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• W2132023404 abstract "The in vivo mutagenic properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole, two products of peroxynitrite oxidation of guanine, are reported. Two oligodeoxynucleotides of identical sequence, but containing either 2-aminoimidazolone or 5-guanidino-4-nitroimidazole at a specific site, were ligated into single-stranded M13mp7L2 bacteriophage genomes. Wild-type AB1157 Escherichia coli cells were transformed with the site-specific 2-aminoimidazolone- and 5-guanidino-4-nitroimidazole-containing genomes, and analysis of the resulting progeny phage allowed determination of the in vivo bypass efficiencies and mutational signatures of the DNA lesions. 2-Aminoimidazolone was efficiently bypassed and 91% mutagenic, producing almost exclusively G to C transversion mutations. In contrast, 5-guanidino-4-nitroimidazole was a strong block to replication and 50% mutagenic, generating G to A, G to T, and to a lesser extent, G to C mutations. The G to A mutation elicited by 5-guanidino-4-nitroimidazole implicates this lesion as a novel source of peroxynitrite-induced transition mutations in vivo. For comparison, the error-prone bypass DNA polymerases were overexpressed in the cells by irradiation with UV light (SOS induction) prior to transformation. SOS induction caused little change in the efficiency of DNA polymerase bypass of 2-aminoimidazolone; however, bypass of 5-guanidino-4-nitroimidazole increased nearly 10-fold. Importantly, the mutation frequencies of both lesions decreased during replication in SOS-induced cells. These data suggest that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole in DNA are substrates for one or more of the SOS-induced Y-family DNA polymerases and demonstrate that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole are potent sources of mutations in vivo. The in vivo mutagenic properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole, two products of peroxynitrite oxidation of guanine, are reported. Two oligodeoxynucleotides of identical sequence, but containing either 2-aminoimidazolone or 5-guanidino-4-nitroimidazole at a specific site, were ligated into single-stranded M13mp7L2 bacteriophage genomes. Wild-type AB1157 Escherichia coli cells were transformed with the site-specific 2-aminoimidazolone- and 5-guanidino-4-nitroimidazole-containing genomes, and analysis of the resulting progeny phage allowed determination of the in vivo bypass efficiencies and mutational signatures of the DNA lesions. 2-Aminoimidazolone was efficiently bypassed and 91% mutagenic, producing almost exclusively G to C transversion mutations. In contrast, 5-guanidino-4-nitroimidazole was a strong block to replication and 50% mutagenic, generating G to A, G to T, and to a lesser extent, G to C mutations. The G to A mutation elicited by 5-guanidino-4-nitroimidazole implicates this lesion as a novel source of peroxynitrite-induced transition mutations in vivo. For comparison, the error-prone bypass DNA polymerases were overexpressed in the cells by irradiation with UV light (SOS induction) prior to transformation. SOS induction caused little change in the efficiency of DNA polymerase bypass of 2-aminoimidazolone; however, bypass of 5-guanidino-4-nitroimidazole increased nearly 10-fold. Importantly, the mutation frequencies of both lesions decreased during replication in SOS-induced cells. These data suggest that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole in DNA are substrates for one or more of the SOS-induced Y-family DNA polymerases and demonstrate that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole are potent sources of mutations in vivo. Oxidative damage of DNA is implicated as a cause of aging (1Sastre J. Pallardo F.V. Vina J. Free Radic. Biol. Med. 2003; 35: 1-8Crossref PubMed Scopus (283) Google Scholar, 2Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7349) Google Scholar, 3Mandavilli B.S. Santos J.H. Van Houten B. Mutat. Res. 2002; 509: 127-151Crossref PubMed Scopus (264) Google Scholar), carcinogenesis (4Klaunig J.E. Kamendulis L.M. