Translesion Synthesis past Acrolein-derived DNA Adduct, γ-Hydroxypropanodeoxyguanosine, by Yeast and Human DNA Polymerase η
2003; Elsevier BV; Volume: 278; Issue: 2 Linguagem: Inglês
10.1074/jbc.m207774200
ISSN1083-351X
AutoresIrina G. Minko, M. Todd Washington, Manorama Kanuri, Louise Prakash, Satya Prakash, R. Stephen Lloyd,
Tópico(s)DNA and Nucleic Acid Chemistry
Resumoγ-Hydroxy-1,N 2-propano-2′deoxyguanosine (γ-HOPdG) is a major deoxyguanosine adduct derived from acrolein, a known mutagen. In vitro, this adduct has previously been shown to pose a severe block to translesion synthesis by a number of polymerases (pol). Here we show that both yeast and human pol η can incorporate a C opposite γ-HOPdG at ∼190- and ∼100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at ∼8- and ∼19-fold lower efficiency. Although DNA synthesis past γ-HOPdG by yeast pol η was relatively accurate, the human enzyme misincorporated nucleotides opposite the lesion with frequencies of ∼10−1 to 10−2. Because γ-HOPdG can adopt both ring closed and ring opened conformations, comparative replicative bypass studies were also performed with two model adducts, propanodeoxyguanosine and reduced γ-HOPdG. For both yeast and human pol η, the ring open reduced γ-HOPdG adduct was less blocking than γ-HOPdG, whereas the ring closed propanodeoxyguanosine adduct was a very strong block. Replication of DNAs containing γ-HOPdG in wild type and xeroderma pigmentosum variant cells revealed a somewhat decreased mutation frequency in xeroderma pigmentosum variant cells. Collectively, the data suggest that pol η might potentially contribute to both error-free and mutagenic bypass of γ-HOPdG. γ-Hydroxy-1,N 2-propano-2′deoxyguanosine (γ-HOPdG) is a major deoxyguanosine adduct derived from acrolein, a known mutagen. In vitro, this adduct has previously been shown to pose a severe block to translesion synthesis by a number of polymerases (pol). Here we show that both yeast and human pol η can incorporate a C opposite γ-HOPdG at ∼190- and ∼100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at ∼8- and ∼19-fold lower efficiency. Although DNA synthesis past γ-HOPdG by yeast pol η was relatively accurate, the human enzyme misincorporated nucleotides opposite the lesion with frequencies of ∼10−1 to 10−2. Because γ-HOPdG can adopt both ring closed and ring opened conformations, comparative replicative bypass studies were also performed with two model adducts, propanodeoxyguanosine and reduced γ-HOPdG. For both yeast and human pol η, the ring open reduced γ-HOPdG adduct was less blocking than γ-HOPdG, whereas the ring closed propanodeoxyguanosine adduct was a very strong block. Replication of DNAs containing γ-HOPdG in wild type and xeroderma pigmentosum variant cells revealed a somewhat decreased mutation frequency in xeroderma pigmentosum variant cells. Collectively, the data suggest that pol η might potentially contribute to both error-free and mutagenic bypass of γ-HOPdG. γ-hydroxy-1,N 2-propano-2′deoxyguanosine deoxyguanosine 1,N 2-propanodeoxyguanosine DNA polymerase xeroderma pigmentosum variant Acrolein (Fig. 1), the simplest α,β-unsaturated aldehyde, is an environmental contaminant and a product of inborn metabolism. In organisms, acrolein is generated via a number of pathways, such as the oxidation of polyamines and lipid peroxidation (1Chung 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, 2Pan J. Chung F.-L. Chem. Res. Toxicol. 2002; 15: 367-372Crossref PubMed Scopus (75) Google Scholar). Like many other bifunctional aldehydes, acrolein reacts with DNA bases to form several DNA adducts, among which the γ-hydroxy-1,N 2-propano-2′deoxyguanosine (γ-HOPdG)1 was identified as a major deoxyguanosine (dG) derivative (3Chung F.