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Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases*

  • Linlin Zhao
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

    Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Matthew G. Pence
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

    Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Plamen P. Christov
    Affiliations
    Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

    Department of Chemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Zdzislaw Wawrzak
    Affiliations
    Northwestern University Synchrotron Research Center, Life Sciences Collaborative Access Team, Argonne, Illinois 60439
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  • Jeong-Yun Choi
    Affiliations
    Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Gyeonggi-do 440-746, Republic of Korea
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  • Carmelo J. Rizzo
    Affiliations
    Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

    Department of Chemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Martin Egli
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

    Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • F.Peter Guengerich
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, 638B Robinson Research Bldg., 2200 Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-4349
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

    Department of Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01 ES010546 (to F. P. G.), R01 ES010375 (to F. P. G. and M. E.), P01 ES05355 (to M. E. and C. J. R.), T32 ES007028 (to F. P. G. and M. G. P.), and P30 ES000267 (to M. E., F. P. G., and C. J. R.) from the United States Public Health Service. This work was also supported by National Research Foundation Grant 2010-0006538 from the Ministry of Education, Science and Technology Korea (to J.-Y. C.).
Open AccessPublished:August 21, 2012DOI:https://doi.org/10.1074/jbc.M112.403253
      N2,3-Ethenoguanine (N2,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N2,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N2,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N2,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N2,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N2,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N2,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N2,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N2,3-ϵG.

      Introduction

      The integrity of DNA is continually challenged by environmental factors (e.g. UV irradiation and radiation), exogenous and endogenous chemicals, and suboptimal repair processes (
      • Lord C.J.
      • Ashworth A.
      The DNA damage response and cancer therapy.
      ). DNA damage produces modified DNA bases (i.e. DNA lesions or DNA adducts), abasic sites, DNA inter- and intrastrand cross-links, and DNA-protein cross-links that, if not properly repaired, can lead to genomic instability and ultimately disease (e.g. cancer).
      DNA polymerases (pols)
      The abbreviations used are: pol
      DNA polymerase
      1,N6-ϵA
      1,N6-ethenoadenine
      1,N2-ϵG
      1,N2-ethenoguanine
      3,N4-ϵC
      3,N4-ethenocytidine
      2′-F-dG
      2′-fluoro-2′-deoxyarabinoguanosine
      2′-F-N2,3-ϵdG
      2′-F-N2,3-ϵ-2′-deoxyarabinoguanosine
      ddC
      dideoxy-CMP
      dNTP
      deoxyribonucleotide triphosphate
      Dpo4
      S. solfataricus P2 DNA polymerase IV
      ϵ
      etheno
      N2,3-ϵG
      N2,3-ethenoguanine
      UPLC
      ultraperformance liquid chromatography
      hREV1
      human REV1
      dG
      2′-deoxyguanosine.
      are crucial in maintaining genome integrity. Fifteen human DNA pols, varying in their functions in replication, repair, and tolerance of DNA damage, are known (
      • Bebenek K.
      • Kunkel T.A.
      Functions of DNA polymerases.
      ). The Y-family DNA polymerases (pol η, pol ι, pol κ, and REV1) are specialized in translesion synthesis (
      • Hubscher U.
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      ,
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      ). For example, pol η is known for its unique role in correctly bypassing UV irradiation-induced cyclobutane pyrimidine dimer (
      • Johnson R.E.
      • Washington M.T.
      • Prakash S.
      • Prakash L.
      Fidelity of human DNA polymerase η.
      ,
      • Yang W.
      Surviving the sun: Repair and bypass of DNA UV lesions.
      ). Pol ι, on the other hand, is unable to copy past cyclobutane pyrimidine dimer but can proficiently insert T or C opposite adducted purines that are impaired in their capability of forming Watson-Crick base pairs (
      • Nair D.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
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      An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-ι active site.
      ,
      • Nair D.T.
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      Hoogsteen base pair formation promotes synthesis opposite the 1,N6-ethenodeoxyadenosine lesion by human DNA polymerase ι.
      ,
      • Pence M.G.
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      • Zink C.N.
      • Hollis T.
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      ). Pol κ has a specialized role in bypassing bulky N2-G adducts (
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      • Nohmi T.
      • Seki C.
      • Kamiya K.
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      Translesional DNA synthesis through a C8-guanyl adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in vitro: REV1 inserts dC opposite the lesion, and DNA polymerase κ potentially catalyzes extension reaction from the 3′-dC terminus.
      ) and interstrand cross-links (
      • Minko I.G.
      • Harbut M.B.
      • Kozekov I.D.
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      • Jakobs P.M.
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      Role for DNA polymerase κ in the processing of N2-N2-guanine interstrand cross-links.
      ) and is distinct in its moderate processivity, extending beyond the lesion, possibly due to the use of its N-clasp domain. REV1 is highly selective for inserting C opposite normal (
      • Swan M.K.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Structure of the human Rev1-DNA-dNTP ternary complex.
      ) and adducted template G (
      • Fukuda H.
      • Takamura-Enya T.
      • Masuda Y.
      • Nohmi T.
      • Seki C.
