Mammalian Translesion DNA Synthesis across an Acrolein-derived Deoxyguanosine Adduct
2003; Elsevier BV; Volume: 278; Issue: 16 Linguagem: Inglês
10.1074/jbc.m212535200
ISSN1083-351X
AutoresIn‐Young Yang, Holly Miller, Zhigang Wang, Ekaterina G. Frank, Haruo Ohmori, Fumio Hanaoka, Masaaki Moriya,
Tópico(s)CRISPR and Genetic Engineering
Resumoα-OH-PdG, an acrolein-derived deoxyguanosine adduct, inhibits DNA synthesis and miscodes significantly in human cells. To probe the cellular mechanism underlying the error-free and error-prone translesion DNA syntheses, in vitro primer extension experiments using purified DNA polymerases and site-specific α-OH-PdG were conducted. The results suggest the involvement of pol η in the cellular error-prone translesion synthesis. Experiments with xeroderma pigmentosum variant cells, which lack pol η, confirmed this hypothesis. The in vitro results also suggested the involvement of pol ι and/or REV1 in inserting correct dCMP opposite α-OH-PdG during error-free synthesis. However, none of translesion-specialized DNA polymerases catalyzed significant extension from a dC terminus when paired opposite α-OH-PdG. Thus, our results indicate the following. (i) Multiple DNA polymerases are involved in the bypass of α-OH-PdG in human cells. (ii) The accurate and inaccurate syntheses are catalyzed by different polymerases. (iii) A modification of the current eukaryotic bypass model is necessary to account for the accurate bypass synthesis in human cells. α-OH-PdG, an acrolein-derived deoxyguanosine adduct, inhibits DNA synthesis and miscodes significantly in human cells. To probe the cellular mechanism underlying the error-free and error-prone translesion DNA syntheses, in vitro primer extension experiments using purified DNA polymerases and site-specific α-OH-PdG were conducted. The results suggest the involvement of pol η in the cellular error-prone translesion synthesis. Experiments with xeroderma pigmentosum variant cells, which lack pol η, confirmed this hypothesis. The in vitro results also suggested the involvement of pol ι and/or REV1 in inserting correct dCMP opposite α-OH-PdG during error-free synthesis. However, none of translesion-specialized DNA polymerases catalyzed significant extension from a dC terminus when paired opposite α-OH-PdG. Thus, our results indicate the following. (i) Multiple DNA polymerases are involved in the bypass of α-OH-PdG in human cells. (ii) The accurate and inaccurate syntheses are catalyzed by different polymerases. (iii) A modification of the current eukaryotic bypass model is necessary to account for the accurate bypass synthesis in human cells. During the last several years, many new DNA polymerases (pol) 1The abbreviations used are: polDNA polymeraseα-OH-PdGthe 6R and 6S isomers of 3H-6-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-oneBSAbovine serum albuminDTTdithiothreitolexo3′→5′ exonucleaseγ-OH-PdGthe 8R and 8S isomers of 3H-8-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-onePCNAproliferating cell nuclear antigenXPVxeroderma pigmentosum variantmXPVmouse XPV cDNA have been discovered in prokaryotes and eukaryotes (1Friedberg E.C. Gerlach V.L. Cell. 1999; 98: 413-416Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 2Johnson R.E. Washington M.T. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12224-12226Crossref PubMed Scopus (131) Google Scholar, 3Woodgate R. Genes Dev. 1999; 13: 2191-2195Crossref PubMed Scopus (234) Google Scholar). Several of these polymerases, such as eukaryotic pol η, pol κ, pol ι, pol ζ, and REV1 and Escherichia coli pol IV and pol V, are thought to be involved in translesion DNA synthesis. With the exception of pol ζ, which belongs to the B family, they share extensive sequence homology and comprise a new polymerase family designated the Y family (4Ohmori H. Friedberg E.C. Fuchs R.P.P. Goodman M.F. Hanaoka F. Hinkle D. Kunkel T.A. Lawrence C.W. Livneh Z. Nohmi T. Prakash L. Prakash S. Todo T. Walker G.C. Wang Z. Woodgate R. Mol. Cell. 2001; 8: 7-8Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). These polymerases are different from replicative polymerases in several aspects, i.e. they replicate more efficiently across altered bases and catalyze both accurate and inaccurate translesion DNA syntheses, they have more flexible and larger catalytic pockets (5Ling H. Boudsocq F. Woodgate R. Yang W. 