-L. Young R. Hecht S.S. Cancer Res. 1984; 44: 990-995PubMed Google Scholar, 4Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis.in: Singer B. Bartsch H. IARC Scientific Publications No. 150. International Agency for Research on Cancer, Lyon, France1999: 1-15Google Scholar). Importantly, γ-HOPdG has been detected in DNA from mammalian tissues (5McDiarmid M.A. Iype P.T. Kolodner K. Jacobson-Kram D. Strickland P.T. Mutat. Res. 1991; 248: 93-99Crossref PubMed Scopus (36) Google Scholar, 6Nath R.G. Chung F.-L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7491-7495Crossref PubMed Scopus (220) Google Scholar, 7Penn A. Nath R.G. Pan J. Chen L.C. Widmer K. Henk W. Chung F.-L. Environ. Health. Perspect. 2001; 109: 219-224PubMed Google Scholar), suggesting that this adduct is generated in vivo. The γ-HOPdG adduct is formed by conjugate addition of acrolein to N2 of dG to produceN 2-(3-oxopropyl)dG. Ring closure at N1 leads to the formation of the cyclic adduct (Fig. 1). In the nucleoside and presumably in single-stranded DNA, γ-HOPdG predominantly exists in the cyclic form, such that at physiological pH, the ring open species cannot be detected spectrophotometrically (8Nechev L.V. Harris C.M. Harris T.M. Chem. Res. Toxicol. 2000; 13: 421-429Crossref PubMed Scopus (70) Google Scholar). However, in the presence of a reducing agent, the acyclic form can be trapped as theN 2-(3-hydroxypropyl) adduct (Fig. 1). Another dG derivative, 1,N 2-propanodeoxyguanosine (PdG) (Fig. 1), whose structure is similar to that of the ring closed γ-HOPdG, has been extensively exploited as a model compound for the γ-HOPdG and other exocyclic dG adducts in both structural and biological studies. NMR spectroscopy of the PdG-adducted oligodeoxynucleotides has revealed that when placed opposite dC, PdG adopts a syn orientation within the duplex and introduces a localized structural perturbation that is pH- and sequence-dependent (9Singh U.S. Moe J.G. Reddy G.R. Weisenseel J.P. Marnett L.J. Stone M.P. Chem. Res. Toxicol. 1993; 6: 825-836Crossref PubMed Scopus (60) Google Scholar, 10Weisenseel J.P. Reddy G.R. Marnett L.J. Stone M.P. Chem. Res. Toxicol. 2002; 15: 127-139Crossref PubMed Scopus (32) Google Scholar). The inability of PdG to form normal Watson-Crick hydrogen bonds severely blocks DNA synthesis both in vitro (11Hashim M.F. Marnett L.J. J. Biol. Chem. 1996; 271: 9160-9165Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 12Hashim M.F. Schnetz-Boutaud N. Marnett L.J. J. Biol. Chem. 1997; 272: 20205-20212Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and in vivo(13Benamira M. Singh U. Marnett L.J. J. Biol. Chem. 1992; 267: 22392-22400Abstract Full Text PDF PubMed Google Scholar, 14Moriya M. Zhang W. Johnson F. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11899-11903Crossref PubMed Scopus (233) Google Scholar, 15Burcham P.C. Marnett L.J. J. Biol. 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A. 1994; 91: 11899-11903Crossref PubMed Scopus (233) Google Scholar) and in the nucleotide excision repair-deficient human cells (16Yang I.-Y. Johnson F. Grollman A.P. Moriya M. Chem. Res. Toxicol. 2002; 15: 160-164Crossref PubMed Scopus (60) Google Scholar), respectively. In both strains, G to T transversions predominated. Recently, the structure of the γ-HOPdG-containing oligodeoxynucleotide was solved by NMR spectroscopy (17de los Santos C. Zaliznyak T. Johnson F. J. Biol. Chem. 2001; 276: 9077-9082Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). These data have indicated that within the duplex, γ-HOPdG exists primarily in the ring open form. In such a conformation, the modified base participates in standard Watson-Crick base pairing by adopting a regular anti orientation around the glycosidic torsion angle, with the N 2-propyl chain in the minor groove pointing toward the solvent (17de los Santos C. Zaliznyak T. Johnson F. J. Biol. Chem. 