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 239-267Crossref PubMed Scopus (1275) Google Scholar, 5Hussain S.P. Hofseth L.J. Harris C.C. Nat. Rev. Cancer. 2003; 3: 276-285Crossref PubMed Scopus (1417) Google Scholar, 6Olinski R. Gackowski D. Foksinski M. Rozalski R. Roszkowski K. Jaruga P. Free Radic. Biol. Med. 2002; 33: 192-200Crossref PubMed Scopus (257) Google Scholar), and a variety of noncancerous diseases such as Alzheimer disease and cardiovascular disease (7Cooke M.S. Evans M.D. Dizdaroglu M. Lunec J. FASEB J. 2003; 17: 1195-1214Crossref PubMed Scopus (2363) Google Scholar) and in the progression to acquired immunodeficiency syndrome in human immunodeficiency virus-infected patients (8Jaruga P. Jaruga B. Gackowski D. Olczak A. Halota W. Pawlowska M. Olinski R. Free Radic. Biol. Med. 2002; 32: 414-420Crossref PubMed Scopus (83) Google Scholar). The reactive species responsible for DNA damage are generated by common endogenous processes (9De Bont R. Van Larebeke N. Mutagenesis. 2004; 19: 169-185Crossref PubMed Scopus (694) Google Scholar) such as respiration and inflammation (10Turrens J.F. Biosci. Rep. 1997; 17: 3-8Crossref PubMed Scopus (754) Google Scholar, 11Ohshima H. Tatemichi M. Sawa T. Arch. Biochem. Biophys. 2003; 417: 3-11Crossref PubMed Scopus (552) Google Scholar). During inflammation, an assortment of reactive oxygen and nitrogen intermediates are generated by activated immune system cells (11Ohshima H. Tatemichi M. Sawa T. Arch. Biochem. Biophys. 2003; 417: 3-11Crossref PubMed Scopus (552) Google Scholar), and reaction of these molecules with DNA produces dozens of oxidized nucleobase derivatives (12Bjelland S. Seeberg E. Mutat. Res. 2003; 531: 37-80Crossref PubMed Scopus (400) Google Scholar). The radicals nitric oxide (·NO) and superoxide (O2˙¯) are produced by macrophages and neutrophils (two types of inflammatory cells) upon immune response activation (13Xia Y. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6954-6958Crossref PubMed Scopus (599) Google Scholar, 14Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Crossref PubMed Scopus (336) Google Scholar). These radicals combine in a diffusion-limited reaction to form peroxynitrite (ONOO–) 1The abbreviations used are: ONOO–, peroxynitrite; 8-oxoG, 7,8-dihydro-8-oxoguanine; Iz, 2-aminoimidazolone; NI, 5-guanidino-4-nitroimidazole; ODN, oligodeoxynucleotide; THF, tetrahydrofuran; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; REAP, restriction endonuclease and post-labeling analysis of mutation frequency; WT, wild type; pol I, DNA polymerase I; Kf, Klenow fragment of DNA polymerase I; Kf (exo–), Klenow fragment of DNA polymerase I without exonuclease activity; pol α, calf thymus polymerase α; pol β, human polymerase β; pol II, DNA polymerase II; pol IV, DNA polymerase IV; pol V, DNA polymerase V; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. (15Burney S. Caulfield J.L. Niles J.C. Wishnok J.S. Tannenbaum S.R. Mutat. Res. 1999; 424: 37-49Crossref PubMed Scopus (463) Google Scholar), a powerful oxidizing and nitrating agent capable of damaging a variety of biomolecules (16Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar, 17Radi R. Beckman J.S. Bush K.M. Freeman B.A. Arch. Biochem. Biophys. 1991; 288: 481-487Crossref PubMed Scopus (2051) Google Scholar, 18Pryor W.A. Jin X. Squadrito G.