Responses to the Major Acrolein-derived Deoxyguanosine Adduct inEscherichia coli
2001; Elsevier BV; Volume: 276; Issue: 12 Linguagem: Inglês
10.1074/jbc.m008918200
ISSN1083-351X
AutoresIn‐Young Yang, Munfarah Hossain, Holly Miller, Sonia Khullar, Francis Johnson, Arthur P. Grollman, Masaaki Moriya,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoAcrolein, a reactive α,β-unsaturated aldehyde found ubiquitously in the environment and formed endogenously in mammalian cells, reacts with DNA to form an exocyclic DNA adduct, 3H-8-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (γ-OH-PdG). The cellular processing and mutagenic potential of γ-OH-PdG have been examined, using a site-specific approach in which a single adduct is embedded in double-strand plasmid DNA. Analysis of progeny plasmid reveals that this adduct is excised by nucleotide excision repair. The apparent level of inhibition of DNA synthesis is ∼70% in Escherichia coli ΔrecA, uvrA. The block to DNA synthesis can be overcome partially byrecA-dependent recombination repair. Targeted G → T transversions were observed at a frequency of 7 × 10−4/translesion synthesis. Inactivation ofpolB, dinB, and umuD,C genes coding for "SOS" DNA polymerases did not affect significantly the efficiency or fidelity of translesion synthesis. In vitroprimer extension experiments revealed that the Klenow fragment of polymerase I catalyzes error-prone synthesis, preferentially incorporating dAMP and dGMP opposite γ-OH-PdG. We conclude from this study that DNA polymerase III catalyzes translesion synthesis across γ-OH-PdG in an error-free manner. Nucleotide excision repair, recombination repair, and highly accurate translesion synthesis combine to protect E. coli from the potential genotoxicity of this DNA adduct. Acrolein, a reactive α,β-unsaturated aldehyde found ubiquitously in the environment and formed endogenously in mammalian cells, reacts with DNA to form an exocyclic DNA adduct, 3H-8-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (γ-OH-PdG). The cellular processing and mutagenic potential of γ-OH-PdG have been examined, using a site-specific approach in which a single adduct is embedded in double-strand plasmid DNA. Analysis of progeny plasmid reveals that this adduct is excised by nucleotide excision repair. The apparent level of inhibition of DNA synthesis is ∼70% in Escherichia coli ΔrecA, uvrA. The block to DNA synthesis can be overcome partially byrecA-dependent recombination repair. Targeted G → T transversions were observed at a frequency of 7 × 10−4/translesion synthesis. Inactivation ofpolB, dinB, and umuD,C genes coding for "SOS" DNA polymerases did not affect significantly the efficiency or fidelity of translesion synthesis. In vitroprimer extension experiments revealed that the Klenow fragment of polymerase I catalyzes error-prone synthesis, preferentially incorporating dAMP and dGMP opposite γ-OH-PdG. We conclude from this study that DNA polymerase III catalyzes translesion synthesis across γ-OH-PdG in an error-free manner. Nucleotide excision repair, recombination repair, and highly accurate translesion synthesis combine to protect E. coli from the potential genotoxicity of this DNA adduct. 