2001; 276: 9077-9082Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The structural differences between PdG and γ-HOPdG within the duplex have led to the hypothesis that the latter lesion would be less blocking for replication and less mutagenic than the former. Biological studies aimed to test the cytotoxic and mutagenic effects of acrolein-modified DNAs and of site-specific γ-HOPdG adduct have generated conflicting results. It is known that acrolein itself causes mutations in both bacterial (18Marnett 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) and mammalian (19Curren R.D. Yang L.L. Conklin P.M. Grafstrom R.C. Harris C.C. Mutat. Res. 1988; 209: 17-22Crossref PubMed Scopus (89) Google Scholar) systems and has tumor-initiating activity (20Cohen S.M. Garland E.M., St. John M. Okamura T. Smith R.A. Cancer Res. 1992; 52: 3577-3581PubMed Google Scholar). When a DNA vector was treated with acrolein and propagated in human cells, the majority of mutations were single, tandem, and multiple base substitutions that predominantly occurred in G:C base pairs (21Kawanishi M. Matsuda T. Nakayama A. Takebe H. Matsui S. Yagi T. Mutat. Res. 1998; 417: 65-73Crossref PubMed Scopus (86) Google Scholar). However in bacteria, γ-HOPdG, the major acrolein-derived dG adduct, is not a strong block for DNA synthesis nor a miscoding lesion (22Yang 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, 23VanderVeen L.A. Hashim M.F. Nechev L.V. Harris T.M. Harris C.M. Marnett L.J. J. Biol. Chem. 2001; 276: 9066-9070Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 24Kanuri M. Minko I.G. Nechev L.V. Harris T.M. Harris C.M. Lloyd R.S. J. Biol. Chem. 2002; 277: 18257-18265Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Analyses of mutations caused by γ-HOPdG in wild type Escherichia coli and inpolB, dinB, and umuDC deficient strains revealed that in the absence of these "SOS" polymerases, the efficiency and accuracy of the translesion synthesis were not significantly affected (22Yang 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). In contrast to the prokaryotic data, γ-HOPdG caused mutations at an overall frequency of 7.4 × 10−2/translesion synthesis when a single-stranded, site-specifically modified vector was propagated in COS-7 cells (24Kanuri M. Minko I.G. Nechev L.V. Harris T.M. Harris C.M. Lloyd R.S. J. Biol. Chem. 2002; 277: 18257-18265Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Interestingly, both the frequencies and types of mutations were remarkably similar to those reported for the PdG adduct (14Moriya M. Zhang W. Johnson F. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11899-11903Crossref PubMed Scopus (233) Google Scholar, 16Yang I.-Y. Johnson F. Grollman A.P. Moriya M. Chem. Res. Toxicol. 2002; 15: 160-164Crossref PubMed Scopus (60) Google Scholar). However, γ-HOPdG was shown to be only marginally miscoding (≤1% base substitution) when double-stranded vector was utilized (16Yang I.-Y. Johnson F. Grollman A.P. Moriya M. Chem. Res. Toxicol. 2002; 15: 160-164Crossref PubMed Scopus (60) Google Scholar). In this investigation, a number of cell lines including HeLa, a nucleotide excision repair-deficient xeroderma pigmentosum group A, and polymerase η-deficient xeroderma pigmentosum variant were examined. Although replication across γ-HOPdG in vivo was predominantly error-free (from 93 to 100% of the translesional events), the adduct was shown to be a severe block and a miscoding lesion during in vitro DNA synthesis by a number of polymerases. Particularly, replication across γ-HOPdG by the Klenow exo− fragment of E. coli polymerase I was significantly inhibited and extremely error-prone (22Yang 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, 23VanderVeen L.A. Hashim M.F. Nechev L.V. Harris T.M. Harris C.M. Marnett