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11173-11177Crossref PubMed Scopus (363) Google Scholar), including DNA (19Juedes M.J. Wogan G.N. Mutat. Res. 1996; 349: 51-61Crossref PubMed Scopus (173) Google Scholar). Under physiological conditions, ONOO– rapidly combines with CO2 to form nitrosoperoxycarbonate (ONOOCO2−), which subsequently undergoes homolysis to produce carbonate radical (CO3˙¯) and nitrogen dioxide (·NO2) (20Lymar S.V. Hurst J.K. Chem. Res. Toxicol. 1996; 9: 845-850Crossref PubMed Scopus (184) Google Scholar, 21Lymar S.V. Hurst J.K. J. Am. Chem. Soc. 1995; 117: 8867-8868Crossref Scopus (495) Google Scholar, 22Lymar S.V. Hurst J.K. Chem. Res. Toxicol. 1998; 11: 714-715Crossref PubMed Scopus (60) Google Scholar). These radicals are believed to be responsible for the oxidation and nitration of DNA caused by exposure to ONOO– (23Shafirovich V. Dourandin A. Huang W. Geacintov N.E. J. Biol. Chem. 2001; 276: 24621-24626Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 24Dedon P.C. Tannenbaum S.R. Arch. Biochem. Biophys. 2004; 423: 12-22Crossref PubMed Scopus (513) Google Scholar, 25Pamir B. Wogan G.N. Chem. Res. Toxicol. 2003; 16: 487-492Crossref PubMed Scopus (9) Google Scholar). Because guanine possesses the lowest redox potential of the four DNA nucleobases (E7 = 1.27 V versus normal hydrogen electrode) (26Steenken S. Jovanovic S.V. J. Am. Chem. Soc. 1997; 119: 617-618Crossref Scopus (1281) Google Scholar), it is preferentially oxidized by ONOO– compared with the other natural nucleobases (27Burney S. Niles J.C. Dedon P.C. Tannenbaum S.R. Chem. Res. Toxicol. 1999; 12: 513-520Crossref PubMed Scopus (145) Google Scholar). Several products are formed directly from guanine residues in DNA including 7,8-dihydro-8-oxoguanine (8-oxoG), 8-nitroguanine (8-NO2-G), 2-aminoimidazolone (Iz), and 5-guanidino-4-nitroimidazole (NI) (Fig. 1) (28Niles J.C. Wishnok J.S. Tannenbaum S.R. J. Am. Chem. Soc. 2001; 123: 12147-12151Crossref PubMed Scopus (56) Google Scholar). The lesions 8-oxoG and 8-NO2-G are highly susceptible to further oxidation (27Burney S. Niles J.C. Dedon P.C. Tannenbaum S.R. Chem. Res. Toxicol. 1999; 12: 513-520Crossref PubMed Scopus (145) Google Scholar) and yield a variety of additional products (29Lee J.M. Niles J.C. Wishnok J.S. Tannenbaum S.R. Chem. Res. Toxicol. 2002; 15: 7-14Crossref PubMed Scopus (37) Google Scholar, 30Tretyakova N.Y. Niles J.C. Burney S. Wishnok J.S. Tannenbaum S.R. Chem. Res. Toxicol. 1999; 12: 459-466Crossref PubMed Scopus (99) Google Scholar). Although many of these guanine-derived oxidation products have been characterized for their in vivo mutagenic potential (31Henderson P.T. Delaney J.C. Gu F. Tannenbaum S.R. Essigmann J.M. Biochemistry. 2002; 41: 914-921Crossref PubMed Scopus (132) Google Scholar, 32Henderson P.T. Delaney J.C. Muller J.G. Neeley W.L. Tannenbaum S.R. Burrows C.J. Essigmann J.M. Biochemistry. 2003; 42: 9257-9262Crossref PubMed Scopus (203) Google Scholar), Iz and NI have received little attention. To assess the biological significance and consequences of oxidatively damaged DNA, it is essential that these lesions be characterized for their genotoxic and mutagenic potential. In the work presented here, we report the in vivo genotoxic and mutagenic properties of Iz and NI. Oligodeoxynucleotides (ODNs), site-specifically modified with Iz or NI, were synthesized using a method described previously (33Ikeda H. Saito I. J. Am. Chem. Soc. 1999; 121: 10836-10837Crossref Scopus (45) Google Scholar) and a procedure developed in our laboratory (34Neeley W.L. Henderson P.T. Essigmann J.M. Org. Lett. 2004; 6: 245-248Crossref PubMed Scopus (20) Google Scholar). The biological impact of unique Iz and NI lesions was addressed under normal and SOS-induced conditions in wild-type AB1157 Escherichia coli cells using viral vectors containing the modified ODNs. Both lesions were