8α and 8β isomers of 3H-8-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one 6α and 6β isomers of 3H-6-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one dimethoxytrityl group double-strand(ed) 1,N 6-ethenodeoxyadenosine 3′→5′-exonuclease-deficient Klenow fragment pyrimido[1,2-α]purin-10(3H)-one 1,N 2-(1,3-propano)-2′-deoxyguanosine single-strand(ed) heteroduplex translesion DNA synthesis polymerase Exocyclic base adducts are formed when various endogenous and exogenous bifunctional agents react with DNA. Such lesions include etheno, ethano, propeno, propano, and bicyclic dG, dA, and/or dC adducts (1Singer B. Bartsch H. Exocycllic DNA Adducts in Mutagenesis and Carcinogenesis.IARC Scientific Publications No. 150,International Agency for Research on Cancer. Lyon, France1999Google Scholar, 2Smith R.A. Williamson D.S. Cerny R.L. Cohen S.M. Cancer Res. 1990; 50: 3005-3012PubMed Google Scholar). Etheno and ethano adducts are formed when DNA reacts with chloroacetaldahyde, 1-substituted oxiranes, and/or enal epoxides; propeno and propano adducts, in reaction with malondialdehyde and enals, respectively; and bicyclic adducts, in reactions withp-benzoquinone, a stable metabolite of benzene (1Singer B. Bartsch H. Exocycllic DNA Adducts in Mutagenesis and Carcinogenesis.IARC Scientific Publications No. 150,International Agency for Research on Cancer. Lyon, France1999Google Scholar). Etheno, propeno, and propano base adducts have been detected in DNA isolated from human and animal tissues (3Chung F.-L. Nath R.G. Nagao M. Nishikawa A. Zhou G.-D. Randerath K. Mutat. Res. 1999; 424: 71-81Crossref PubMed Scopus (142) Google Scholar, 4Marnett L.J. Mutat. Res. 1999; 424: 83-95Crossref PubMed Scopus (952) Google Scholar, 5Nair J. Barbin A. Velic I. Bartsch H. Mutat. Res. 1999; 424: 59-69Crossref PubMed Scopus (187) Google Scholar). The reaction of peroxidation products of polyunsaturated fatty acids with DNA is suspected to be the major source of these endogenous adducts (3Chung F.-L. Nath R.G. Nagao M. Nishikawa A. Zhou G.-D. Randerath K. Mutat. Res. 1999; 424: 71-81Crossref PubMed Scopus (142) Google Scholar, 4Marnett L.J. Mutat. Res. 1999; 424: 83-95Crossref PubMed Scopus (952) Google Scholar, 5Nair J. Barbin A. Velic I. Bartsch H. Mutat. Res. 1999; 424: 59-69Crossref PubMed Scopus (187) Google Scholar). The ring structure of exocyclic DNA adducts prevents the formation of normal Watson-Crick hydrogen bonds. These adducts miscode in in vitro primer extension studies conducted with purified DNA polymerases (1Singer B. Bartsch H. Exocycllic DNA Adducts in Mutagenesis and Carcinogenesis.IARC Scientific Publications No. 150,International Agency for Research on Cancer. Lyon, France1999Google Scholar, 6Hashim M.F. Marnett L.J. J. Biol. Chem. 1996; 271: 9160-9165Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 7Singer B. Kusmierek J.T. Folkman W. Chavez F. Dosanjh M.K. Carcinogenesis. 1991; 12: 745-747Crossref PubMed Scopus (69) Google Scholar, 8Simha D. Palejwala V.A. Humayun M.Z. Biochemistry. 1991; 30: 8727-8735Crossref PubMed Scopus (46) Google Scholar, 9Zhang W. Johonson F. Grollman A.P. Shibutani S. Chem. Res. Toxicol. 1995; 8: 157-163Crossref PubMed Scopus (52) Google Scholar) and induce point mutations in Escherichia coli and mammalian cells (1Singer B. Bartsch H. Exocycllic DNA Adducts in Mutagenesis and Carcinogenesis.IARC Scientific Publications No. 150,International Agency for Research on Cancer. Lyon, France1999Google Scholar, 10Basu A.K. Wood M.L. Niederhofer L.J. Ramos L.A. Essigmann J.M. Biochemistry. 1993; 32: 12793-12801Crossref PubMed Scopus (202) Google Scholar, 11Benamira M. Singh U. Marnett L.J. J. Biol. Chem. 1992; 267: 22392-22400Abstract Full Text PDF PubMed Google Scholar, 12Burcham P.C. Marnett L.J. J. Biol. Chem. 1994; 269: 28844-28850Abstract Full Text PDF PubMed Google Scholar, 13Cheng K.C. Preston B.D. Cahill D.S. Dosanjh M.K. Singer B. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9974-9978Crossref PubMed Scopus (147) Google Scholar, 14Fink S.P. Reddy R. Marnett L.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8652-8657Crossref PubMed Scopus (151) Google Scholar, 15Moriya M. Zhang W. Johnson F. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11899-11903Crossref PubMed Scopus (232) Google Scholar, 16Pandya G.A. Moriya M. Biochemistry. 