      • Kamiya K.
      • Sugimura T.
      • Masutani C.
      • Hanaoka F.
      • Nakagama H.
      Translesional DNA synthesis through a C8-guanyl adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in vitro: REV1 inserts dC opposite the lesion, and DNA polymerase κ potentially catalyzes extension reaction from the 3′-dC terminus.
      ,
      • Choi J.Y.
      • Guengerich F.P.
      Kinetic analysis of translesion synthesis opposite bulky N2- and O6-alkyl guanine DNA adducts by human DNA polymerase REV1.
      ). Crystal structures of Y-family pols provide insight into their diverse functions in bypassing normal and adducted templates (
      • Yang W.
      • Woodgate R.
      What a difference a decade makes: insights into translesion DNA synthesis.
      ). Pol ι adopts an induced fit mechanism by flipping template purines into the syn conformation, forming Hoogsteen base pairs (
      • Nair D.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-ι active site.
      ,
      • Nair D.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Hoogsteen base pair formation promotes synthesis opposite the 1,N6-ethenodeoxyadenosine lesion by human DNA polymerase ι.
      ,
      • Nair D.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Human DNA polymerase ι incorporates dCTP opposite template G via a G.C+ Hoogsteen base pair.
      ,
      • Pence M.G.
      • Choi J.Y.
      • Egli M.
      • Guengerich F.P.
      Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase ι.
      ). REV1 features pairing between dCTP and template G but uses its G-loop to hydrogen bond with the template G and an Arg in another segment (N-digit) to ensure the incorporation of dCTP (
      • Swan M.K.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Structure of the human Rev1-DNA-dNTP ternary complex.
      ). A high degree of functional and structural differences underlies the diverse but specialized roles in lesion bypass by Y-family human DNA polymerases (
      • Sale J.E.
      • Lehmann A.R.
      • Woodgate R.
      Y-family DNA polymerases and their role in tolerance of cellular DNA damage.
      ).
      Etheno (ϵ) DNA adducts comprise a series of exocyclic adducts, including 1,N6-ethenoadenine (1,N6-ϵA), 3,N4-ethenocytidine (3,N4-ϵC), N2,3-ethenoguanine (N2,3-ϵG), and 1,N2-ethenoguanine (1,N2-ϵG) (Fig. 1). These were first identified as reaction products of nucleobases with chloroacetaldehyde (
      • Kochetko N.K.
      • Shibaev V.N.
      • Kost A.A.
      New reaction of adenine and cytosine derivatives, potentially useful for nucleic acids modification.
      ) and were used as fluorescent analogs in biochemical studies and as probes for nucleic acid structures (
      • Secrist 3rd, J.A.
      • Barrio J.R.
      • Leonard N.J.
      • Weber G.
      Fluorescent modification of adenosine-containing coenzymes: biological activities and spectroscopic properties.
      ,
      • Sattsangi P.D.
      • Leonard N.J.
      • Frihart C.R.
      1,N2-Ethenoguanine and N2,3-ethenoguanine synthesis and comparison of electronic spectral properties of these linear and angular triheterocycles related to Y bases.
      ,
      • Leonard N.J.
      Etheno-bridged nucleotides in enzyme reactions and protein binding.
      ). The ϵ DNA adducts were subsequently recognized as reaction products of DNA with reactive metabolites of several genotoxic chemicals, including the carcinogens vinyl chloride and vinyl carbamate (an oxidation product of urethane). The detection of etheno DNA adducts in various tissues of unexposed rodents (
      • Fedtke N.
      • Boucheron J.A.
      • Walker V.E.
      • Swenberg J.A.
      Vinyl chloride-induced DNA adducts. 2. Formation and persistence of 7-(2′-oxoethyl)guanine and N2,3-ethenoguanine in rat-tissue DNA.
      ) and humans (
      • Gonzalez-Reche L.M.
      • Koch H.M.
      • Weiss T.
      • Müller J.
      • Drexler H.
      • Angerer J.
      Analysis of ethenoguanine adducts in human urine using high performance liquid chromatography-tandem mass spectrometry.
      ) led to the discovery of the endogenous pathways of formation (e.g. via reaction with trans-4-hydroperoxy-2-nonenal, a lipid peroxidation product (
      • Lee S.H.
      • Arora J.A.
      • Oe T.
      • Blair I.A.
      4-Hydroperoxy-2-nonenal-induced formation of 1,N2-etheno-2′-deoxyguanosine adducts.
      )). In livers of unexposed rats or humans, the steady-state amounts of N2,3-ϵG, 1,N2-ϵG, and 1,N6-ϵA have been estimated to be ∼36, 30, and 12 lesions/cell, respectively (
      • Swenberg J.A.
      • Lu K.
      • Moeller B.C.
      • Gao L.
      • Upton P.B.
      • Nakamura J.
      • Starr T.B.
      Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment.
      ).
      The mutagenic potentials of etheno adducts have been established in in vitro bypass assays (
      • Hang B.
      • Chenna A.
      • Guliaev A.B.
      • Singer B.
      Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea.
      ,
      • Shibutani S.
      • Suzuki N.