bypassed by the E. coli replication machinery and were substrates for SOS-induced error-prone DNA polymerase bypass. Furthermore, each lesion was potently mutagenic during DNA replication. Oligodeoxynucleotides—DNA synthesis reagents were purchased from Glen Research. Unmodified ODNs were purchased from IDT, Inc. and were purified by PAGE. The 19-mer ODN sequence used was 5′-GCG AAG ACC GXA GCG TCC G-3′, where X is G, Iz, NI, or a tetrahydrofuran (THF) abasic site. The 19-mer containing the THF abasic site analog was prepared as described (31Henderson P.T. Delaney J.C. Gu F. Tannenbaum S.R. Essigmann J.M. Biochemistry. 2002; 41: 914-921Crossref PubMed Scopus (132) Google Scholar). The 19-mer containing Iz was prepared as described previously (33Ikeda H. Saito I. J. Am. Chem. Soc. 1999; 121: 10836-10837Crossref Scopus (45) Google Scholar). Briefly, 8-methoxy-2′-deoxyguanosine was incorporated into the 19-mer ODN by the phosphoramidite method, and the ODN was deprotected and cleaved from the solid support with concentrated NH4OH at 55 °C for 15 h. The 8-methoxy-2′-deoxyguanosine-containing ODN was purified by PAGE, and the 8-methoxy-2′-deoxyguanosine was subsequently converted to Iz by photoirradiation with 365 nm light in the presence of riboflavin. The Iz-containing 19-mer was purified by anion exchange HPLC on a Dionex NucleoPac PA-100 (4 × 250 mm) analytical column using 10% CH3CN in water (solvent A) and aqueous 1.5 m NH4OAc (solvent B) as solvents. A flow rate of 1.0 ml/min was used, and solvent B was increased from 10 to 25% over 2.5 min and then increased from 25 to 100% over 30 min. The purified 19-mer was characterized using MALDI-TOF mass spectrometry (calculated Mr, 5824.8; found Mr, 5825.8) and enzymatic digestion to nucleosides followed by HPLC analysis. For the enzymatic digestion, 50 mm Tris-Cl, pH 7.0, 0.1 mm ZnSO4, 18 units of nuclease P1, 12 units of alkaline phosphatase (both enzymes from Roche Applied Science), and 2 nmol of Iz-containing 19-mer in 50 μl were incubated at room temperature for 30 min and then immediately analyzed by HPLC. For the HPLC analysis, a Supelco Supercosil LC-18-DB (250 × 2.1 mm, 5 μm) column was used with aqueous 150 mm NH4OAc as solvent A and CH3CN as solvent B. A flow rate of 0.25 ml/min was used, and solvent B was increased from 0 to 15% over 40 min. Five peaks were observed with UV-visible spectra consistent with the nucleosides Iz, C, G, T, and A. The ODN containing NI was prepared by incorporating 5-bromo-4-nitroimidazole into the 19-mer sequence by the phosphoramidite method and subsequently treating the 19-mer with 0.5 m guanidine in THF and then with concentrated NH4OH to produce the 19-mer containing NI (34Neeley W.L. Henderson P.T. Essigmann J.M. Org. Lett. 2004; 6: 245-248Crossref PubMed Scopus (20) Google Scholar). The NI-containing 19-mer was purified by C18 reversed phase HPLC (34Neeley W.L. Henderson P.T. Essigmann J.M. Org. Lett. 2004; 6: 245-248Crossref PubMed Scopus (20) Google Scholar) and by anion exchange HPLC as described above and characterized using MALDI-TOF mass spectrometry (calculated Mr, 5882.9; found Mr, 5882.7) and enzymatic digestion with snake venom phosphodiesterase (ICN Biomedical) and alkaline phosphatase (34Neeley W.L. Henderson P.T. Essigmann J.M. Org. Lett. 2004; 6: 245-248Crossref PubMed Scopus (20) Google Scholar) followed by HPLC analysis (same method as described for the 19-mer containing Iz). For the HPLC analysis, five peaks were observed with UV-visible spectra consistent with the nucleosides NI, C, G, T, and A. Genome Construction—Genomes were constructed in triplicate (Fig. 2) on a 10-pmol scale as described previously (31Henderson P.T. Delaney J.C. Gu F. Tannenbaum S.R. Essigmann J.M. Biochemistry. 