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Kunkel T.A. Nature. 2000; 404: 1011-1013Crossref PubMed Scopus (327) Google Scholar, 12Ohashi E. Bebenek K. Matsuda T. Feaver W.J. Gerlach V.L. Friedberg E.C. Ohmori H. Kunkel T.A. J. Biol. Chem. 2000; 275: 39678-39684Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 13Tissier A. McDonald J.P. Frank E.G. Woodgate R. Genes Dev. 2000; 14: 1642-1650PubMed Google Scholar, 14Zhang Y. Yuan F. Xin H. Wu X. Rajpal D.K. Yang D. Wang Z. Nucleic Acids Res. 2000; 28: 4147-4156Crossref PubMed Google Scholar). Their ability to catalyze translesion synthesis has been studied extensivelyin vitro using various DNA lesions as substrates, but knowledge of their roles in translesion synthesis in mammalian cells is still very fragmentary. Among these polymerases, pol η, which is defective in cells of xeroderma pigmentosum variant (XPV) patients, was shown to catalyze accurate and efficient translesion synthesis across certain UV photoproducts (15Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (385) Google Scholar), whereas human pol ζ (16Gibbs P.E.M. McGregor W.G. Maher V.M. Nisson P. Lawrence C.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6876-6880Crossref PubMed Scopus (295) Google Scholar) and REV1 (17Gibbs P.E.M. Wang X.-D. Li Z. McManus T.P. McGregor W.G. Lawrence C.W. Maher V.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4186-4191Crossref PubMed Scopus (162) Google Scholar) are involved in inaccurate syntheses across UV photoproducts. One recent study using pol κ-defective mouse cells has shown that the enzyme is involved in the error-free translesion synthesis across a benzo[a]pyrene-dG adduct(s) (18Ogi T. Shinkai Y. Tanaka K. Ohmori H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15548-15553Crossref PubMed Scopus (199) Google Scholar). Two eukaryotic translesion synthesis pathways have been proposed (19Guo D. Wu X. Rajpal D.K. Taylor J.-S. Wang Z. Nucleic Acids Res. 2001; 29: 2875-2883Crossref PubMed Google Scholar, 20Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar, 21Prakash S. Prakash L. Genes Dev. 2002; 16: 1872-1883Crossref PubMed Scopus (293) Google Scholar, 22Tissier A. Frank E.G. McDonald J.P. Iwai S. Hanaoka F. Woodgate R. EMBO J. 2000; 19: 5259-5266Crossref PubMed Scopus (184) Google Scholar, 23Wang Z. Mol. Interv. 2001; 1: 269-281PubMed Google Scholar). In one pathway, both insertion and extension steps are catalyzed by one DNA polymerase. In the other pathway, extension is catalyzed by a DNA polymerase, such as pol ζ or pol κ, which is different from the one inserting a nucleotide opposite a DNA lesion. DNA polymerase the 6R and 6S isomers of 3H-6-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one bovine serum albumin dithiothreitol 3′→5′ exonuclease the 8R and 8S isomers of 3H-8-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one proliferating cell nuclear antigen xeroderma pigmentosum variant mouse XPV cDNA In this research, we conducted translesion synthesis studies in vitro and in vivo to probe the cellular bypass mechanism for an acrolein-derived dG adduct. Acrolein, the simplest member of the α,β-unsaturated aldehyde family, is widely found in the environment and is also produced endogenously. It initiates urinary bladder carcinogenesis in rats (24Cohen S.M. Garland E.M. St. John M. Okamura T. Smith R.A. Cancer Res. 1992; 52: 3577-3581PubMed Google Scholar) and is mutagenic in bacteria (25Marnett L.J. Hurd H.K. Hollstein M.C. Levin D.E. Esterbauer H. Ames B.N. Mutat. 