      • Matsumoto Y.
      • Grollman A.P.
      Miscoding properties of 3,N4-etheno-2′-deoxycytidine in reactions catalyzed by mammalian DNA polymerases.
      ,
      • Zang H.
      • Goodenough A.K.
      • Choi J.Y.
      • Irimia A.
      • Loukachevitch L.V.
      • Kozekov I.D.
      • Angel K.C.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      DNA adduct bypass polymerization by Sulfolobus solfataricus DNA polymerase Dpo4. Analysis and crystal structures of multiple base pair substitution and frameshift products with the adduct 1,N2-ethenoguanine.
      ,
      • Choi J.Y.
      • Zang H.
      • Angel K.C.
      • Kozekov I.D.
      • Goodenough A.K.
      • Rizzo C.J.
      • Guengerich F.P.
      Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases.
      ,
      • Singer B.
      • Kuśmierek J.T.
      • Folkman W.
      • Chavez F.
      • Dosanjh M.K.
      Evidence for the mutagenic potential of the vinyl-chloride induced adduct, N2,3-etheno-deoxyguanosine, using a site-directed kinetic assay.
      ) and site-specific mutagenesis in bacteria (
      • Basu A.K.
      • Wood M.L.
      • Niedernhofer L.J.
      • Ramos L.A.
      • Essigmann J.M.
      Mutagenic and genotoxic effects of three vinyl chloride-induced DNA lesions: 1,N6-ethenoadenine, 3,N4-ethenocytosine, and 4-amino-5-(imidazol-2-yl)imidazole.
      ,
      • Cheng K.C.
      • Preston B.D.
      • Cahill D.S.
      • Dosanjh M.K.
      • Singer B.
      • Loeb L.A.
      The vinyl-chloride DNA derivative N2,3-ethenoguanine produces G→A transitions in Escherichia coli.
      ), Chinese hamster ovary cells (
      • Akasaka S.
      • Guengerich F.P.
      Mutagenicity of site-specifically located 1,N2-ethenoguanine in Chinese hamster ovary cell chromosomal DNA.
      ), and simian kidney cells (
      • Moriya M.
      • Zhang W.
      • Johnson F.
      • Grollman A.P.
      Mutagenic potency of exocyclic DNA adducts: marked differences between Escherichia coli and simian kidney cells.
      ). N2,3-ϵG has been less well studied in terms of its replication and repair mechanisms because of the lability of its glycosidic bond. In a polyribo(G/N2,3-ϵG) template, both C and T were incorporated opposite N2,3-ϵG by avian myeloblastosis virus reverse transcriptase (
      • Singer B.
      • Spengler S.J.
      • Chavez F.
      • Kuśmierek J.T.
      The vinyl chloride-derived nucleoside, N2,3-ethenoguanosine, is a highly efficient mutagen in transcription.
      ). N2,3-ϵ-Deoxyguanosine triphosphate was reported to be inserted opposite T by Escherichia coli DNA polymerase I (Klenow fragment), Drosophila melanogaster polymerase α-primase complex, and human immunodeficiency virus-I reverse transcriptase (
      • Singer B.
      • Kuśmierek J.T.
      • Folkman W.
      • Chavez F.
      • Dosanjh M.K.
      Evidence for the mutagenic potential of the vinyl-chloride induced adduct, N2,3-etheno-deoxyguanosine, using a site-directed kinetic assay.
      ). An indirect assay in E. coli showed an estimated mutation frequency of 13% for N2,3-ϵG, resulting in G to A transitions (
      • Cheng K.C.
      • Preston B.D.
      • Cahill D.S.
      • Dosanjh M.K.
      • Singer B.
      • Loeb L.A.
      The vinyl-chloride DNA derivative N2,3-ethenoguanine produces G→A transitions in Escherichia coli.
      ). The long half-life of N2,3-ϵG in rat liver and lung (150 days) and in rat kidney (75 days) in vinyl chloride-exposed rats suggests inefficient repair of this lesion. In human glycosylase assays (in vitro), N2,3-ϵG was released at a much slower rate compared with 1,N6-ϵA and 3,N4-ϵC (
      • Dosanjh M.K.
      • Chenna A.
      • Kim E.
      • Fraenkel-Conrat H.
      • Samson L.
      • Singer B.
      All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase.
      ). The mutagenicity and persistence of N2,3-ϵG suggest a high miscoding potential in vivo. N2,3-ϵG is generally considered to contribute to the carcinogenesis of vinyl chloride and inflammation-driven malignancies (
      • Nair U.
      • Bartsch H.
      • Nair J.
      Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: a review of published adduct types and levels in humans.
      ). The dominance of GC to AT transitions in five of six K-ras (oncogene) tumors found in vinyl chloride workers (
      • Swenberg J.A.
      • Lu K.
      • Moeller B.C.
      • Gao L.
      • Upton P.B.
      • Nakamura J.
      • Starr T.B.
      Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment.
      ) suggests the importance of G adducts, and the miscoding pattern of 1,N2-ϵG is not consistent with this transition (
      • Hang B.
      • Chenna A.
      • Guliaev A.B.
      • Singer B.
      Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea.
      ,
      • Shibutani S.
      • Suzuki N.
      • Matsumoto Y.
      • Grollman A.P.
      Miscoding properties of 3,N4-etheno-2′-deoxycytidine in reactions catalyzed by mammalian DNA polymerases.
      ,
      • Zang H.
      • Goodenough A.K.
      • Choi J.Y.
      • Irimia A.
      • Loukachevitch L.V.
      • Kozekov I.D.
      • Angel K.C.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      DNA adduct bypass polymerization by Sulfolobus solfataricus DNA polymerase Dpo4. Analysis and crystal structures of multiple base pair substitution and frameshift products with the adduct 1,N2-ethenoguanine.
      ,
      • Choi J.Y.
      • Zang H.
      • Angel K.C.
      • Kozekov I.D.
      • Goodenough A.K.
      • Rizzo C.J.
      • Guengerich F.P.
      Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases.
      ,
      • Singer B.
      • Kuśmierek J.T.
      • Folkman W.
      • Chavez F.
      • Dosanjh M.K.
      Evidence for the mutagenic potential of the vinyl-chloride induced adduct, N2,3-etheno-deoxyguanosine, using a site-directed kinetic assay.
      ,
      • Basu A.K.
      • Wood M.L.
      • Niedernhofer L.J.
      • Ramos L.A.
      • Essigmann J.M.
      Mutagenic and genotoxic effects of three vinyl chloride-induced DNA lesions: 1,N6-ethenoadenine, 3,N4-ethenocytosine, and 4-amino-5-(imidazol-2-yl)imidazole.
      ,
      • Cheng K.C.
      • Preston B.D.
      • Cahill D.S.
      • Dosanjh M.K.
      • Singer B.
      • Loeb L.A.
      The vinyl-chloride DNA derivative N2,3-ethenoguanine produces G→A transitions in Escherichia coli.
      ,
      • Akasaka S.
      • Guengerich F.P.
      Mutagenicity of site-specifically located 1,N2-ethenoguanine in Chinese hamster ovary cell chromosomal DNA.
      ,
      • Moriya M.
      • Zhang W.
      • Johnson F.
      • Grollman A.P.
      Mutagenic potency of exocyclic DNA adducts: marked differences between Escherichia coli and simian kidney cells.
      ,
      • Singer B.
      • Spengler S.J.
      • Chavez F.
      • Kuśmierek J.T.
      The vinyl chloride-derived nucleoside, N2,3-ethenoguanosine, is a highly efficient mutagen in transcription.
      ).
      We recently investigated the miscoding of N2,3-ϵG using a stabilized 2′-fluoro-substituted analog, 2′-fluoro-N2,3-ϵ-2′-deoxyarabinoguanosine (2′-F-N2,3-ϵdG) (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ). The presence of the electronegative fluorine atom destabilizes the transition state leading to an oxocarbenium-like intermediate and hydrolysis of the glycosidic bond. This analog was site-specifically incorporated into oligonucleotides; the stability of 2′-F-N2,3-ϵdG permitted steady-state kinetics, primer extension assays, and crystallographic studies. Catalytic insertions opposite 2′-F-N2,3-ϵdG were examined using five DNA polymerases, including Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), the replicative bacteriophage pol T7 DNA exonuclease, (E. coli pol I) Klenow fragment exonuclease, yeast pol η, and human DNA pol κ, where a consistent miscoding pattern (2′-F-N2,3-ϵdG:T) was found. Crystal structures of Dpo4 with 2′-F-N2,3-ϵdG paired with dCTP showed a Watson-Crick-like structure, whereas the complex with 2′-F-N2,3-ϵdG:T revealed a “sheared” base pair (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ).
      To further understand the miscoding potential of N2,3-ϵG by Y-family human DNA polymerases, which are highly relevant to translesion synthesis, we carried out a series of primer extension and steady state-kinetic analyses using human pol ι, human pol η, and human REV1 with a template containing 2′-F-N2,3-ϵdG. The extension products formed by pol ι were identified using LC-MS/MS. A consistent mispairing pattern was observed (2′-F-N2,3-ϵdG:T), and base pairing mechanisms were revealed in two pol ι crystal structures with either dCTP or dTTP paired with 2′-F-N2,3-ϵdG but with individual differences.

      DISCUSSION

      The DNA adduct N2,3-ϵG is a ubiquitous modification produced from endogenous processes (e.g. lipid peroxidation) or exposure to environmental pollutants (e.g. vinyl chloride or urethane). We recently developed an isostere approach to incorporate the stabilized analog (2′-F-N2,3-ϵdG) into oligonucleotides and investigated the miscoding potential of N2,3-ϵG using several prokaryotic and eukaryotic DNA pols (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ). In the present work, we extended our previous investigation into the other three human Y-family DNA pols and provided the structural basis of the most error-prone bypass enzyme, pol ι.
      Primer extension gel analysis generated a qualitative comparison of the capability of bypassing 2′-F-N2,3-ϵdG by Y-family pols (Fig. 1). The order of bypassing efficiency (from the percentage of total product extended) is pol η > pol κ > REV1 ≈ pol ι. Compared with pol κ and pol ι, the higher activity of pol η copying past N2,3-ϵG observed here is similar to that seen previously for other etheno adducts, i.e. 1,N2-ϵG (
      • Choi J.Y.