L.J. J. Biol. Chem. 2001; 276: 9066-9070Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). γ-HOPdG also strongly blocked DNA synthesis by two major eukaryotic polymerases, pol δ and pol ε (24Kanuri M. Minko I.G. Nechev L.V. Harris T.M. Harris C.M. Lloyd R.S. J. Biol. Chem. 2002; 277: 18257-18265Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In the presence of proliferating cell nuclear antigen, little bypass of the adduct by pol δ was achieved, and it appeared to be highly mutagenic (24Kanuri M. Minko I.G. Nechev L.V. Harris T.M. Harris C.M. Lloyd R.S. J. Biol. Chem. 2002; 277: 18257-18265Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). We hypothesized therefore that in mammalian cells, specialized, translesion DNA synthesis polymerases (25Ohmori H. Friedberg E.C. Fuchs R.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, 26Wang Z. Mutat. Res. 2001; 486: 59-70Crossref PubMed Scopus (85) Google Scholar) are involved in promoting replication across γ-HOPdG. Among DNA polymerases proficient in translesion synthesis, yeast polymerase η (a product of the RAD30 gene) (27Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (694) Google Scholar) and its human counterpart (a product of the RAD30A (XPV , POLH) gene) (28Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Crossref PubMed Scopus (672) Google Scholar, 29Masutani 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 (1148) Google Scholar) both possess a unique ability to replicate efficiently and accurately past a cis-syn cyclobutane pyrimidine dimer (30Washington M.T. Johnson R.E. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3094-3099PubMed Google Scholar, 31Johnson R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), the predominant DNA lesion caused by ultraviolet irradiation. In the yeast Saccharomyces cerevisiae, deletion of RAD30 confers moderate sensitivity to UV irradiation and leads to increased UV-induced mutagenesis (32McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar). Mutations in the human RAD30A gene cause the variant form of xeroderma pigmentosum (XPV), suggesting that predisposition of XPV individuals to sunlight-induced skin cancer is due to the lack of accurate translesion DNA synthesis across UV-induced DNA lesions (28Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Crossref PubMed Scopus (672) Google Scholar, 29Masutani 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 (1148) Google Scholar, 33Broughton B.C. Cordonnier A. Kleijer W.J. Jaspers N.G.J. Fawcett H. Raams A. Garritsen V.H. Stary A. Avril M.-F. Boudsocq F. Masutani C. Hanaoka F. Fuchs R.P. Sarasin A. Lehmann A.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 815-820Crossref PubMed Scopus (142) Google Scholar). Yeast and human pol η also efficiently bypass a product of oxidative DNA damage, the 7,8-dihydro-8-oxoguanine, and do so in a predominantly error-free manner (34Haracska L., Yu, S.-L. Johnson R.E. Prakash L. Prakash S. Nat. Genet. 2000; 25: 458-461Crossref PubMed Scopus (304) Google Scholar). In addition, several other DNA lesions were reported to be substrates for human (35Haracska L. Prakash S. Prakash L. Mol. Cell. Biol. 2000; 20: 8001-8007Crossref PubMed Scopus (118) Google Scholar, 36Zhang Y. Yuan F., Wu, X. Rechkoblit O. Taylor J.-S. Geacintov N.E. Wang Z. Nucleic Acids Res. 2000; 28: 4717-4724Crossref PubMed Google Scholar, 37Levine R.L. Miller H. Grollman A. Ohashi E. Ohmori H. Masutani C. Hanaoka F. Moriya M. J. Biol. 