2002; 41: 914-921Crossref PubMed Scopus (132) Google Scholar, 32Henderson P.T. Delaney J.C. Muller J.G. Neeley W.L. Tannenbaum S.R. Burrows C.J. Essigmann J.M. Biochemistry. 2003; 42: 9257-9262Crossref PubMed Scopus (203) Google Scholar, 35Delaney J.C. Essigmann J.M. Chem. Biol. 1999; 6: 743-753Abstract Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, the single-stranded M13 DNA was linearized by cleavage with EcoRI (30 units/pmol DNA, 23 °C for 8 h) at a hairpin containing a single EcoRI site (35Delaney J.C. Essigmann J.M. Chem. Biol. 1999; 6: 743-753Abstract Full Text PDF PubMed Scopus (58) Google Scholar). The genome was recircularized by annealing in the presence of the 5′-phosphorylated 19-mer insert and two “scaffold” ODNs (sequences that are partially complementary to the 5′ and 3′ sides of the insert and the genomic DNA termini) and incubating with 22.5 units/μl T4 DNA ligase (New England Biolabs) for 2 h at 16 °C in a volume of 55 μl (31Henderson P.T. Delaney J.C. Gu F. Tannenbaum S.R. Essigmann J.M. Biochemistry. 2002; 41: 914-921Crossref PubMed Scopus (132) Google Scholar). Two short scaffolds are used to leave a single-stranded gap at the site of the lesion, thereby facilitating efficient ligation regardless of the lesion structure. The scaffold DNA was removed using the exonuclease activity of T4 DNA polymerase (Amersham Biosciences) by treating with 0.25 units/μl for 1 h at 16 °C in a volume of 65 μl (36Moriya M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1122-1126Crossref PubMed Scopus (432) Google Scholar, 37Moriya M. Zhang W. Johnson F. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11899-11903Crossref PubMed Scopus (233) Google Scholar). Under these conditions, scaffold digestion was complete as determined by using a radiolabeled scaffold and analyzing the reaction with PAGE and phosphorimaging. The genome constructs were diluted to 115 μl with H2O, extracted with 100 μl of phenol:chloroform:isoamyl alcohol (25:24:1) (Invitrogen) and desalted with Sephadex G50 fine resin (Amersham Biosciences). The stability of the lesion-containing 19-mers was assessed using MALDI-TOF mass spectrometry. The 19-mers were exposed to the conditions used for genome construction with the exception that M13 DNA, other ODNs, and enzymes were excluded. The mass spectra of the 19-mers were essentially unchanged after exposure to the mock genome construction conditions. The amount of circular, 19-mer insert-containing genome in each sample was quantified by agarose gel electrophoresis and phosphorimaging. This was accomplished by annealing 1 pmol of a 5′ 32P-labeled 30-mer probe ODN (5′-TCC CAG TCA CGA CGT TGT AAA ACG ACG GCC-3′) to 0.1 pmol of each genome construct in a region of the genome that did not include the lesion-containing 19-mer insert. The annealing solution consisted of 100 mm NaCl, 4.2% Ficoll, 0.042% bromphenol blue, and 0.042% xylene cyanol FF in a volume of 15.5 μl. The genomeprobe mixtures (15.5 μl) were run on a 0.9% agarose gel in 1× Tris borate/EDTA buffer for 4 h at 100 V, after which the free probe front was excised. The gel was run for an additional 3 h and then transferred onto a glass plate and dried under a box fan for 36 h. The amount of circular genome was quantified by phosphorimaging and the genome construct solution volumes adjusted such that each solution used for cell transformation contained an equal concentration of circular DNA. Preparation of Electrocompetent Cells—Two 150-ml aliquots of LB medium were each inoculated with 1.5 ml from separate overnight cultures of wild-type AB1157 E. coli and grown on a shaker at 37 °C to an A600 of ∼0.4. Each culture was centrifuged, resuspended in 25 ml of 10 mm MgSO4, and transferred to a large (150 × 15 mm) Petri dish. The SOS system was induced in the cells by irradiating with 