      • Zang H.
      • Angel K.C.
      • Kozekov I.D.
      • Goodenough A.K.
      • Rizzo C.J.
      • Guengerich F.P.
      Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases.
      ), 1,N6-ϵA (
      • Hang B.
      • Chenna A.
      • Guliaev A.B.
      • Singer B.
      Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea.
      ,
      • Levine R.L.
      • Miller H.
      • Grollman A.
      • Ohashi E.
      • Ohmori H.
      • Masutani C.
      • Hanaoka F.
      • Moriya M.
      Translesion DNA synthesis catalyzed by human pol η and pol κ across 1,N6-ethenodeoxyadenosine.
      ), and 3,N4-ϵC (
      • Singer B.
      • Medina M.
      • Zhang Y.
      • Wang Z.
      • Guliaev A.B.
      • Hang B.
      8-(Hydroxymethyl)-3,N4-etheno-C, a potential carcinogenic glycidaldehyde product, miscodes in vitro using mammalian DNA polymerases.
      ). With regard to DNA polymerases, the extension pattern is particularly similar to that of bypass of 1,N2-ϵG; i.e. pol η readily extended the primer into full-length products, whereas pol ι and pol κ showed some single base incorporation (
      • Choi J.Y.
      • Zang H.
      • Angel K.C.
      • Kozekov I.D.
      • Goodenough A.K.
      • Rizzo C.J.
      • Guengerich F.P.
      Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases.
      ).
      Steady-state kinetic analysis established the preferred base incorporated opposite the lesion and provided a kinetic rationale for primer extension experiments (Table 1). For all four human Y-family DNA pols, the correct base C is marginally preferred opposite 2′-F-N2,3-ϵdG with similar relative efficiencies in comparison with the insertion of C opposite a regular G (Table 1). The misinsertion of T is consistent for all four human Y-family DNA pols as well as for several other prokaryotic and eukaryotic DNA polymerases (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ). The highest absolute value of catalytic efficiency (kcat/Km) seen (for pol η) is in line with primer extension results, which may be partly explained by the more open active site of pol η compared with other polymerases (
      • Silverstein T.D.
      • Johnson R.E.
      • Jain R.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Structural basis for the suppression of skin cancers by DNA polymerase η.
      ). The pattern of fidelity for pol η bypassing different etheno lesions is similar: both error-free and error-prone syntheses have been observed. Pol η inserted a C opposite N2,3-ϵG in a marginally error-free manner with a misinsertion frequency of 0.63 for T (Table 1). Similarly, pol η copied past 1,N6-ϵA in the order of preference T > C > A > G (
      • Levine R.L.
      • Miller H.
      • Grollman A.
      • Ohashi E.
      • Ohmori H.
      • Masutani C.
      • Hanaoka F.
      • Moriya M.
      Translesion DNA synthesis catalyzed by human pol η and pol κ across 1,N6-ethenodeoxyadenosine.
      ). The order was G > A > C for 1,N2-ϵG (
      • Choi J.Y.
      • Zang H.
      • Angel K.C.
      • Kozekov I.D.
      • Goodenough A.K.
      • Rizzo C.J.
      • Guengerich F.P.
      Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases.
      ) and A ≈ G > C ≈ T for 3,N4-ϵC (
      • Singer B.
      • Medina M.
      • Zhang Y.
      • Wang Z.
      • Guliaev A.B.
      • Hang B.
      8-(Hydroxymethyl)-3,N4-etheno-C, a potential carcinogenic glycidaldehyde product, miscodes in vitro using mammalian DNA polymerases.
      ). Pol ι has the highest misincorporation frequency (although C is preferred 1.5-fold compared with T), which is consistent with the view that pol ι generally catalyzes error-prone bypass (
      • Hubscher U.
      • Maga G.
      • Spadari S.
      Eukaryotic DNA polymerases.
      ). The incorporation patterns seen for pol ι bypassing other etheno DNA adducts are as follows: pol ι somewhat prefers to incorporate C opposite 1,N6-ϵA (
      • Nair D.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Hoogsteen base pair formation promotes synthesis opposite the 1,N6-ethenodeoxyadenosine lesion by human DNA polymerase ι.
      ) and inserts both C and T opposite 1,N2-ϵG with almost the same catalytic efficiencies (
      • Choi J.Y.
      • Zang H.
      • Angel K.C.
      • Kozekov I.D.
      • Goodenough A.K.
      • Rizzo C.J.
      • Guengerich F.P.
      Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases.
      ). The fact that REV1 prefers to catalyze dCTP insertion is not surprising in that REV1 utilizes its G-loop to hydrogen bond with template G and an Arg in another segment (N-digit) to ensure the incorporation of dCTP (
      • Swan M.K.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Structure of the human Rev1-DNA-dNTP ternary complex.