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To further explore the bypass mechanism, comparative studies were also performed with two model DNA adducts: PdG, which mimics the cyclic form of γ-HOPdG, andN 2-(3-hydroxypropyl)dG, which is similar to γ-HOPdG in its ring open form. In addition, the mutagenic potential of γ-HOPdG was tested in vivo in both human fibroblasts and pol η-deficient XPV cells utilizing a site-specifically modified single-stranded pMS2 vector. T4 DNA ligase, T4 polynucleotide kinase, andEcoRV were obtained from New England BioLabs (Beverly, MA). S1 nuclease and proteinase K were purchased from Invitrogen. [γ-32P]ATP was purchased from PerkinElmer Life Sciences. Bio-Spin columns were purchased from Bio-Rad. Centricon 100 concentrators were obtained from Amicon Inc. (Beverly, CA). Slide-A-Lyzer Dialysis Cassettes were obtained from Pierce. Single-stranded pMS2 DNA was a generous gift from Dr. M. Moriya (State University of New York, Stony Brook, NY). SV40-transformed cTAG derived from XP4BE cells and SV80 normal human fibroblasts were obtained from Dr. Marila Cordeiro-Stone (University of North Carolina, Chapel Hill, NC). The E. coliDH10B cells used for amplification of transformed DNA isolated from mammalian cells were purchased from Invitrogen. 12-mer oligodeoxynucleotide modified with γ-HOPdG, 5′-GCTAGC(γ-HOPdG)AGTCC-3′, was kindly provided by Dr. T. M. Harris and Dr. C. M. Harris (Vanderbilt University, Nashville, TN), and it was prepared by a previously described procedure (8Nechev L.V. Harris C.M. Harris T.M. Chem. Res. Toxicol. 2000; 13: 421-429Crossref PubMed Scopus (70) Google Scholar). The 24-mer oligodeoxynucleotide, 5′-GCAGTATCGCGC(PdG)CGGCATGAGCT-3′, adducted with PdG was synthesized as described (41Marinelli E.R. Johnson F. Iden C.R. Yu P.L. Chem. Res. Toxicol. 1990; 3: 49-58Crossref PubMed Scopus (51) Google Scholar) and was a generous gift from Dr. L. J. Marnett (Vanderbilt University, Nashville, TN). Nondamaged 12- and 24-mer with a dG in place of γ-HOPdG or PdG, respectively, were purchased from Midland Certified Reagent Co. (Midland, TX). All of the other oligodeoxynucleotides were synthesized by the Molecular Biology Core Laboratory of the National Institute of Environmental Health Sciences Toxicology Center at the University of Texas Medical Branch (Galveston, TX) and purified by electrophoresis through a 15% denaturing PAGE (in the presence of 7 m urea). Construction of site-specifically modified linear templates forin vitro replication assays was done according to the previously described procedure (24Kanuri M. Minko I.G. Nechev L.V. Harris T.M. Harris C.M. Lloyd R.S. J. Biol. Chem. 2002; 277: 18257-18265Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Sequences of the resulting oligodeoxynucleotides were identical: 5′-GCTAGCGAGTCCGCGCCAAGCTTGGGCTGCAGCAGGTC-3′, where the underlined G is either γ-HOPdG or nonadducted dG and 5′-GCAGTATCGCGCGCGGCATGAGCTGCGCCAAGCTTGGGCTGCAGCAGGTC-3′, where the underlined G is either PdG or nonadducted dG. To obtain theN 2-(3-hydroxypropyl)dG-containing DNA substrate, 10 μl of 1 m NaBH4 dissolved in 1m Hepes buffer (pH 7.4) were added twice to 200 μl of the γ-HOPdG-adducted 38-mer oligodeoxynucleotide (1–2 μm). Each addition of the reducing agent was followed by incubation at room temperature for 4 h. DNA was then dialyzed against 10 mm Tris-HCl (pH 7.0), 1 mm EDTA overnight using Slide-A-Lyzer Dialysis Cassette (3,500 molecular weight cut off). To confirm the completeness of reduction, the polypeptide trapping technique was utilized (42Kurtz A.J. Dodson M.L. Lloyd R.S. Biochemistry. 