254 nm light (45 J/m2 of energy), immediately transferring the cells to two 125-ml aliquots of 2× YT medium, and growing them for 40 min at 37 °C with shaking. Uninduced cells were treated identically, except without exposure to UV light. The 2× YT cultures were centrifuged, combined, and washed twice with 175 ml of deionized water. The electrocompetent cells were resuspended in 4 ml of a 10% solution of glycerol in water, stored at 4 °C, and used the following day. Translesion Bypass Efficiency—An equal amount of each circular genome construct was mixed with 0.025 pmol of internal standard (wild-type circular single-stranded M13mp7L2 DNA) and 100 μl of electrocompetent cells. The cell/genome mixtures were electroporated and transferred to 10 ml of LB, generating at least 105 independent transformed cells as determined by plating of an aliquot onto agar plates. The cultures were incubated at room temperature for at least 30 min and then incubated on a roller drum for 4 h at 37 °C to amplify the progeny phage. The cells were spun down and the progeny phage-containing supernatant retained. In this system, successful replication of the genomes by the E. coli host leads to the production of progeny phage. When plated on a lawn of NR9050 indicator E. coli in the presence of isopropyl 1-thio-β-d-galactopyranoside and X-gal (Gold Biotechnology), progeny phage derived from genomes containing the 19-mer insert produce blue plaques if no mutation, a point mutation, or an in-frame insertion or deletion mutation occurs at the lesion site, whereas progeny phage from WT M13mp7L2 genomes, genomes lacking an insert (genetic engineering mutants), and lesion-induced out-of-frame insertions and deletions produce clear plaques (31Henderson P.T. Delaney J.C. Gu F. Tannenbaum S.R. Essigmann J.M. Biochemistry. 2002; 41: 914-921Crossref PubMed Scopus (132) Google Scholar). Thus, the amplified progeny phage were diluted and plated such that ∼1000–2000 total plaques were produced per Petri dish, and the number of blue and clear plaques per plate was counted. The number of normalized blue plaques resulting from each lesion-containing genome relative to that of the normalized guanine control indicated the bypass efficiency of a lesion (see “Results” for details). The normalization allowed direct comparison of lesion bypass by DNA polymerase(s) between genomes containing different inserts, regardless of unavoidable variation in factors such as electroporation efficiency and the total number of plaques on each plate. Mutation Type and Frequency—Electrocompetent cells were transformed with each genome construct, as described in the previous section, except that the internal standard was excluded. As before, the electroporations produced at least 105 independent transformed cells. Amplified progeny phage (100 μl) and 10 μl of an overnight growth of SCS110 E. coli were added to 10 ml of LB medium and grown for 6 h at 37 °C on a roller drum. The cultures were centrifuged, and the phage-containing supernatant was retained. Single-stranded phage DNA was isolated from 700 μl of each sample using a QIAPrep Spin M13 kit (Qiagen). The region containing the lesion site was amplified by PCR as described previously (31Henderson P.T. Delaney J.C. Gu F. Tannenbaum S.R. Essigmann J.M. Biochemistry. 2002; 41: 914-921Crossref PubMed Scopus (132) Google Scholar), except 0.7 μm of each primer was used, and the resultant 101-mer PCR product was purified using a QIAPrep PCR purification kit (Qiagen). The restriction endonuclease and post-labeling analysis of mutation frequency (REAP) assay was used to determine the identity of the base at the site formerly occupied by the lesion (31Henderson P.T. Delaney J.C. Gu F. Tannenbaum S.R. Essigmann J.M. Biochemistry. 