      ). When comparisons are made with the catalytic efficiency of dCTP insertions opposite native G in the template, the order of relative efficiency is pol κ (0.24) > REV1 (0.11) > pol η (0.027) ≈ pol ι (0.026) (Table 1), suggesting that 2′-F-N2,3-ϵdG affects the DNA syntheses of the four Y-family pols to a similar extent.
      LC-MS/MS analysis of the primer extension products by pol ι provided further insight into the nature of the bases inserted beyond the lesion in these error-prone reactions. With pol ι, approximately half of the products contained T with a high fidelity extension beyond the lesion (Fig. 3 and Table 2). The observation of almost equal amounts of products containing C and T opposite 2′-F-N2,3-ϵdG (with LC-MS/MS analysis) is in line with results from kinetic analysis (Table 1). The much lower amount of total extended products (6%) compared with Dpo4 (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ) agrees with the low bypass efficiency of pol ι seen in the primer extension gel analysis (Fig. 2). These extension products are similar to the products generated by Dpo4 (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ); however, the pattern of miscoding is considerably different from that generated by 1,N2-ϵG, which yields mainly products with G inserted by human pol η (
      • Choi J.Y.
      • Zang H.
      • Angel K.C.
      • Kozekov I.D.
      • Goodenough A.K.
      • Rizzo C.J.
      • Guengerich F.P.
      Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases.
      ) and −1 deletion products by Dpo4 (
      • Zang H.
      • Goodenough A.K.
      • Choi J.Y.
      • Irimia A.
      • Loukachevitch L.V.
      • Kozekov I.D.
      • Angel K.C.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      DNA adduct bypass polymerization by Sulfolobus solfataricus DNA polymerase Dpo4. Analysis and crystal structures of multiple base pair substitution and frameshift products with the adduct 1,N2-ethenoguanine.
      ).
      The hydrogen bonding patterns of 2′-F-N2,3-ϵdG:C and 2′-F-N2,3-ϵdG:T base pairs seen in the crystal structures provided molecular explanations for the error-free and error-prone bypass of pol ι. The distance of 2.5 Å is a clear indication that a hydrogen bond is established between the O6 atom of 2′-F-N2,3-ϵdG and the N4 atom of dCTP. The possibility of a second hydrogen bond also exists, i.e. between the N7 atom of 2′-F-N2,3-ϵdG and the N3 atom of dCTP, provided that the N3 atom of dCTP is protonated. The tendency for protonation of the N3 atom of dCTP has been discussed in several other pol ι·DNA structures with both native and adducted purines in the templates (
      • Nair D.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Hoogsteen base pair formation promotes synthesis opposite the 1,N6-ethenodeoxyadenosine lesion by human DNA polymerase ι.
      ,
      • Nair D.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Human DNA polymerase ι incorporates dCTP opposite template G via a G.C+ Hoogsteen base pair.
      ,
      • Pence M.G.
      • Choi J.Y.
      • Egli M.
      • Guengerich F.P.
      Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase ι.
      ). Although the N3 atom of free cytosine has a pKa ∼4.5, the local molecular environment could elevate the pKa to 6.2–7.2 at a terminal position or >8.5 at an internal position in DNA triple helices (
      • Asensio J.L.
      • Lane A.N.
      • Dhesi J.
      • Bergqvist S.
      • Brown T.
      The contribution of cytosine protonation to the stability of parallel DNA triple helices.
      ,
      • Plum G.E.
      • Breslauer K.J.
      Thermodynamics of an intramolecular DNA triple-helix: a calorimetric and spectroscopic study of the pH and salt dependence of thermally-induced structural transitions.
      ). Nair et al. (
      • Nair D.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Hoogsteen base pair formation promotes synthesis opposite the 1,N6-ethenodeoxyadenosine lesion by human DNA polymerase ι.
      ) suggested that an elevation of the pKa of dCTP could be due to the base-stacking and long range electrostatic interactions of the active site residues Asp-126 and Glu-127. The one hydrogen bond observed in the 2′-F-N2,3-ϵdG:T pair is an indication that the 2′-F-N2,3-ϵdG:T pair might be less stable compared with the 2′-F-N2,3-ϵdG:C pair. Our crystallization attempts are consistent with this view in that pol ι-1 type crystals (with dCTP) grew more easily and diffracted to higher resolution than the pol ι-2 crystal (with dTTP). Collectively, the difference in hydrogen bonding may explain a slightly higher catalytic efficiency for dCTP by pol ι.
      The typical strategy that pol ι uses a Hoogsteen base pairing mechanism to accommodate native and adducted purines was once again demonstrated in both the pol ι-1 and pol ι-2 structures albeit with different hydrogen bonding schemes. The similarity of the two structures consists of their use of the Hoogsteen edge of 2′-F-N2,3-ϵdG to hydrogen bond with the incoming nucleotide. The conformation of 2′-F-N2,3-ϵdG:C also resembles G:dCTP (
      • Nair D.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Human DNA polymerase ι incorporates dCTP opposite template G via a G.C+ Hoogsteen base pair.
      ) and N2-ethylG:dCTP (
      • Pence M.G.
      • Blans P.
      • Zink C.N.
      • Hollis T.
      • Fishbein J.C.