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Acrolein reacts with dG residues in DNA to form two pairs of stereoisomeric exocyclic propano adducts (Fig.1), namely the 8R and 8S isomers of 3H-8-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (γ-OH-PdG) and the 6R and 6S isomers of 3H-6-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (α-OH-PdG). γ-OH-PdG predominates over α-OH-PdG (30Chung F.-L. Nath R.G. Nagao M. Nishikawa A. Zhou G.-D. Randerath K. Mutat. Res. 1999; 424: 71-81Crossref PubMed Scopus (143) Google Scholar, 31Chung F.-L. Young R. Hecht S.S. Cancer Res. 1984; 44: 990-995PubMed Google Scholar, 32Nath R.G. Ocando J.E. Chung F.-L. Cancer Res. 1996; 56: 452-456PubMed Google Scholar) and has been detected in DNA isolated from human and animal tissue (30Chung F.-L. Nath R.G. Nagao M. Nishikawa A. Zhou G.-D. Randerath K. Mutat. Res. 1999; 424: 71-81Crossref PubMed Scopus (143) Google Scholar, 33Nath R.G. Chung F.-L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7491-7495Crossref PubMed Scopus (220) Google Scholar). Lipid peroxidation is suspected to be the major endogenous source (30Chung F.-L. Nath R.G. Nagao M. Nishikawa A. Zhou G.-D. Randerath K. Mutat. Res. 1999; 424: 71-81Crossref PubMed Scopus (143) Google Scholar). Comparative genotoxic studies with a site-specific adduct in human cells have shown that γ-OH-PdG is less blocking than is α-OH-PdG (34Yang I.-Y. Chan G. Miller H. Huang Y. Torres M.C. Johnson F. Moriya M. Biochemistry. 2002; 41: 13826-13832Crossref PubMed Scopus (89) Google Scholar) and is bypassed with high fidelity (34Yang I.-Y. Chan G. Miller H. Huang Y. Torres M.C. Johnson F. Moriya M. Biochemistry. 2002; 41: 13826-13832Crossref PubMed Scopus (89) Google Scholar, 35Yang I.-Y. Johnson F. Grollman A.P. Moriya M. Chem. Res. Toxicol. 2002; 15: 160-164Crossref PubMed Scopus (60) Google Scholar). α-OH-PdG, on the other hand, miscodes substantially in human cells with a frequency of 10–12% per bypass synthesis, with G→T being predominant (34Yang I.-Y. Chan G. Miller H. Huang Y. Torres M.C. Johnson F. Moriya M. Biochemistry. 2002; 41: 13826-13832Crossref PubMed Scopus (89) Google Scholar). As α-OH-PdG strongly inhibits DNA synthesis (34Yang I.-Y. Chan G. Miller H. Huang Y. Torres M.C. Johnson F. Moriya M. Biochemistry. 2002; 41: 13826-13832Crossref PubMed Scopus (89) Google Scholar), it is likely that the translesion polymerases are involved in bypassing this adduct. This leads to the following questions: (i) which translesion polymerase is responsible for the correct and incorrect syntheses; and (ii) whether these syntheses are catalyzed by one polymerase or by different polymerases. Here, we show the following. (i) Multiple DNA polymerases are involved in the bypass synthesis. (ii) Pol η participates in incorrect synthesis. (iii) The current eukaryotic bypass model (19Guo D. Wu X. Rajpal D.K. Taylor J.-S. Wang Z. Nucleic Acids Res. 2001; 29: 2875-2883Crossref PubMed Google Scholar, 20Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar, 21Prakash S. Prakash L. Genes Dev. 2002; 16: 1872-1883Crossref PubMed Scopus (293) Google Scholar, 22Tissier A. Frank E.G. McDonald J.P. Iwai S. Hanaoka F. Woodgate R. EMBO J. 2000; 19: 5259-5266Crossref PubMed Scopus (184) Google Scholar, 23Wang Z. Mol. Interv. 2001; 1: 269-281PubMed Google Scholar) does not seem to account for the error-free bypass of this adduct. The procedures for the synthesis, purification, and characterization of oligonucleotides containing α-OH-PdG have been described (37Huang Y. Torres M.C. Johnson F. Bioorg. Chem. 2003; (in press)Google Scholar). The 13-mer (5′-CTCCTCXATACCT-3′) and 28-mer (5′-CTGCTCCTCXATACCTACACGCTAGAAC-3′), in which X represents α-OH-PdG, were the same oligonucleotides as those used in our previous study (34Yang I.-Y. Chan G. Miller H. Huang Y. Torres M.C. Johnson F. Moriya M. Biochemistry. 