2002; 41: 914-921Crossref PubMed Scopus (132) Google Scholar, 32Henderson P.T. Delaney J.C. Muller J.G. Neeley W.L. Tannenbaum S.R. Burrows C.J. Essigmann J.M. Biochemistry. 2003; 42: 9257-9262Crossref PubMed Scopus (203) Google Scholar, 35Delaney J.C. Essigmann J.M. Chem. Biol. 1999; 6: 743-753Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 38Kroeger K.M. Jiang Y.L. Kow Y.W. Goodman M.F. Greenberg M.M. Biochemistry. 2004; 43: 6723-6733Crossref PubMed Scopus (46) Google Scholar). Genome Construction—Convertible nucleoside phosphoramidites were used to introduce each oxidized DNA lesion into a 19-mer ODN at a defined site. Following incorporation into the 19-mer by automated DNA synthesis, 8-methoxyguanine was converted to Iz by photoirradiation in the presence of riboflavin (33Ikeda H. Saito I. J. Am. Chem. Soc. 1999; 121: 10836-10837Crossref Scopus (45) Google Scholar). Using a procedure recently developed in our laboratory, NI was formed within an ODN from the convertible nucleoside 5-bromo-4-nitroimidazole by treatment of the ODN with guanidine (34Neeley W.L. Henderson P.T. Essigmann J.M. Org. Lett. 2004; 6: 245-248Crossref PubMed Scopus (20) Google Scholar). Each ODN was characterized by MALDI-TOF mass spectrometry and by nuclease and phosphatase digestion followed by HPLC analysis. Single-stranded M13mp7L2 viral genomes containing Iz or NI at a specific site in the genome were constructed as shown in Fig. 2. An aliquot of each genome construct, annealed to a radiolabeled ODN probe, was analyzed by agarose gel electrophoresis and phosphorimaging to assess the yield of circular insert-containing genome. Following quantification, the genome construct solutions were normalized such that each contained an equivalent amount of circular insert-containing genome. The stability of each lesion to the genome construction conditions was confirmed by MALDI-TOF mass spectrometry after subjecting the lesion-containing ODNs to the same conditions. Translesion Bypass Efficiency—A viral plaque assay based on lacZ α-complementation was employed for the determination of the lesion bypass efficiency (Fig. 3). In this system, phage produced from genomes containing the 19-mer insert cause the formation of blue plaques when plated on isopropyl 1-thio-β-d-galactopyranoside/X-gal indicator plates if either no mutation or a point mutation occurs at the lesion site, and phage produced from WT M13mp7L2 genomes produce clear plaques. The number of phage that form blue plaques varies with the efficiency of lesion bypass, and the rate of replication of the WT M13mp7L2 genomes is assumed to be independent of the identity of the insert-containing genomes. Thus, a genome containing a lesion that partially blocks replication yields a lower proportion of progeny phage relative to a genome containing a freely bypassed lesion. Because the same amount of internal standard (WT M13mp7L2 genome) was used in each mixture, the blue to clear ratio of each mixture can be directly compared after being normalized to reflect the identical amount of internal standard in each mixture. The bypass efficiency of a lesion is scaled relative to guanine, which is defined as having a bypass efficiency of 100%. From this logic the equation given in Fig. 3 results, where the term B(L)/B(G) is the definition of relative bypass efficiency and the term C(G)/C(L) is the normalization factor. A limitation of this assay is that lesion-induced out-of-frame insertion and deletion mutations would also produce clear plaques and therefore not be counted as bypass events. However, as discussed in the next section the lesions studied here induced negligible amounts of observable insertion and deletion mutations and so a correction for these events was not performed. In normal WT AB1157 E. coli, the bypass efficiency of Iz was 60 ± 5% (Fig. 4). By contrast, NI was a much stronger replication block and was bypassed with an efficiency of only 7.0 ± 1.6%. As a control, we also determined the bypass efficiency of a synthetic THF abasic site. The THF lesion was bypassed with an efficiency of 5.8 ± 0.7% in agreement with previous work showing that this lesion is a block to DNA replication (31Henderson P.T. Delaney J.C. Gu F. Tannenbaum S.R. Essigmann J.M. Biochemistry. 