      • Perrino F.W.
      Lesion bypass of N2-ethylguanine by human DNA polymerase ι.
      ) at the pol ι active site.
      However, the base pair conformations seen here are quite different from what has been observed at the active site of Dpo4 (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ). Specifically, the 2′-F-N2,3-ϵdG:C pair adopts a Watson-Crick-like conformation, and the 2′-F-N2,3-ϵdG:T structure contains a sheared base pair at the Dpo4 active site (Fig. 6, C and D, and Ref.
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ). That 2′-F-N2,3-ϵdG was observed to be positioned in the anti conformation by Dpo4 is likely due to the relatively open active site compared with pol ι (
      • Sale J.E.
      • Lehmann A.R.
      • Woodgate R.
      Y-family DNA polymerases and their role in tolerance of cellular DNA damage.
      ) (surface view shown in Fig. 6, A and B). Particularly, the residues adjacent to the template base are bulkier (Leu-62, Val-64, and Gln-59) in pol ι compared with Dpo4 (Ala-42, Ala-44, and Val-32) (
      • Nair D.T.
      • Johnson R.E.
      • Prakash S.
      • Prakash L.
      • Aggarwal A.K.
      Replication by human DNA polymerase-ι occurs by Hoogsteen base-pairing.
      ). In the Dpo4 structure, hydrophobic interactions are likely to exist between Val-32 and the imidazole ring of 2′-F-N2,3-ϵdG (anti). Conversely, residues (Leu-62, Val-64, and Gln-59) may force 2′-F-N2,3-ϵdG to rotate into the syn conformation, which would otherwise clash with these residues if the lesion were positioned in the anti conformation. Despite these conformational differences, similar extents of T misinsertion are observed in both cases.
      Figure thumbnail gr6
      FIGURE 6A comparison of the active site conformations of pol ι (A) and Dpo4 (B) (protein shown in surface view) is shown. The conformations of 2′-F-N2,3-ϵdG paired with incoming nucleotides at the Dpo4 active site are shown in C (dCTP) and D (dTTP) (from Ref.
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ).
      As mentioned in the Introduction, Singer and co-workers (
      • Singer B.
      • Kuśmierek J.T.
      • Folkman W.
      • Chavez F.
      • Dosanjh M.K.
      Evidence for the mutagenic potential of the vinyl-chloride induced adduct, N2,3-etheno-deoxyguanosine, using a site-directed kinetic assay.
      ,
      • Cheng K.C.
      • Preston B.D.
      • Cahill D.S.
      • Dosanjh M.K.
      • Singer B.
      • Loeb L.A.
      The vinyl-chloride DNA derivative N2,3-ethenoguanine produces G→A transitions in Escherichia coli.
      ,
      • Singer B.
      • Spengler S.J.
      • Chavez F.
      • Kuśmierek J.T.
      The vinyl chloride-derived nucleoside, N2,3-ethenoguanosine, is a highly efficient mutagen in transcription.
      ) reported three studies on the miscoding of N2,3-ϵG more than 20 years ago. These studies were limited by the general methods available for studying miscoding at the time as well as the inherent lability of the glycosidic bond of N2,3-ϵdG. The uncorrected mutation frequency for N2,3-ϵdG inserted into an M13 phage system was only 0.5%, but in that study (
      • Cheng K.C.
      • Preston B.D.
      • Cahill D.S.
      • Dosanjh M.K.
      • Singer B.
      • Loeb L.A.
      The vinyl-chloride DNA derivative N2,3-ethenoguanine produces G→A transitions in Escherichia coli.
      ), an in vitro study with a polyribo(G/N2,3-ϵG) template and a reverse transcriptase (
      • Singer B.
      • Spengler S.J.
      • Chavez F.
      • Kuśmierek J.T.
      The vinyl chloride-derived nucleoside, N2,3-ethenoguanosine, is a highly efficient mutagen in transcription.
      ), and a study on “reverse” incorporation of N2,3-ϵdG triphosphate (
      • Singer B.
      • Kuśmierek J.T.
      • Folkman W.
      • Chavez F.
      • Dosanjh M.K.
      Evidence for the mutagenic potential of the vinyl-chloride induced adduct, N2,3-etheno-deoxyguanosine, using a site-directed kinetic assay.
      ), the general pattern was pairing of N2,3-ϵG with T and C. This pattern, despite any deficiencies in the earlier work, is similar to those seen in our own studies (Ref.
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      and the present work). The N2,3-ϵG:T wobble pairing proposed in that early work (
      • Singer B.
      • Spengler S.J.
      • Chavez F.
      • Kuśmierek J.T.
      The vinyl chloride-derived nucleoside, N2,3-ethenoguanosine, is a highly efficient mutagen in transcription.
      ) had no experimental basis and has not been observed in our crystal structures with Dpo4 (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ) or human pol ι (Fig. 4).
      More recently, theoretical studies (
      • Srinivasadesikan V.
      • Sahu P.K.
      • Lee S.L.
      Model calculations for the misincorporation of nucleotides opposite five-membered exocyclic DNA adduct: N2,3-ethenoguanine.