1996; 35: 11487-11492Crossref PubMed Scopus (179) Google Scholar, 17Palejwala V.A. Simha D. Humayun M.Z. Biochemistry. 1991; 30: 8736-8743Crossref PubMed Scopus (76) Google Scholar). Exocyclic etheno adducts are thought to be responsible for the mutations observed in the tumor suppressor p53 gene of humans and animals exposed to vinyl chloride (18Hollstein M. Marion M.J. Lehman T. Welsh J. Harris C.C. Martel-Planche G. Kusters I. Montesano R. Carcinogenesis. 1994; 15: 1-3Crossref PubMed Scopus (196) Google Scholar, 19Levine R.L. Yang I.-Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). Acrolein, the simplest member of the α,β-unsaturated aldehyde family, is found widely in the environment and is formed in cells via lipid peroxidation (3Chung F.-L. Nath R.G. Nagao M. Nishikawa A. Zhou G.-D. Randerath K. Mutat. Res. 1999; 424: 71-81Crossref PubMed Scopus (142) Google Scholar). Acrolein was shown to initiate urinary bladder carcinogenesis in rats (20Cohen S.M. Garland E.M. St. John M. Okamura T. Smith R.A. Cancer Res. 1992; 52: 3577-3581PubMed Google Scholar). Acrolein reacts with dG residues in DNA to form two sets of stereoisomeric propano adducts (see Fig. 1): 8α and 8β isomers of 3H-8-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (γ-OH-PdG)1 (I) and 6α and 6β isomers of 3H-6-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (α-OH-PdG) (II). The γ-OH-PdG isomers are in greatest abundance (3Chung F.-L. Nath R.G. Nagao M. Nishikawa A. Zhou G.-D. Randerath K. Mutat. Res. 1999; 424: 71-81Crossref PubMed Scopus (142) Google Scholar, 21Nath R.G. Ocando J.E. Chung F.-L. Cancer Res. 1996; 56: 452-456PubMed Google Scholar). Chung et al. (3Chung F.-L. Nath R.G. Nagao M. Nishikawa A. Zhou G.-D. Randerath K. Mutat. Res. 1999; 424: 71-81Crossref PubMed Scopus (142) Google Scholar, 21Nath R.G. Ocando J.E. Chung F.-L. Cancer Res. 1996; 56: 452-456PubMed Google Scholar, 22Nath R.G. Chung F.-L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7491-7495Crossref PubMed Scopus (220) Google Scholar) detected background acrolein- and crotonaldehyde (methylacrolein)-derived adducts in experimental animals and human tissues at levels ranging between 0.01 and 7.53 μmol/mol of guanines. These authors also reported that oxidative stress, enhanced lipid peroxidation, and decreased levels of glutathione increase markedly the tissue level of propano adducts (3Chung F.-L. Nath R.G. Nagao M. Nishikawa A. Zhou G.-D. Randerath K. Mutat. Res. 1999; 424: 71-81Crossref PubMed Scopus (142) Google Scholar). 1,N 2-Propanodeoxyguanosine adducts appear to be ubiquitous in cellular DNA and may contribute to so-called spontaneous mutagenesis, thereby playing a role in aging and cancer. In this paper, we establish a genotoxic mechanism for γ-OH-PdG in E. coliand describe the processing of this adduct in bacterial cells. We have developed a novel experimental approach that allows us to explore cellular responses to DNA adducts at a mechanistic level (23Pandya G.A. Yang I.-Y. Grollman A.P. Moriya M. J. Bacteriol. 2000; 182: 6598-6604Crossref PubMed Scopus (17) Google Scholar). The plasmid vector used in this study employs strand-specific markers tagged with mismatches. An oligonucleotide containing a single DNA adduct is incorporated into heteroduplex (HD) DNA containing short stretches of mismatches at several locations. The modified DNA is introduced into mismatch repair-deficient hosts, and adduct-related events are measured. Progeny plasmid are analyzed for their marker sequences. Linkage analysis of marker sequences allows us to group progeny according to in vivo processing events, including excision repair, translesion DNA synthesis, and recombination repair. Restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were purchased from