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Chem. Res. Toxicol. 1998, 11, 64-69
The Major Mitomycin C-DNA Monoadduct Is Cytotoxic but Not Mutagenic in Escherichia coli Leilani A. Ramos,† Roselyn Lipman,‡ Maria Tomasz,‡ and Ashis K. Basu*,† Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, and Department of Chemistry, Hunter College, City University of New York, New York, New York 10021 Received September 4, 1997X
To determine the mutagenic and genotoxic properties of the major guanine N2-adduct formed by the antitumor drug mitomycin C, we have synthesized a decanucleotide, d(TTACGMCTATCT), containing the adduct, which was inserted into a gapped bacteriophage M13 genome. Analysis of the constructed genome indicated that 41% ligation of the adducted 10-mer occurred on both sides of the gap, whereas the control 10-mer ligated with 34% efficiency. After transfection of the adducted single-stranded M13 DNA into Escherichia coli, the adduct was found to be highly genotoxic. Viability of the adducted genome in a repair-competent strain was only 7%, which increased to 12% and 15% upon induction of SOS by irradiating the cells with 254-nm light at 20 and 50 J/m2, respectively. Even lower viability of 2%, 4.6%, and 0.2% was observed in uvrA, uvrB, and uvrC strains, respectively, which increased up to 10-fold with SOS. An examination of the surviving phage populations revealed that the adduct was not detectably mutagenic. No mutants from the repair-proficient strain were detected after analysis of more than 2500 progeny phage. Only 0.2% of the survivors were mutants in the uvrA strain. It is uncertain, however, if they were induced by the adduct, since all the mutants showed untargeted mutations. We conclude that the major guanine N2-adduct formed by mitomycin C is cytotoxic but not appreciably mutagenic in E. coli.
Introduction Mitomycin C (MC, 1)1 is an antitumor antibiotic used clinically in cancer chemotherapy (1). Its antitumor activity has been attributed to its ability to form monoadducts and cross-links in DNA (2). Upon reductive activation, MC alkylates DNA in both monofunctional and bifunctional manners, generating, in the latter case, inter- or intrastrand DNA cross-links (2-8). The major covalent adducts formed by MC both in vitro and in vivo have been isolated and characterized (5, 6, 8; see ref 9 for a review). The structures of these adducts are displayed in Scheme 1, which includes two monoadducts (2 and 3) and two bisadducts (4 and 5). Bisadduct 4 represents interstrand cross-links, while bisadduct 5 originates from intrastrand cross-links in DNA. The MC moiety in all these adducts is attached covalently to the N2-position of guanine (Gua). The relative proportions of formation of these adducts are dependent on the conditions of reductive activation of MC (6, 7). These adducts have also been detected in intact cells (10, 11). Recently, in addition to the four guanine N2-adducts formed by MC, a guanine N7-adduct of 2,7-diaminomitosene, a reduction product of MC, has been isolated (12). The antitumor action of the drugs that covalently bind to DNA is believed to involve selective killing of tumor * To whom correspondence should be addressed. Phone: (860) 4863965. Fax: (860) 486-2981. E-mail:
[email protected]. † University of Connecticut. ‡ Hunter College, City University of New York. X Abstract published in Advance ACS Abstracts, December 15, 1997. 1 Abbreviations: MC, mitomycin C; DMC, 10-decarbamoylmitomycin C; MC-dG and DMC-dG, deoxyguanosine N2-monoadduct of MC and DMC, respectively; dG, 2′-deoxyguanosine; TEAA, triethylammonium acetate; SVD, snake venom phosphodiesterase; BSA, bovine serum albumin; MF, mutation frequency; GHD, gapped heteroduplex.