2002; 41: 914-921Crossref PubMed Scopus (132) Google Scholar, 32Henderson P.T. Delaney J.C. Muller J.G. Neeley W.L. Tannenbaum S.R. Burrows C.J. Essigmann J.M. Biochemistry. 2003; 42: 9257-9262Crossref PubMed Scopus (203) Google Scholar, 39Reuven N.B. Tomer G. Livneh Z. Mol. Cell. 1998; 2: 191-199Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The bypass efficiencies of the lesions were also determined in cells with the SOS system induced. Under these conditions, the bypass efficiency of Iz did not change significantly (71 ± 7%); however, the bypass efficiency of NI increased a remarkable 8-fold to 57 ± 1%. The bypass efficiency of the THF lesion increased to 30 ± 2%, demonstrating that the SOS system was indeed induced. Mutation Type and Frequency—The mutational signature of each lesion in WT AB1157 E. coli was determined using the REAP assay (Fig. 5). Insertion and deletion mutations that do not compromise the BbsI recognition site or the PCR primer sites are detectable by the REAP assay during the PAGE purification step and were negligible in this work (less than 1%, data not shown). In normal cells, the mutation frequency of Iz was 91% versus 50% for NI (Fig. 6). The mutations induced by Iz were essentially all G→ C mutations, as predicted by molecular orbital calculations and in vitro primer extension experiments (40Kino K. Sugiyama H. Chem. Biol. 2001; 8: 369-378Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 41Kino K. Saito I. Sugiyama H. J. Am. Chem. Soc. 1998; 120: 7373-7374Crossref Scopus (171) Google Scholar). NI caused 8.9 ± 0.5% G→ C mutations and a roughly equivalent amount of G→ A and G→ T mutations (19 ± 2% and 22 ± 3%, respectively). The mutation type and frequency for each lesion was also determined in SOS-induced cells to investigate the effect of the SOS response system on the coding properties of these lesions. Interestingly, Iz and NI had somewhat lower overall mutation frequencies of 84 and 33%, respectively. The coding properties of Iz were more degenerate under SOS-induced conditions. Whereas Iz caused 2.0 ± 0.1% G→ A and 1.1 ± 0.2% G→ T mutations without SOS induced, the lesion now induced 3.4 ± 0.5% G→ A and 5.5 ± 0.8% G→ T mutations. The mutational signature of NI also differed in SOS-induced cells, as the lesion induced many fewer G→ A and G→ C mutations (13 ± 2% and 2.5 ± 0.6%, respectively). To assess the biological fate of Iz and NI in DNA, we have determined the relative bypass efficiency and the mutation type and frequency of each lesion in vivo. The Iz derivative is a major and ubiquitous in vitro product of guanine oxidation. In addition to forming as a result of ONOO– oxidation (28Niles J.C. Wishnok J.S. Tannenbaum S.R. J. Am. Chem. Soc. 2001; 123: 12147-12151Crossref PubMed Scopus (56) Google Scholar), this lesion also form" @default.
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• W2132023404 date "2004-10-01" @default.
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• W2132023404 title "In Vivo Bypass Efficiencies and Mutational Signatures of the Guanine Oxidation Products 2-Aminoimidazolone and 5-Guanidino-4-nitroimidazole" @default.
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• W2132023404 doi "https://doi.org/10.1074/jbc.m407117200" @default.
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