New England BioLabs. [γ-32P]ATP (3000 Ci/mmol) and polymerase chain reaction-grade dNTPs were obtained from Amersham Pharmacia Biotech and Roche Molecular Biochemicals, respectively. The 3′→5′-exonuclease-deficient Klenow fragment (KF exo−) was purified from E. coli CJ374 containing pCJ141, an expression vector (gift from C. Joyce, Yale University), as described previously (24Joyce C.M. Grindley N.D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1830-1834Crossref PubMed Scopus (136) Google Scholar). The synthesis of oligodeoxyribonucleotides containing γ-OH-PdG or 1,N 2-(1,3-propano)-2′-deoxyguanosine (PdG) (IV in Fig. 1) has been described previously (25Khullar S. Varaprasad C.V. Johnson F. J. Medicinal Chem. 1999; 42: 947-950Crossref PubMed Scopus (49) Google Scholar, 26Marinelli E.R. Johnson F. Iden C.R. Yu P.L. Chem. Res. Toxicol. 1990; 3: 49-58Crossref PubMed Scopus (51) Google Scholar). Oligonucleotides containing the precursor of γ-OH-PdG (N 2-[3,4-dihydroxybutyl] dG) or PdG were purified twice by high pressure liquid chromatography using a Waters μBondapak C18 column (3.9 × 300 mm) and acetonitrile in 100 mm triethylammonium acetate buffer, pH 6.8, at a flow rate of 1 ml/min with the dimethoxytrityl group (DMT) on and off. The acetonitrile gradient was 16–33% with DMT-on DNA and 0–20% with DMT-off DNA and applied over 40 min. To generate γ-OH-PdG, oligonucleotides containingN 2-[3,4-dihydroxybutyl] dG were incubated in 100 mm sodium periodate for >6 h at room temperature (25Khullar S. Varaprasad C.V. Johnson F. J. Medicinal Chem. 1999; 42: 947-950Crossref PubMed Scopus (49) Google Scholar). γ-OH-PdG-containing oligonucleotides were separated from the parental oligonucleotides by high pressure liquid chromatography as described above for DMT-off DNA. All oligonucleotides were subjected to electrophoresis in a denaturing 20% polyacrylamide gel. Bands were detected by UV shadowing. Oligonucleotides were excised from the gel then purified over a Sep-Pak column (Waters). Purified oligonucleotides were subjected to electrospray mass spectrometry analysis. The results were as follows: 13-mer with γ-OH-PdG m/z: observed, 3901.8 ± 0.47; calculated, 3901.6; 28-mer with γ-OH-PdGm/z: observed, 8510.15 ± 0.23; calculated, 8509.6; and 28-mer with PdG m/z: observed, 8494.37 ± 0.58; calculated, 8493.6. E. coli strains used are shown in Table I; MO strains were constructed by P1 transduction (27Miller J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 263-278Google Scholar) and are mismatch repair-deficient. Plasmids pS and pA have been described (23Pandya G.A. Yang I.-Y. Grollman A.P. Moriya M. J. Bacteriol. 2000; 182: 6598-6604Crossref PubMed Scopus (17) Google Scholar). These plasmids differ in DNA sequence at three regions but are otherwise identical; hence, HD DNA prepared from these plasmids contains three mismatched regions (see Fig. 3). A mismatched region involving a BamHI site is located 150 nucleotides upstream from the adduct site, the others, containing NheI and SpeI/AatII sites, are located ∼220 and 3100 nucleotides, respectively, downstream from the adduct (see Fig. 3). As described later, these mismatches serve as strand-specific markers.Table IE. coli strainsStrainRelevant genotypeSource and/or referenceAB1157EGSC 1-aE. coli Genetic Stock Center, Yale, CT.AB1886As AB1157, butuvrA6EGSCES1481mutS215∷Tn10EGSC, (33Siegel E.C. Wain S.L. Meltzer S.F. Binion M.L. Steinberg J.L. Mutat. Res. 1982; 93: 25-33Crossref PubMed Scopus (44) Google Scholar)MO199As AB1157, butmutS215∷Tn10AB1157 x P1(ES1481)MO211As RK1517, butΔ(recA-srl)306∷Tn10RK1517 x P1(MO937)MO220As NR9232, butmutS201∷Tn5NR9232 x P1(RK1517)MO221mutD5, zaf13∷Tn10,ΔumuDC∷cam,mutS201∷Tn5MO220 x P1(YG7210)MO230As AB1886, butΔumuDC∷camAB1886 x P1(YG7210)MO231As MO230, but ΔdinB∷kanMO230 x P1(YG7210)MO232As MO231, butΔ(araD-polB)∷ΩMO231 x P1(SH2101)MO233As MO232, but mutS215∷Tn10MO232 x P1(ES1481)MO234As AB1886, butmutS215∷Tn10AB1886 x P1(ES1481)MO933As MV1932, but mutS201∷Tn5(23Pandya G.A. Yang I.