cells, and studies with several anticancer drugs suggest that bifunctionality, and hence cross-linking, plays a crucial role (e.g., see ref 13). Even though DNA crosslinks are believed to be responsible for the antitumor effect of MC, various studies indicate that 10-decarbamoyl-MC (DMC, 6), which is incapable of forming DNA cross-links, also possesses potent antibiotic and cytotoxic effects (14-18). Interestingly, both MC and DMC form a guanine monoadduct of identical structure (3) in DNA upon reductive activation (19). It is conceivable, therefore, that one or both of the monoadducts formed by MC (2 and 3) are cytotoxic. Although some studies suggest that both monoadducts and cross-links are repaired by excision nucleases such as UvrABC in Escherichia coli (20), the comparative kinetics of repair of the monoadduct and cross-link have not been directly determined. Like many other antitumor agents, MC is a mutagen and carcinogen, and its potent direct-acting mutagenicity was shown in several bioassays including the Salmonella typhimurium assay (Ames assay) (21, 22) and SOS chromotest (23). In the Ames assay MC-induced mutagenesis is detectable only in the presence of the plasmid pKM101. Furthermore, MC mutagenesis is unique as it reverts only S. typhimurium strains that contain a functional excision repair system (22). That excision repair plays a role in mutagenic processing of MC has also been concluded from the observation that MC is more mutagenic in excision repair-proficient strains of E. coli than the repair-deficient strains (24). Despite such studies, the adducts responsible for its mutagenicity have not been identified. In a more recent investigation, Maccubbin et al. exposed a shuttle vector to monofunctionally activated MC under conditions which predomi-
S0893-228x(97)00163-X CCC: $15.00 © 1998 American Chemical Society Published on Web 01/19/1998
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Chem. Res. Toxicol., Vol. 11, No. 1, 1998 65
Scheme 1. Formation of Adducts of Mitomycin C and DNA
nantly generate MC-dG (25). Following replication of the adducted plasmid in human Ad293 cells, a variety of mutations were detected. Nevertheless, such random mutagenesis studies cannot eliminate the possibility that a minor adduct was responsible for the observed mutagenesis. To address which particular DNA-bound form of MC is responsible for a specific biological effect, we have initiated studies of cytotoxicity and mutagenesis caused by site-specific single adducts of MC. Earlier, we investigated the in vitro effects of the major monoadducts 2 and 3 and showed that both monoadducts are strong blocks of DNA synthesis (26). In the current work we have constructed an M13 genome containing monoadduct 2 at a preselected site and evaluated the viability and mutagenesis in E. coli.
Materials and Methods Materials. MC was obtained from Bristol-Myers Squibb Co. (Wallingford, CT). All DNA synthesis reagents were purchased from Applied Biosystems, Inc. Radiochemicals were from DuPont New England Nuclear (Boston, MA). DNA sequencing kits were purchased from Amersham Life Sciences Inc. (Arlington Hts, IL). T4 Polynucleotide kinase and T4 DNA ligase were obtained from Bethesda Research Laboratory (Gaithersburg, MD). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). The scaffold 50-mer and the “anti” 50mer were synthesized at the Biotechnology Center, University of Connecticut (Storrs, CT). Sources for all other materials used were given in a previous publication (27). E. coli strains: GW5100 (JM103, P1-) was from G. Walker; DL7 (AB1157, lac∆U169, uvr+) and the isogenic repair-deficient strains DL6 (uvrA), DL5 (uvrB), and DL4 (uvrC), which carry a chromosomal lac deletion, were from J. Essigmann (both Massachusetts Institute of Technology, Cambridge, MA). 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 (Beckman RPSC, C-3 Ultrapore, 10 × 250 mm, for oligonucleotide separations and Beckman C-18 ODS Ultrasphere, 4.6 × 250 mm, for nucleoside and MC-dG adduct analysis). Elution conditions were similar to those described in a prior publication (28). Quantitative analysis of nonradiolabeled oligonucleotides was based on