2002; 41: 13826-13832Crossref PubMed Scopus (89) Google Scholar). The 13-mer and 28-mer were used in the translesion synthesis studies in vivo (human cells) and in vitro, respectively. The 16-mer (5′-GTTCTAGCGTGTAGGT-3′), 18-mer (5′-GTTCTAGCGTGTAGGTAT) and 19-mer (5′-GTTCTAGCGTGTAGGTATN-3′, in which N stands for A, G, C, or T) were employed as primers in the experiments of read-through nucleotide incorporation opposite α-OH-PdG and primer extension from a terminus opposite α-OH-PdG, respectively. The 28-mer template contained the entire sequence of the 13-mer. All unmodified as well as modified oligonucleotides were purified by electrophoresis in denaturing 20% polyacrylamide gel and formed a single band following purification. Human Pol η (38Masutani C. Kusumoto R. Iwai S. Hanaoka F. EMBO J. 2000; 19: 3100-3109Crossref PubMed Google Scholar), pol κ (39Ohashi E. Ogi T. Kusumoto R. Iwai S. Masutani C. Hanaoka F. Ohmori H. Genes Dev. 2000; 14: 1589-1594PubMed Google Scholar), pol ι (13Tissier A. McDonald J.P. Frank E.G. Woodgate R. Genes Dev. 2000; 14: 1642-1650PubMed Google Scholar), REV1 (40Lin W. Xin H. Zhang Y. Wu X. Yuan F. Wang Z. Nucleic Acids Res. 1999; 27: 4468-4475Crossref PubMed Scopus (164) Google Scholar), calf thymus pol δ (41Ng L. Tan C.K. Downey K.M. Fisher P.A. J. Biol. Chem. 1991; 266: 11699-11704Abstract Full Text PDF PubMed Google Scholar), and Saccharomyces cerevisiae pol ζ (19Guo D. Wu X. Rajpal D.K. Taylor J.-S. Wang Z. Nucleic Acids Res. 2001; 29: 2875-2883Crossref PubMed Google Scholar) were purified as described. The 3′→5′ exonuclease (exo)-proficient Klenow enzyme was obtained from New England BioLabs (Beverly, MA); human PCNA was a gift from Paul A. Fisher (State University of New York, Stony Brook, NY). The 28-mer template and a 5′-32P-end-labeled primer were mixed at a molar ratio of 1:2, heated at 70 °C for 5 min, and annealed by slow cooling. Reaction mixtures (10 μl) contained 40 mmbis-Tris (pH6.8), 6 mm MgCl2, 10 mmdithiothreitol (DTT), 40 μg/ml bovine serum albumin (BSA), and 14 ng/μl PCNA for pol δ; 40 mm Tris-HCl (pH 8.0), 30 mm KCl, 5 mm MgCl2, 10 mm DTT, and 250 μg/ml BSA for pol η and pol κ (42Levine R.L. Miller H. Grollman A. Ohashi E. Ohmori H. Masutani C. Hanaoka F. Moriya M. J. Biol. Chem. 2001; 276: 18717-18721Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar); 25 mm KH2PO4 (pH 7.0), 5 mm MgCl2, 5 mm DTT, 100 μg/ml BSA and 10% glycerol for REV1 (43Zhang Y. Wu X. Rechkoblit O. Geacintov N.E. Taylor J.-S. Wang Z. Nucleic Acids Res. 2002; 30: 1630-1638Crossref PubMed Scopus (116) Google Scholar) and pol ζ (19Guo D. Wu X. Rajpal D.K. Taylor J.-S. Wang Z. Nucleic Acids Res. 2001; 29: 2875-2883Crossref PubMed Google Scholar); 40 mmTris-HCl (pH 8.0), 5 mm MgCl2, 10 mm β-mercaptoethanol (replacing DDT used in the original buffer), 250 μg/ml BSA and 2.5% glycerol for pol ι (44Vaisman A. Woodgate R. EMBO J. 2001; 20: 6520-6529Crossref PubMed Scopus (107) Google Scholar); and 10 mm Tris-HCl (pH 7.5), 5 mm MgCl2, and 7.5 mm DTT for the Klenow enzyme. The final concentration of dNTP was 10 μm for incorporation experiments and 100 μm each in extension and read-through experiments. A primed template was added at a concentration of 40 nm. The amounts of polymerases added are indicated in the legends to Figs. Figure 3, Figure 4, Figure 5, Figure 6, Figure 7. Reactions with pol δ were incubated at 30 °C for 30 min, and those with the other enzymes were at 37 °C for 10 min. Following reaction, 7 μl of a formamide dye mixture (95% formamide, 0.1% xylene cyanol, 0.1% bromphenol blue, and 20 mm EDTA) was added, and aliquots (4 μl) were subjected to electrophoresis in denaturing (8 m urea) 20% polyacrylamide gel at 2300 V for 2.5 h. Radioactive bands were detected and, if necessary, quantified by a PhosphorImager and ImageQuant software (Amersham Biosciences).Figure 4Incorporation of a nucleotide opposite α-OH-PdG by pol δ/PCNA.32P-5′-end-labeled 18-mer primer/28-mer template complex (40 nm), 0.75 units of pol δ, 140 ng of PCNA, and 10 μm (left) or 100 μm(right) dNTP were used. The other conditions were the same as those in Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Resumption of DNA synthesis by pol δ/PCNA.32P-5′-end-labeled primers of various lengths (19Guo D. Wu X. Rajpal D.K. Taylor J.