      ) have predicted that G should be the base most likely to pair with N2,3-ϵG followed by T > A > C, a prediction that is clearly inconsistent with the results obtained with all DNA polymerases thus far (TABLE 1, TABLE 2) (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ). The pairing patterns predicted in the theoretical study (
      • Srinivasadesikan V.
      • Sahu P.K.
      • Lee S.L.
      Model calculations for the misincorporation of nucleotides opposite five-membered exocyclic DNA adduct: N2,3-ethenoguanine.
      ) are also inconsistent with our N2,3-ϵG:C and N2,3-ϵG:T structures observed in the Dpo4 (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ) and human pol ι (Fig. 4) crystals.
      As mentioned in the Introduction, the goal of the 2′-fluoro substitution was to stabilize the glycosidic bond by destabilizing the transition state leading to an oxocarbenium-like intermediate in hydrolysis. The substitution was clearly successful in stabilizing the residue in oligonucleotides (
      • Zhao L.
      • Christov P.P.
      • Kozekov I.D.
      • Pence M.G.
      • Pallan P.S.
      • Rizzo C.J.
      • Egli M.
      • Guengerich F.P.
      Replication of N2,3-ethenoguanine by DNA polymerases.
      ). Although miscoding by N2,3-ϵG (specifically, 2′-F-N2,3-ϵdG) was clearly demonstrated relative to both dG and 2′-F-dG (Table 1), it should be noted that the substitution of fluorine for hydrogen at the C2′ sugar position is not without effect; i.e. the substitution caused up to a 12-fold change in kcat/Km (primarily in the Km parameter) among four Y-family DNA polymerases: an ∼8-fold decrease of kcat/Km with pol κ and a ∼12-fold increase of kcat/Km with pol ι but no changes with pol η or REV1. Therefore, 2′-fluoro substitution seems to slightly interfere with pol ι activity but to facilitate pol κ activity, which might be related to a possible stabilizing effect of 2′-fluorine to exert a (intra- and/or inter-residual) pseudo-hydrogen bonding interaction with purine H8 as shown previously with 2′-fluoroarabinonucleic acid (
      • Berger I.
      • Tereshko V.
      • Ikeda H.
      • Marquez V.E.
      • Egli M.
      Crystal structures of B-DNA with incorporated 2′-deoxy-2′-fluoro-arabino-furanosyl thymines: implications for conformational preorganization for duplex stability.
      ,
      • Li F.
      • Sarkhel S.
      • Wilds C.J.
      • Wawrzak Z.
      • Prakash T.P.
      • Manoharan M.
      • Egli M.
      2′-Fluoroarabino- and arabinonucleic acid show different conformations, resulting in deviating RNA affinities and processing of the heteroduplexes with RNA by RNase H.
      ,
      • Anzahaee M.Y.
      • Watts J.K.
      • Alla N.R.
      • Nicholson A.W.
      • Damha M.J.
      Energetically important C–H···F–C pseudohydrogen bonding in water: evidence and application to rational design of oligonucleotides with high binding affinity.
      ). Such a conformational effect (preferentially to an anti conformation) by 2′-F at dG might affect catalysis differently with the various polymerases by interfering with the (syn-anti) Hoogsteen base pairing adopted by pol ι but facilitating the (anti-anti) Watson-Crick base pairing utilized by pol κ (albeit not with pol η). Nevertheless, these points regarding the influence of the fluorine do not affect our conclusions about the miscoding properties of N2,3-ϵG reported here.
      In conclusion, we have utilized a recently developed stabilized analog, 2′-F-N2,3-ϵdG, to discern the mutation potential of a ubiquitous but unstable DNA lesion, N2,3-ϵdG. Kinetic and extension analyses allow qualitative and quantitative assessments of the miscoding pattern of this lesion for Y-family DNA polymerases, which are particularly relevant to translesion synthesis. Structural insights provided the molecular bases of error-free and error-prone synthesis by pol ι. The consistency of T misinsertion with all polymerases studied thus far underscores the miscoding potential of N2,3-ϵG. The miscoding for T suggests the relevance of N2,3-ϵG to vinyl chloride-induced angiosarcomas in which prevailing GC to AT transition mutations were found in the second base of codon 13 of the K-ras gene (
      • Marion M.J.
      • De Vivo I.
      • Smith S.
      • Luo J.C.
      • Brandt-Rauf P.W.
      The molecular epidemiology of occupational carcinogenesis in vinyl chloride exposed workers.
      ). Our study supports the hypothesis that N2,3-ϵG may contribute to the carcinogenesis of vinyl chloride and inflammation-driven malignancies (
      • Swenberg J.A.
      • Lu K.
      • Moeller B.C.
      • Gao L.
      • Upton P.B.
      • Nakamura J.
      • Starr T.B.
      Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment.
      ,
      • Nair U.
      • Bartsch H.
      • Nair J.
      Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: a review of published adduct types and levels in humans.
      ).

      Acknowledgments

      The use of the Advanced Photon Source and Life Sciences Collaborative Access Team Sector 21 was supported by United States Department of Energy Grant DE-AC02-06CH11357 and Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor Grant 085P1000817.

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