absorbance measurements in buffer solutions as described previously (27). The oligonucleotides were denatured by heating at 90 °C for 2 min and immediately loaded and electrophoresed on a 16% polyacrylamide-7 M urea gel in 89 mM Tris-borate, 2 mM EDTA (pH 8.0). The gels were electrophoresed in an IBI sequencing gel apparatus until the bromophenol blue dye ran out of the gel. Relative proportions of oligonucleotide products in the autoradiograms were analyzed by a Bio-Rad model 620 video densitometer equipped with a Hewlett-Packard 3396 integrator. Nucleoside and MC-dG Composition Analysis. Unmodified or monoadducted oligonucleotides (1 A260 unit) were digested with SVD (1 unit) and E. coli alkaline phosphatase (0.5 unit) in 0.2 mL of 0.1 M Tris-2 mM MgCl2, pH 8.2, buffer, at 45 °C for 4 h. The digest was directly analyzed by HPLC. Peak areas were integrated. Molar ratios of each peak were calculated by dividing a peak area by 254 (M-1 cm-1) of the corresponding nucleoside or nucleoside-MC adduct (dC, 6300; dG, 13 000; dT, 6600; dA, 13 300; adducts 2 and 3, 24 000) (28). Synthesis of a Decamer Containing MC at a Specific Site. A mixture of two complementary oligonucleotides, 5′TTACGTATCT-3′ (10-mer) and 5′-TAm5CGTAA-3′ (7-mer; 1 mM each in mononucleotide units), and MC (4 mM) in 0.1 M potassium phosphate, pH 7.4, buffer (0.45 mL) was briefly heated at 50 °C and then allowed to cool slowly to 0 °C. An anaerobic Na2S2O4 solution (40 mM) in the same buffer was prepared at the same time by purging the buffer with argon before, during, and after the addition of the solid Na2S2O4. An aliquot of this solution was added in one portion to the oligonucleotide-drug mixture to give a 2 mM Na2S2O4 concentration, and the solution was stirred, exposed to air, for 1 h at 0 °C. The reaction mixture was chromatographed immediately on a Sephadex G-50 column (2.5 × 56 cm), using 0.02 M NH4HCO3 as eluant. The first eluting UV-absorbing peak contained modified and unmodified oligonucleotides as a mixture. After lyophilization of this fraction, it was separated into individual components by HPLC using a semipreparative column. The collected HPLC fractions were desalted by passage through a 2.5- × 56-cm Sephadex G-25 (fine) column, with 0.02 M NH4HCO3 as eluant. The collected G-25 fractions were pooled, concentrated, and subjected to 16% polyacrylamide gel electrophoresis in the presence of 7 M urea. The modified decamer that migrated more slowly than the unmodified 10-mer was visualized by UV shadowing and excised. The product was eluted in 1× TE buffer and desalted on a C-18 Sep-Pak cartridge (Waters).
66 Chem. Res. Toxicol., Vol. 11, No. 1, 1998 Scheme 2. Diagram of Site-Specific Incorporation of MC-dG Monoadduct 2 into a Single-Stranded Viral Genome