-S. Wang Z. Nucleic Acids Res. 2001; 29: 2875-2883Crossref PubMed Google Scholar, 20Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar, 21Prakash S. Prakash L. Genes Dev. 2002; 16: 1872-1883Crossref PubMed Scopus (293) Google Scholar, 22Tissier A. Frank E.G. McDonald J.P. Iwai S. Hanaoka F. Woodgate R. EMBO J. 2000; 19: 5259-5266Crossref PubMed Scopus (184) Google Scholar, 23Wang Z. Mol. Interv. 2001; 1: 269-281PubMed Google Scholar, 24Cohen S.M. Garland E.M. St. John M. Okamura T. Smith R.A. Cancer Res. 1992; 52: 3577-3581PubMed Google Scholar, 25Marnett L.J. Hurd H.K. Hollstein M.C. Levin D.E. Esterbauer H. Ames B.N. Mutat. Res. 1985; 148: 25-34Crossref PubMed Scopus (556) Google Scholar, 26Parent R.A. Caravello H.E. San R.H.C. J. Appl. Toxicol. 1996; 16: 103-108Crossref PubMed Scopus (23) Google Scholar) were annealed to a modified 28-mer template, and the primer extension reaction was performed using pol δ (0.75 units) and PCNA (140 ng) as described in the legend to Fig 3. X represents α-OH-PdG.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Translesion syntheses catalyzed by pol η and pol κ.32P-5′-end-labeled 16-mer primer/28-mer template complex (40 nm) was incubated with various amounts of a DNA polymerase in the presence of 100 μm each of four dNTPs at 37 °C for 10 min. Reaction products were analyzed in denaturing 20% polyacrylamide gel. X indicates the position of α-OH-PdG.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Translesion DNA polymerase-catalyzed nucleotide incorporation opposite α-OH-PdG (A) and extension from termini opposite α-OH-PdG (B). A,32P-5′-end-labeled 18-mer primer/28-mer template complex (40 nm) was incubated with a DNA polymerase in the presence of 10 μm of one dNTP at 37 °C for 10 min. Concentrations of polymerases were 3.63 nm pol η, 3.1 nm pol κ, 2.5 nm pol ι, 14.4 nmREV1, and 5.4 nm pol ζ in a 10-μl reaction mixture.B, 5′ 32P-labeled 19-mer primer/28-mer template complex (40 nm) was incubated with a DNA polymerase in the presence of four dNTPs (100 μm each) at 37 °C for 10 min. Concentrations of DNA polymerases were the same as those used inpanel A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The SV40-transformed human XPV cell lines CTag (45King S.A. Wilson S.J. Farber R.A. Kaufmann W.K. Cordeiro-Stone M. Exp. Cell Res. 1995; 217: 100-108Crossref PubMed Scopus (17) Google Scholar) and XP30RO(sv) (46Cleaver J.E. Afzal V. Feeney L. McDowell M. Sadinski W. Volpe J.P.G. Busch D.B. Coleman D.M. Ziffer D.W. Yu Y. Nagasawa H. Little J.B. Cancer Res. 1999; 59: 1102-1108PubMed Google Scholar) were obtained from M. Cordeiro-Stone (University of North Carolina, Chapel Hill, NC) and J. Cleaver (University of California, San Francisco, CA), respectively. CTag and XP30RO(sv) were established from XP4BE and XP30RO (GM3617), respectively. XP4BE and XP30RO cells contain a four-nucleotide (positions 289–292) and a 13-nucleotide (positions 343–355) deletion, respectively, in the coding region of one allele of the XPVgene and produce severely truncated proteins due to the new stop codons generated (47Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Crossref PubMed Scopus (672) Google Scholar, 48Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Crossref PubMed Scopus (1147) Google Scholar). The other allele is not transcribed in either cell line. Cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (100 μg/ml), and streptomycin (100 μg/ml) at 37 °C in 5% CO2. An expression vector containing a mouse XPV cDNA (mXPV) was constructed as follows. A NotI fragment