substitution 3.4 1.3 2.4
429 (97.7) 230 (98.3)
8 (1.8) 3 (1.3)
1 (0.23) 1 (0.43)
1 (0.23) 0 (0)
2.3 1.7 2.0
a
Clear plaques analyzed by oligonucleotide hybridization followed by DNA sequencing. b The number in parentheses represents the percentage of each targeted event.
the hybridized filter provided the locations of the radioactively labeled probe, which were compared to the actual plates. The plaques that did not match any spot on the film were considered putative mutants and subjected to DNA sequencing. The conditions for hybridization were stringent enough such that the probe annealed only to the nonmutants whereas it failed to anneal to any progeny containing a single base substitution.
Results Construction of M13 Genomes. The construction of site-specifically modified M13 genomes was accomplished by a strategy originally developed by Lawrence and coworkers (23), which is routinely used in our laboratory (Scheme 1) (16, 21, 24). In short, the hairpin region of the single-stranded M13mp7L2 DNA was digested with EcoRI. The linear DNA was recircularized noncovalently by annealing a scaffold 50-mer so that an 11-mer gap, complementary to 5′-CCATCGCTACC-3′, was generated. The 5′-phosphorylated control and adducted 11-mers were ligated to this gap. Prior to ligation, both adducted and control 11-mers were examined for purity. The mobility of the adducted 11-mer was significantly retarded with respect to that of the control 11-mer, and subsequent analysis with a phosphorimager ensured that each 11-mer was pure (data not shown). Ligation efficiency was determined by running a portion of the constructs on an agarose gel followed by densitometry analysis. For the first set of constructs, the efficiency of ligation was ∼25% for both control and adducted DNA.
In a subsequent experiment, it was 29 and 18% for the control and dGAP-adducted DNA, respectively. On the basis of the ligation efficiency, the same amount of circular genome was used for transformations. Viability of the Site-Specifically Modified M13 Genomes. A single dGAP reduced the viability of the M13 genome significantly. In uninduced cells, the average extent of survival was 1.3% of the control, which increased to 1.8% with SOS (with 20 J/m2 UV irradiation). It is worth mentioning that in a prior work with sitespecifically located dGAP in ssDNA, we have observed extents of survival that were as high as 20-30% in uninduced cells and 30-40% in SOS-induced cells (16). This suggests that viability may be dependent on the local DNA sequence. Frame Shift Mutagenesis. The M13 construct generated after ligation of the 11-mer is a +1 derivative of M13mp7, which gave clear plaques in the presence of IPTG and X-gal. A -1 (or +2) frame shift should restore the reading frame, resulting in blue plaques. Initially, we used this phenotypic screening to identify the putative one-base deletion mutants. To our surprise, sequencing of the DNA isolated from the blue plaques revealed that one-base deletions occurred, but not at the adducted G. The deletions occurred only at the adjacent C residues with a marked preference for deletion of the 3′-C (Table 1). The C deletions occurred at a frequency of 0.3-0.4% in uninduced cells (Table 1). Statistical analysis using a P value approach suggested that upon induction of SOS
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Scheme 2. A Model To Account for Mutagenesis and Error-Free Bypass by E. coli and Human DNA Polymerasesa
a
The bold arrows represent the predominant steps in both organisms.
an increase of 0.5-2% in the frequency of C deletions occurred. The C deletion frequency with SOS, therefore, was 3-6-fold of the same in uninduced cells. Of the four transfections of control constructs, only in one case was deletion of the 3′-C detectable at a frequency of