Construction of Site-Specifically Modified M13 Genomes. For construction of site-specifically modified genomes, we followed the procedure developed by Lawrence and coworkers (Scheme 2) (29, 30). Bacteriophage M13mp7L2 (200 µg) was digested with a large excess of EcoRI (2400 units) for 2 h at 25 °C in 1 mL of 100 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 50 mM NaCl. Agarose gel electrophoresis indicated no visible band for the remaining circular DNA (Figure 1). Two-fold molar excess of a scaffold 50-mer was annealed to the linear ss DNA at a concentration of 100 ng/mL by heating at 67 °C for 4 min followed by slow cooling to room temperature over a period of 3-4 h. Fifty-fold molar excess of the modified or unmodified decanucleotide was ligated into the gap of this annealed DNA in the presence of 20 units of T4 DNA ligase in 40 mM TrisHCl buffer (pH 7.8), 8 mM MgCl2, 16 mM dithiothreitol, and 1 mM ATP at 16 °C overnight. A “mock ligation” was also carried out in which no decamer was included. To remove the 50-mer scaffold from the M13 DNA, each DNA solution was heated at 100 °C for 2 min and rapidly cooled to 0 °C. Prior to heating, a 10-fold molar excess of a 50-mer that contained the DNA sequence complementary to the scaffold oligomer was added to the DNA solution to ensure that the scaffold, once denatured, did not reanneal on the M13 DNA. To monitor whether removal of the scaffold was quantitative, an aliquot of gapped heteroduplex (GHD) was subjected to the same steps of denaturation and analyzed by agarose gel electrophoresis. Since migration characteristic of linear and circular DNA in an agarose gel is different, we could demonstrate a complete conversion of any unligated circular genome (or a gapped genome) to linear M13 DNA by the above protocol. SOS Induction and Transformation in E. coli. E. coli cells were grown in 100-mL cultures in Luria broth to 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 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 resus-
Ramos et al. pended in 50 mL of 10 mM MgSO4 and treated with UV light (254 nm) (10, 20, or 50 J/m2) in 25-mL aliquots in 150- × 50mm plastic Petri dishes. The cultures were incubated in Luria broth at 37 °C for 40 min (20 min for uvr strains) in order to express SOS functions maximally. Following SOS induction, these cells were centrifuged, deionized, and resuspended in glycerol/water in a similar manner as described above except all manipulations were carried out in subdued light. Before transformation, the constructed genome was subjected to another round of EcoRI treatment to digest any uncut or religated M13mp7L2 DNA. For each transformation, 40 µL of the cell suspension was mixed with 4 µL (500 ng) of DNA solution 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 Gene-Pulser apparatus at 25 µF and 1.8 kV with the pulse controller set at 200 Ω. Immediately after electroporation, 1 mL of SOC medium (31) was added, and the mixture was transferred to a 1.5-mL microcentrifuge tube. Part of the cells were plated following a 45-min recovery at 37 °C in the presence of the plating bacteria E. coli GW5100, IPTG, and X-gal to determine the number of independent transformants. The remainder of the cells were centrifuged at 15000g for 5 min to isolate the phage-containing supernatant. It is important to point out that in contrast to the large phage population generated by transfection of the unligated gapped genome, the same amount of the denatured gap did not produce any progeny phage. Analysis of Transformants for Mutations. Progeny phage were analyzed by a differential oligonucleotide hybridization technique as reported (32). To determine the optimum temperature for hybridization and wash, a mutant clone was constructed, which contained a T at the adduct site of the constructed vector. A 32P-end-labeled 25-mer, 5′-ATGGAATTAGATACGTAACACTGAA-3′, was used as the hybridization probe. The initial temperature for hybridization was calculated by the formula (G + C) × 4 + (A + T) × 2 - 4, and the conditions were modified so that only the perfectly matched probe hybridizes with DNA. The discriminating wash was at 67 °C. Our protocol was made so stringent that 100% of the plaques of the mutant clone containing a T at the adduct site failed to hybridize with the probe.