containing mXPV was isolated from pGEM-mXPV (49Yamada A. Masutani C. Iwai S. Hanaoka F. Nucleic Acids Res. 2000; 28: 2473-2480Crossref PubMed Scopus (76) Google Scholar) and cloned in the correct orientation into the NotI site of pIRESneo2 (Clontech), which has the G418 resistance gene. The construct, pIRES-mXPV, was introduced into CTag cells by the FuGENE6 method (Roche Molecular Biochemicals) according to a manufacturer's protocol. Transfected cells were selected for G418 (Mediatech, Herndon, VA) resistance at 500 μg/ml medium. pIRESneo2 is designed to translate a cloned gene and the G418 resistance gene from the same transcript. As this transcript contains an internal ribosome entry site between the cloned gene and the G418 resistance gene, themXPV gene and the G418 resistance gene are independently translated. Furthermore, translation of the G418 resistance gene is designed to be less efficient than that of the cloned gene. Therefore, all G418-resistant cells are expected to express mXPV. To further assure the collection of mXPV-expressing cells, G418-resistant cells were irradiated with UV at 2J/m2 and then cultured in the presence of 1 mm caffeine (49Yamada A. Masutani C. Iwai S. Hanaoka F. Nucleic Acids Res. 2000; 28: 2473-2480Crossref PubMed Scopus (76) Google Scholar). Almost all cells transfected with the empty pIRESneo2 vector died after 4 days, whereas cells transfected with pIRES-mXPV survived. Following two cycles of this phenotypic selection, surviving cells were used as the host for site-specific experiments. Finally, the transcription of the mXPV gene was confirmed by RT-PCR (reverse transcriptase-polymerase chain reaction) using RNeasy Mini Kit (Qiagen) and SuperScript One-Step RT-PCR Kit (Invitrogen). The shuttle vector, pBTE, was described previously (35Yang I.-Y. Johnson F. Grollman A.P. Moriya M. Chem. Res. Toxicol. 2002; 15: 160-164Crossref PubMed Scopus (60) Google Scholar). This vector is stably maintained in human cells and confers blasticidin S resistance to host human and E. coli cells. Expression of the resistance gene is driven by the SV40 early promoter in human cells and the EM7 bacterial promoter in E. coli. The construction of double-stranded DNA plasmid containing site-specific α-OH-PdG has been described (34Yang I.-Y. Chan G. Miller H. Huang Y. Torres M.C. Johnson F. Moriya M. Biochemistry. 2002; 41: 13826-13832Crossref PubMed Scopus (89) Google Scholar) and is shown in Fig.2 together with the experimental strategy. α-OH-PdG was incorporated into the leading strand template. An important feature of this construct is that the adduct was inserted opposite a unique SnaBI site (5′-TACGTA-3′) with mismatches on both sides of the adduct (Fig. 2); thus, only the unmodified complementary strand contains the SnaBI site. Progeny plasmids derived from the unmodified strand and excision repair events are sensitive to SnaBI digestion, whereas those derived from translesion synthesis are not. Hence, progeny derived from translesion synthesis can be selectively collected for fidelity analysis by digesting with SnaBI prior to E. colitransformation. CTag/pIRES and CTag/pIRES-mXPV cells were seeded at 1 × 106 cells/25-cm2 flask, cultured overnight, then transfected overnight with 1 μg of a DNA construct by the FuGENE6 method. Where indicated, cells were treated with mitomycin C at 1 μg/ml medium for 50 min in an incubator, after which the medium was replaced with a fresh medium, and transfection was begun immediately. The next day, cells were detached by treating with trypsin-EDTA and replated in a 75-cm2 flask. The following day, blasticidin S (Invitrogen) was added to the culture medium at 5 μg/ml. Resistant cells were collected after 5 or 6 days. The progeny plasmid was purified by the method of Hirt (50Hirt B. J. Mol. Biol. 1967; 26: 365-369Crossref PubMed Scopus (3350) Google Scholar) and treated with DpnI (2 units) for 1 h to remove residual input DNA. To establish the apparent efficiency of translesion DNA synthesis,DpnI-treated plasmid was used to transform E. coli. To determine coding events at the site of α-OH-PdG, theDpnI-treated plasmid was digested with SnaBI prior to transformation. One-tenth to one-fifth of the recovered plasmid was electroporated into E. coli DH10B ElectroMAX (25 μl) (Invitrogen) by an E. coli Pulser (Bio-Rad), after which 975 μl of YT (2×) medium (36Yang I.