2002; 41: 7054-7064Crossref PubMed Scopus (42) Google Scholar) modified by A. J. Kurtz for γ-HOPdG-containing DNAs. Briefly, probes of both γ-HOPdG- and reduced γ-HOPdG-adducted oligodeoxynucleotides (50 nm) were incubated with 50 mm lysine-tryptophan-lysine-lysine in the presence of 25 mm NaCNBH3 and 100 mm Hepes (pH 7.4) for 5 h. The reactions were terminated by the addition of an equal volume of 95% (v/v) formamide, 20 mm EDTA, 0.02% (w/v) xylene cyanol, and 0.02% (w/v) bromphenol blue and heating at 90 °C for 2 min. Next, DNAs were resolved through a 15% denaturing PAGE and visualized with PhosphorImager Screen. Under these conditions, no trapping was detected in reactions with γ-HOPdG-containing oligodeoxynucleotide, whereas the γ-HOPdG-containing DNA was completely complexed with the polypeptide. Purifications of yeast pol η and human pol η were done as described in Refs. 27Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (694) Google Scholar and 31Johnson R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, respectively. The 21-mer oligodeoxynucleotides were used as primers for in vitro polymerase reactions. Their sequences were: 5′-CCTGCTGCAGCCCAAGCTTGG-3′, which is complementary to the 38-mer γ-HOPdG-containing template DNAs from positions −9 to −29 relative to the site of lesion (−9 primer) as well as complementary to the PdG-adducted 50-mer from positions −15 to −35 (−15 primer); 5′-AGCCCAAGCTTGGCGCGGACT-3′ and 5′-AGCTTGGCGCAGCTCATGCCG-3′, which are complementary from the position −1 to −21 to the γ-HOPdG-containing template and the PdG-containing template, respectively (−1 primers); and 5′-GCCCAAGCTTGGCGCGGACTC-3′ and 5′-GCTTGGCGCAGCTCATGCCGC-3′, which overlap the lesion site in modified templates (0 primers). Primer oligodeoxynucleotides were phosphorylated with T4 polynucleotide kinase using [γ-32P]ATP and purified using P-6 Bio-Spin columns supplied with 10 mm Tris-HCl buffer (pH 7.4). The γ-32P-labeled primers were mixed with the oligodeoxynucleotide substrates at a molar ratio of 1:2 in the presence of 25 mm Tris-HCl buffer (pH 7.6), 50 mm NaCl, heated at 90 °C for 2 min, and cooled to room temperature overnight. Primer extension and single-nucleotide incorporation experiments with yeast pol η were carried out as described (27Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (694) Google Scholar) and with human pol η as in Ref. 31Johnson R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar. Briefly, the reaction mixture (10 μl) contained 5 nm primer annealed to a template, 25 mmTris-HCl buffer (pH 7.5), 10 mm NaCl, 5 mmMgCl2, 10% glycerol, 100 μg/ml of bovine serum albumin, 5 mm dithiothreitol, 100 μm of each of the four dNTPs (primer extension experiments), or 10 μmindividually (single-nucleotide incorporation experiments), and yeast or human pol η at the concentrations as indicated in the figure legends. The reactions were incubated at 22 °C and terminated by the addition of 4× excess of stop solution consisting of 95% (v/v) formamide, 20 mm EDTA, 0.02% (w/v) xylene cyanol, and 0.02% (w/v) bromphenol blue. The reaction products were resolved through a 20% denaturing PAGE and visualized by a PhosphorImager screen. Steady-state kinetic assays were carried out under the same conditions as the DNA polymerase assays except that 1 nm yeast or human pol η and 20 nm DNA substrates were used with various concentrations of one of the four nucleotides. The reactions were quenched after 5 min. Quantitative analyses were performed using a PhosphorImager screen and Image-Quant 5.0 software (Molecular Dynamics, Sunnyvale, CA). Calculations of rates of nucleotide incorporation were done as described in Ref. 43Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (226) Google Scholar. The rates of nucleotide incorporation were graphed as a function of nucleotide concentration, and thek cat and K m parameters were obtained from the best fit of the data to the Michaelis-Menten equation. The 12-mer oligodeoxynucleotides containing either γ-HOPdG or a nondamaged dG were phosphorylated at the 5′ end with ATP and inserted into single-stranded pMS2 shuttle vector as described earlier (24Kanuri M. Minko I.G. Nechev L.V. Harris T.M. Harris C.M. Lloyd R.S. J. Biol. Chem. 2002; 277: 18257-18265Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The two ligated samples were designated pMS2(dG) and