Results Synthesis and Characterization of an Oligonucleotide Containing MC-dG (2) at a Specific Site. We reported that substoichiometric amounts of Na2S2O4 and aerobic conditions result quantitatively in monofunctional activation of MC, leading exclusively to monoadduct formation (Scheme 1, 2) and that Gua in the 5′-CpG sequence in a duplex structure is required for optimal monoalkylation yields (28). Also, the yield of alkylation by MC is enhanced at G‚m5C base pairs (29). We, therefore, annealed the decamer 5′-TTACGTATCT-3′ with 5′-TAm5CGTAA-3′ to provide a duplex substrate for the alkylation. The guanines in both strands were alkylated, as expected, as seen from the product profile on HPLC (data not shown): the adducted decamer was formed in 31% yield. The modified decamer was subjected to denaturing polyacrylamide gel electrophoresis, which eliminated any residual unmodified decamer. Identification of the MC-dG-containing decamer was based on quantitative nucleoside and MC-nucleoside adduct analysis: The purified adducted decamer was digested with SVD and E. coli alkaline phosphatase, and the mixture was directly analyzed by HPLC. The composition was consistent with theoretical calculation. Construction and Characterization of an M13 Genome Containing a Single MC-dG. Following the protocol of Lawrence and co-workers, the hairpin region
Site-Specific MC-dG Monoadduct in E. coli
Chem. Res. Toxicol., Vol. 11, No. 1, 1998 67
Figure 1. Characterization of the M13 constructs. The DNA samples were electrophoresed on 1% agarose gel in TBE buffer and subsequently stained with ethidium bromide (1 µg/mL). Lanes 1 and 2 show M13mp7L2 before and after EcoRI digestion, respectively. Lane 3 shows the EcoRI-digested M13 after annealing the 50-mer scaffold. The M13 constructs generated by ligation of unmodified and modified decamers are shown in lanes 4 and 5, respectively, after denaturation of the scaffold, whereas lane 6 shows the same for the mock ligation product.
of M13mp7L2 was digested with EcoRI (30, 31). This was recircularized noncovalently by annealing a scaffold 50-mer, the two ends of which were complementary to the terminal 20 nucleotides of the linearized vector. The central segment of the oligomer was complementary to 5′-TTACGTATCT, which allowed ligation of 5′-TTACGMCTATCT to the ends of the M13 vector by T4 DNA ligase. Similar steps were used to construct a control unmodified genome. A mock ligation was also carried out in the absence of the decamer, so that the extent of ligation at the two ends of the gap could be determined. A portion of each of these genomes was run on an 1% agarose gel. As shown in Figure 1, lanes 1 and 2 show the migration characteristics of M13mp7L2 DNA before and after digestion with EcoRI. Lane 3 shows generation of approximately 40% of gapped M13 after annealing the scaffold 50-mer on to the linearized DNA. Lanes 4, 5, and 6 show M13 gap ligation products in the presence of the control decamers, MC-adducted decamers, and the mock, respectively, after heat denaturation. Densitometry analysis indicated that the efficiency of recircularization of both the unmodified and modified vectors was 30-35%. Less than 1% of circular DNA was detected in the mock ligation, suggesting that end ligation of the gap by forming a loop of the scaffold is a rare event. To further characterize the constructed genomes, we ligated a 32P-end-labeled modified and control decamer to the gapped M13 genome. Part of the ligated genomes were digested with HinfI and HaeIII before removing the 50mer scaffold. As schematically described in Figure 2, the ligation of the decamer at both the 5′- and 3′-end should generate a 28-mer oligonucleotide upon HinfI and HaeIII digestion, whereas an 18- or 20-mer would result if ligation took place only on the 5′- or 3′-end of the decamer, respectively. Indeed, all three DNA fragments were observed by polyacrylamide gel electrophoresis, and densitometry analysis established that 41% and 34% of the modified and control 10-mer, respectively, were ligated at both ends of the gap. In a separate experiment the adducted decamer was exposed to the conditions of ligation and denaturation of the scaffold and analyzed by HPLC. No detectable degradation of the adduct was noted, suggesting that MC-dG is stable to the conditions of genome construction. Survival and Mutagenesis of MC-dG-Adducted M13 Genome. As shown in Table 1, survival of the M13 genome containing a single adduct was ∼7% of that of the control in E. coli cells with normal repair functions. With SOS, survival increased to 12% and 15% with 20
Figure 2. A. Postligation localization of 32P-radiolabeled decamer following HinfI and HaeIII digestion. Ligation of the decamer at both the 5′- and 3′-end should generate a radiolabeled 28-mer oligonucleotide, whereas an 18- or 20-mer would result if ligation took place only on the 5′- or 3′-end, respectively. B. Autoradiogram showing the migration characteristics of HinfI and HaeIII digestion products. Lanes 1 and 2 show the 18- and 28-mer standards, respectively. Lanes 3 and 4 show the HinfI and HaeIII digestion products from the control and MC-dGadducted genome, respectively. As expected, the MC-dGadducted fragments ran slower than the control fragments.