-Y. Grollman A.P. Moriya M. J. Bacteriol. 2000; 182: 6598-6604Crossref PubMed Scopus (17) Google Scholar)MO934As MO933, but uvrA6,malE3∷Tn10(23Pandya G.A. Yang I.-Y. Grollman A.P. Moriya M. J. Bacteriol. 2000; 182: 6598-6604Crossref PubMed Scopus (17) Google Scholar)MO936As MO934, butmal +(23Pandya G.A. Yang I.-Y. Grollman A.P. Moriya M. J. Bacteriol. 2000; 182: 6598-6604Crossref PubMed Scopus (17) Google Scholar)MO937As MO936, butΔ(recA-srl)306∷Tn10(23Pandya G.A. Yang I.-Y. Grollman A.P. Moriya M. J. Bacteriol. 2000; 182: 6598-6604Crossref PubMed Scopus (17) Google Scholar)MO939As MO933, but Δ(recA-srlR)306∷Tn10MO933 x P1(MO937)MV1185uvrA,malE3∷Tn10Volkert, M.1-bUniversity of Massachusetts, Worcester, MA.MV1932alkA1,tag1Volkert, M. (34Santerre A. Britt A.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2240-2244Crossref PubMed Scopus (67) Google Scholar)NR9232mutD5,zaf13∷Tn10Schaaper, R.1-cNIEHS, NC. (35Schaaper R.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85 Suppl. 4: 8126-8130Crossref Scopus (161) Google Scholar)RK1517As AB1157, butmutS201∷Tn5Kolodner, R.1-dUniversity of California at San Diego, CA.36Au K.G. Cabrera M. Miller J.H. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9163-9166Crossref PubMed Scopus (116) Google Scholar)SH2101Δ(araD-polB)∷ΩGoodman, M.F.1-eUniversity of Southern California, CA.(37Escarceller M. Hicks J. Gudmundsson G. Trump G. Touati D. Lovett S. Foster P.L. McEntee K. Goodman M.F. J. Bacteriol. 1994; 10: 6221-6228Crossref Google Scholar)YG7210As AB1157, but ΔumuDC∷cam ΔdinB∷kanNohmi, T.1-fNational Institute of Health Science, Tokyo. (38Kim S.-R. Maenhaut-Michel G. Yamada M. Yamamoto Y. Matsui K. Sofuni T. Nohmi T. Ohmori H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13792-13797Crossref PubMed Scopus (295) Google Scholar)1-a E. coli Genetic Stock Center, Yale, CT.1-b University of Massachusetts, Worcester, MA.1-c NIEHS, NC.1-d University of California at San Diego, CA.1-e University of Southern California, CA.1-f National Institute of Health Science, Tokyo. Open table in a new tab The scheme for this construction is shown in Fig.2. Detailed procedures have been described previously (19Levine R.L. Yang I.-Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). In brief, double-stranded (ds) pA was digested with EcoRV (Step I), and the linearized plasmid DNA was ligated to a blunt-ended duplex 13-mer, 5′-d(AGGTACGTAGGAG)/ 3′-d(TCCATGCATCCTC), containing aSnaBI site (5′-TACGTA) (Step II). Two constructs, each containing a single insert with opposite orientation, were isolated; one of these, pA106, was used in this study. Single-stranded (ss) pA106 was mixed with EcoRV-digested ds pS (Steps III and IV), treated with NaOH to denature ds pS, then neutralized to form ds DNA. Circular ss pA106 and its complementary strand (derived from ds pS) were annealed to form HD DNA containing a 13-nucleotide gap. An unmodified or modified 13-mer (3′-d(TCCATAXCTCCTC), where X is dG or γ-OH-PdG) phosphorylated at the 5′-termini using T4 polynucleotide kinase and ATP, was annealed into the gap and ligated by T4 DNA ligase. The 13-mers are not fully complementary to the gap sequence, because they form mismatches at and adjacent (5′ and 3′) to the adduct site (Fig. 3). These mismatches, located opposite the SnaBI site, also serve as a strand-specific marker. The ligation mixture was treated with SpeI andEcoRV to remove residual ds pS. Closed circular ds DNA was purified by ultracentrifugation in a CsCl/ethidium bromide solution. DNA was