pMS2(γ-HOPdG). Transfection of pMS2(dG) and pMS2(γ-HOPdG) into cTAG and SV80 cells, isolation of DNA, amplification in E. coli DH10B cells, and differential hybridization analysis were done as previously described (24Kanuri M. Minko I.G. Nechev L.V. Harris T.M. Harris C.M. Lloyd R.S. J. Biol. Chem. 2002; 277: 18257-18265Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Hybridization with the progeny plasmid DNA was performed using [γ-32P]ATP-labeled 18-mer oligodeoxynucleotide probes (5′-GATGCTAGCNAGTCCATC-3′, where N refers to A, T, G, or C). Whatman 541 filters containing hybridized colonies were exposed to X-Omat AR film overnight, and autoradiographs were developed to identify mutation frequency and types of mutations. Representative colonies were subjected to dideoxy sequencing (44Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52674) Google Scholar) to confirm the presence of the mutations. A 20-mer primer (5′-CCATCTTGTTCAATCATGCG-3′) sequence around 100 nucleotides downstream of the adduct was used for sequencing the region containing the 12-mer oligodeoxynucleotide in progeny plasmid DNA. To examine whether yeast pol η was able to replicate past a γ-HOPdG adduct, running start primer extension experiments were performed (Fig. 2 A). A 21-mer primer was annealed to the template DNA so that it allowed the addition of 9 nucleotides before encountering the adduct (−9 primer). On the nondamaged DNA substrate, primers were efficiently extended by yeast pol η (Fig. 2 A, lanes 1–4). On the γ-HOPdG-containing substrate (Fig. 2 A, lanes 5–8), yeast pol η appeared to be capable of bypassing the lesion and forming full-length products. However, DNA synthesis was partially inhibited right before the DNA lesion and opposite from it. To understand better the importance of ring opening during replication, primer extension experiments were carried out using two model DNA substrates: the PdG adduct, which is an analogue of the ring closed form of the γ-HOPdG, and the reduced γ-HOPdG, which is similar to the ring open form of the natural adduct. In the case of the 50-mer PdG-containing substrate, 21-mer primer was positioned on the template so that the incorporation of 15 nucleotides was needed before reaching the lesion (−15 primer). Because both efficiency and accuracy of the DNA synthesis are known to be sequence-dependent (43Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (226) Google Scholar, 45Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (619) Google Scholar), an additional nondamaged control 50-mer DNA template was utilized that had the same sequence as the PdG-adducted template. These data revealed that the PdG adduct was a much stronger block for replication by yeast pol η than γ-HOPdG. Under conditions that allowed an efficient replication of the nondamaged DNA template (Fig. 2 A,lanes 13–16), DNA synthesis on the PdG-adducted template was greatly inhibited one nucleotide before the lesion, and synthesis was completely aborted after incorporating a nucleotide opposite the lesion (Fig. 2 A, lanes 17–20). However, replication by yeast pol η beyond the PdG can be achieved but at much higher concentrations of enzyme (data not shown). With the reduced γ-HOPdG-adducted template (Fig. 2 A, lanes 9–12), the bypass efficiency by yeast pol η seemed to be comparable with that on the γ-HOPdG-adducted template. The specificity of nucleotide incorporation by yeast pol η opposite and beyond the lesions was also tested. To identify the nucleotide that is incorporated by this polymerase opposite the adducted base, single-nucleotide incorporation experiments were carried out using standing start DNA substrates in which 3′ terminus of the primer was located one nucleotide before the lesions (−1 primer
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