and 50 J/m2 UV irradiation of the host cells, respectively. This suggests that MC-dG constitutes a strong block of DNA replication and that SOS partially alleviates the inhibitory effect. The adduct was more toxic in uvr cells, but in each case viability increased severalfold with SOS (Table 1). The toxicity of MC-dG was particularly pronounced in the uvrC strain in which viability dropped to 0.2% in the absence of SOS. Mutational analysis of the progeny phage was carried out by oligonucleotide hybridization. To determine the ideal conditions for hybridization, we constructed a mutant clone in which a T was introduced at the MCdG adduct site. Mutational analysis was targeted primarily on progeny isolated from SOS-induced cells (Table 1), because higher frequency of mutagenesis has always been observed from both damaged and undamaged templates when this error-prone repair was induced (34). However, in the case of the MC-dG-adducted template, no mutants were detected after screening more than 2500 plaques from repair-competent cells (MF < 0.04%). The progeny from uvrA cells showed mutagenesis at a frequency of ∼0.2%. It is uncertain whether they were induced by MC-dG, because no targeted mutations occurred. Two of the four mutants contained base
68 Chem. Res. Toxicol., Vol. 11, No. 1, 1998
Ramos et al.
Table 1. Viability of MC-dG-Modified M13 in E. coli and Analysis of Progeny Phagea relative survival MC-adducted E. coli expt strain no.b DL7 (uvr+) DL6 (uvrA-) DL5 (uvrB-) DL4 (uvrC-)
1 1 2 2 3 4 5 3 5 3 5
-SOS
10 J/m2
100 (8.6 × 100 (9.7 × 104) 100 (6.9 × 105) 100 (2.2 × 104) 100 (2.1 × 106) 100 (2.2 × 104) 100 (2.4 × 109) 100 (2.9 × 105) 100 (4.6 × 104) 100 (2.9 × 106) 100 (2.3 × 109)
100 (5.9 × 100 (6.6 × 103)
104)d
+SOSe 20 J/m2
50 J/m2
103)
100 (3.4 ×
106)
screening for mutants
+SOSc
control
100 (2.2 × 105) 100 (9.5 × 104) 100 (1.3 × 104)
100 (7.1 × 102) 100 (1.6 × 104) 100 (8.4 × 104)
no. of plaques mutants screened found 20 50 10 -SOS J/m2 J/m2 J/m2 -SOS +SOSe -SOS +SOS 5.4 6.8 6.7 7.8 3.3 2.2 0.5 2.2 7.0 0.16 0.29
10.7 12.3
2586
0
14.7 14.9 2.4
493
2001
0
4f
13.5 8.1 8.6 2.2
233 204
424
0 0
0
a Viability (in percent) was determined by comparing the number of infective centers from the adducted DNA with that of the control genome (assumed to be 100% viability). b Each experiment refers to a separate construction of the control and adducted genomes. c SOS was induced by UV irradiation at various levels (10, 20, and 50 J/m2) as described in Materials and Methods. d Transformation efficiencies (per µg) of DNA are shown in parentheses for the control genome. e Plaques from 10 J/m2 treatment were screened. f Mutations found include base substitutions from TTACGTATCT to CTACGTATCT (1) and TTACGTCTCT (1) and frameshifts from TTACGTATCT to TTGACTGTAATGCTTG (2), where the underlined bases were insertions.
substitutions: a T f C transition four bases 5′ to and an A f C transversion two bases 3′ to the adducted G occurred. The other two mutants contained an unusual multiple insertion of TTGACTGTAATGCTTG from the original TTACGTATCT sequence. Because of the high toxicity of MC-dG, we did not have enough progeny from uvrB and uvrC strains for mutational analysis.