concentrated by Centricon 30 (Amicon, Beverly, MA), and the concentration was determined spectrophotometrically. In this HD construct, six and three base mismatches are formed at theSpeI/AatII and SnaBI sites, respectively. At the BamHI and NheI sites, one strand has six extra bases. These four mismatched regions serve as strand-specific markers: A/a (BamHI site), B/b (SnaBI site), C/c (NheI site), and D/d (AatII/SpeI site). The unmodified complementary strand derived from ss pA106 contains the A-B-C-D linkage (Fig.3). γ-OH-PdG was incorporated into the site of b on the strand bearing the a-b-c-d linkage; this strand is the template for leading strand synthesis. Unmodified or modified DNA (12 ng) was introduced into mismatch repair-deficient (mutS) MO strains (50 μl of electrocompetent cells) by electroporation. The mutSmutation assures that the mismatches will not be repaired. 2× YT medium 16 g of tryptone, 10 g of yeast extract, 5 g/1000 ml NaCl, pH 7) (950 μl) was added to the electroporation mixture, then incubated for 20 min at 37 °C. A portion (10–50 μl of a 100× dilution) of the mixture was plated onto a 1× YT-ampicillin (100 μg/ml) plate to determine the number of transformants in the mixture. The remaining mixture was incubated for additional 40 min and then added to 10 ml of 2× YT containing ampicillin. After culturing overnight, progeny plasmid was prepared by the alkaline lysis method and used to transform E. coli DH5α (Life Technologies, Inc.). This second transformation segregates progeny plasmid derived from each strand of the HD DNA. Transformants were inoculated individually in 96-well plates and cultured for several hours. Bacterial cultures were stamped onto filter paper placed on a 1× YT-ampicillin plate and cultured overnight. The filter was treated with 0.5 m NaOH for 11 min, neutralized in 0.5 mTris-HCl, pH 7.4, for 7 min, washed with 1× SSC (150 mmNaCl, 15 mm Na3 citrate, pH 7.2) then with ethanol, and baked at 80 °C for 2 h. Differential oligonucleotide hybridization (16Pandya G.A. Moriya M. Biochemistry. 1996; 35: 11487-11492Crossref PubMed Scopus (179) Google Scholar, 23Pandya G.A. Yang I.-Y. Grollman A.P. Moriya M. J. Bacteriol. 2000; 182: 6598-6604Crossref PubMed Scopus (17) Google Scholar, 28Wallace R.B. Shaffer J. Murphy R.F. Bonner J. Hirose T. Itakura K. Nucleic Acids Res. 1979; 6: 3543-3557Crossref PubMed Scopus (516) Google Scholar) using the32P-labeled probes shown in Fig. 3 was employed to detect strand-specific marker sequences and the base located at the position of the adduct. L and R probes were used to confirm the presence of the 13-mer insert. In several experiments, mitomycin C was used to induce SOS functions inE. coli. Overnight cultures of MO strains were diluted 20-fold with prewarmed 2× YT and cultured for 2 h. Mitomycin C (1–10 μg/ml) was added to the cultures and incubated at 37 °C for 30 min with shaking. Cells were prepared for electroporation by repeated washings with H2O. When a HD construct bearing a single γ-OH-PdG residue is introduced into a host cell, various events may occur (Fig. 4). If the adduct is removed by excision repair before being replicated, the immediate 5′- and 3′-flanking mismatches also are removed. Gap-filling DNA synthesis converts 5′-CXA (b) to 5′-ACG (B); therefore, progeny derived from the repaired strand contain the linkage of a-B-c-d (progeny III) (Fig. 4, step 1). When the construct is replicated in the absence of DNA repair, progeny produced from the unmodified strand contain the A-B-C-D linkage (step 2), whereas those from the modified strand have a-b-c-d (step 4) following translesion synthesis (TLS). When DNA synthesis is blocked by the adduct, the block may be overcome by UmuD′2C/RecA-assisted TLS or recombination repair (daughter