-Y. Hossain M. Miller H. Khullar S. Johnson F. Grollman A. Moriya M. J. Biol. Chem. 2001; 276: 9071-9076Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) was added, and the bacteria were cultured for 40 min at 37 °C. Portions of the transformation mixture were plated onto YT (1×) plates containing blasticidin S (50 μg/ml) and ampicillin (100 μg/ml). After overnight incubation, E. coli transformants were subjected to differential oligonucleotide hybridization (51Pandya G. Moriya M. Biochemistry. 1996; 35: 11487-11492Crossref PubMed Scopus (180) Google Scholar, 52Pandya G.A. Yang I.-Y. Grollman A.P. Moriya M. J. Bacteriol. 2000; 182: 6598-6604Crossref PubMed Scopus (17) Google Scholar) to analyze for mutations in the adducted region. This method permits the detection of specific sequences using oligonucleotide probes. G, T, A, C, and D probes (Fig. 2C) determine coding specificity at the site of α-OH-PdG. The S probe hybridizes to the complementary SnaBI-containing strand. L and R probes confirm the presence of the 13-mer insert. Automated DNA sequence analysis was performed as necessary. To understand the mechanism of the translesion synthesis across α-OH-PdG in human cells, we first conducted in vitroexperiments to select candidate polymerases whose translesion synthesis activity and fidelity are consistent with the in vivoresults, and we then examined the role of one (pol η) of the candidates in human cells. A running start experiment (Fig. 3) using a 16-mer primer and a 28-mer template showed that pol δ bypassed α-OH-PdG very weakly only in the presence of PCNA. Extended products were not observed opposite the adduct, and the majority of the extension was terminated at one base before the adduct site. These results suggest that nucleotide insertion opposite α-OH-PdG and the subsequent extension are poor. When the read-through experiment was catalyzed by exo+ Klenow enzyme, full-length products were rarely observed, and some extended products were observed opposite the adduct. These results suggest that the full-length products observed in the pol δ-catalyzed reaction were generated by true bypass synthesis across the adduct. No stable insertion of a nucleotide opposite α-OH-PdG by pol δ was confirmed by nucleotide incorporation experiments using 10 and 100 μm dNTP (Fig. 4). The results of these experiments indicate that pol δ/PCNA catalyzes bypass of α-OH-PdG very weakly. At this time the fidelity of this bypass synthesis is not known. In subsequent experiments designed to determine the nucleotide distance between the adduct and the primer terminus at which pol δ/PCNA recovered efficient synthesis, we found that exonucleolytic proofreading prevailed over polymerization when the primer terminus was located three nucleotides or less 5′ to the adduct (Fig.5). When the terminus was five nucleotides away, net polymerization efficiency increased. At seven nucleotides, proofreading became marginal, and polymerization was predominant. Therefore, if a translesion polymerase catalyzes DNA synthesis ≥7 nucleotides past α-OH-PdG, the subsequent synthesis can be performed efficiently by pol δ. As α-OH-PdG inhibits DNA synthesis strongly, it is conceivable that translesion polymerases participate in bypassing this adduct. To determine which polymerase(s) plays a role in the accurate and inaccurate bypass syntheses, we first examined the abilities of pol η and pol κ to catalyze a by
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