Discussion The high cytotoxicity of MC has been attributed to MC’s ability to induce covalent cross-links between the two strands of DNA. It has been demonstrated that a single cross-link per genome was sufficient to cause death of a bacterial cell (3). Excellent correlation was found between DNA cross-linking activity and cytotoxicity in mammalian cells within a series of mitomycin derivatives (35). However, several studies have implicated that monofunctional alkylation also represents cytotoxic DNA damage, as discussed in the Introduction. The data in this paper now show clearly that the major monoadduct of MC also is cytotoxic, which is in accord with our previous in vitro investigation (26). Viability of the adducted vector was severalfold lower in uvr strains, and the effect was most pronounced in a uvrC strain. Survival of the modified genome increased with SOS, and yet the level of mutagenesis remained extremely low even in an E. coli strain deficient in excision repair. The biological effects of MC-dG may be consistent with the molecular modeling studies that suggested that the monoadducts can fit in the minor groove of duplex B-DNA without causing significant distortions (5, 36-38). A high-resolution NMR study of the monoadduct in a DNA duplex indicated that the MC residue is situated inside the slightly widened minor groove and that there are extensive noncovalent contacts between the drug and the minor groove of the DNA duplex (39). Thermal melting and molecular modeling studies of a template-primer complex containing monoadduct 2 or 3 at the duplex junction (25) are also consistent with this hypothesis. It is conceivable that the DNA polymerase, albeit stalled, may still be able to “read” such a nondistortive adduct structure as a guanine derivative so that in excess of
99.9% translesion synthesis was error-free, even when the SOS functions have been induced. It is interesting that the uvr gene products influenced the survival of MC-dG-adducted ss DNA, even though nucleotide excision repair is believed to be active only in ds DNA. Could it be due to an incomplete denaturation of the scaffold 50-mer before transfection? To rule out this possibility, we subjected an equal amount of a scaffolded gapped genome to the conditions of denaturation. Upon transfection, the latter did not generate any progeny phage whereas the undenatured scaffolded gap produced a large phage population. This suggests that the denaturation conditions were appropriate for quantitative removal of the scaffold. Nevertheless, this was an external control, and the scaffolded genome containing the ligated 10-mer had additional base pairs. We cannot rule out that a small fraction of the adducted genome contained the scaffold, which led to repair of MC-dG by UvrABC excision nucleases. However, an alternative, and perhaps more convincing, explanation of the uvr effect on viability is that partially double-stranded genomes that arise during conversion of ss M13 to a replicative form are subject to repair by UvrABC. Particularly if the repair of MC-dG is rapid, the observed influence on the viability of the adducted genome is not surprising. The effect of UvrC toward MC-dG is even more intriguing, and further studies are needed. When Gibbs and Lawrence compared ss M13 genomes containing the T-T and U-U photodimers without SOS, increased survival was observed in a uvrA strain relative to the repair-competent strains (40). In addition, viability of the photodimers with SOS was much higher in two different uvr+ strains compared to a uvrA strain (40). Although without SOS the survival data of the MC-dG genome are exactly opposite that for the photodimers, with SOS they followed a similar trend. These effects probably reflect an intricate set of interactions between the multiple repair proteins and the replication apparatus of E. coli. The kinetics of DNA repair probably also play a role. Further studies are ongoing in our laboratory to determine whether the biological property of the adduct in mammalian cells is similar.
Site-Specific MC-dG Monoadduct in E. coli
Acknowledgment. This work was supported by Grant ES07946 from the National Institute of Environmental Health Sciences, NIH (to A.K.B.), and Grant CA28681 from the National Cancer Institute (to M.T.). Facilities at Hunter College were supported by a Research Centers in Minority Institutions award (RR03037) from the Division of Research Resources, NIH.
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