strand gap repair) (29Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, DC1995: 407-414Google Scholar). In recombination repair, the ss gap is filled by strand transfer from the unmodified parental strand (step 5). The 3′-end of the blocked nascent strand is used to replicate the transferred region (step 6). These processes create a Holliday junction. When the Holliday junction is migrated by the RuvA·RuvB complex, a Holliday junction-specific helicase, and resolved by RuvC, a Holliday junction-specific endonuclease (step 7), progeny with the linkage of A-B-C-D (progeny I), a-B-C-d (progeny IV), a-B-C-D (progeny V), and A-B-C-d (progeny VI) are created. Oligonucleotides used for primer extension studies are as follows: 28-mer templates, 5′-CTGCTCCTCXATACCTACACGCTAGAAC, where X is dG, γ-OH-PdG, or PdG; an 18-mer primer for incorporation experiments, 5′-GTTCTAGCGTGTAGGTAT; 19-mer primers for extension experiments, 5′-GTTCTAGCGTGTAGGTATN, where N is dA, dG, dC, or dT; and a 16-mer primer for read-through experiments, 5′-GTTCTAGCGTGTAGGT. All oligonucleotides were purified by electrophoresis in a denaturing 20% polyacrylamide gel. The 5′-32P-end-labeled primers were hybridized to templates at a molar ratio of 1:1.2 in a buffer containing 50 mm Tris-HCl, pH 7.5, 5 mm EDTA, and 500 mm NaCl. Annealing reactions were conducted by heating at 70 °C for 5 min followed by slow cooling. Reaction mixtures (10 μl) contained 50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 65 nmprimer/template, 200 or 300 μm dNTP, and 0–850 nm KF exo−. The concentrations of dNTP were 200 μm for incorporation and extension experiments and 300 μm for read-through experiments. KF exo−was diluted in a solution containing 50 mm Tris-HCl, pH 7.5, 0.5 mg/ml bovine serum albumin, and 10% glycerol. Reactions were conducted at 25 °C for 30 min and stopped by adding 10 μl of formamide dye mixture (90% formamide, 0.001% xylene cyanol, 0.001% bromphenol blue, 20 mm EDTA). Samples were heated at 95 °C for 5 min, and aliquots (1.5 μl) were subjected to electrophoresis in a denaturing 20% polyacrylamide gel (0.4 mm thick, 40 cm long) at 2600 V for 3 h. Gels were analyzed by a PhosphorImager using ImageQuaNT software (Molecular Dynamics). Pathway 1 depicted in Fig. 4 predicts that progeny III derived from plasmids subjected to excision repair will contain the a-B-c-d linkage. The unmodified control construct, which contained three base mismatches and no adduct, yielded <1% of progeny III in the uvrA (MO937) and uvr + (MO939) strains (Table II), indicating that the mismatches were not subjected to nucleotide excision repair. When the adducted construct was introduced into the uvrA strains (MO937 and MO934), progeny III accounted for 1 × 106 and >1 × 104 transformants per transformation in the absence and presence of induced SOS functions, respectively. The lower transformation efficiency of mitomycin C-treated cells is thought to be due to DNA damage. Progeny plasmid DNA was purified following overnight culture of the transformation mixture in the presence of ampicillin and then digested with SnaBI, the site for which is located in marker B (Fig. 3). This digestion removes progeny derived from the unmodified strand and plasmids subjected to excision repair or recombination repair (see Fig.4), facilitating analysis of TLS events. Targeted events were analyzed by differential oligonucleotide hybridization and DNA sequencing. This analysis revealed that almost all targeted events were γ-OH-PdG → dG, indicating accurate TLS. Only one γ-OH-PdG → dT transversion was observed among 282 transformants of SOS-uninduced MO933, yielding a miscoding frequency of 0.35%. The numbers of transformants analyzed were 144 f
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