The Spacious Active Site of a Y-Family DNA Polymerase Facilitates Promiscuous Nucleotide Incorporation Opposite a Bulky Carcinogen-DNA Adduct

Y-family DNA polymerases lack some of the mechanisms that replicative DNA polymerases employ to ensure fidelity, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass certain aberrant bases, such as those produced by exposure to carcinogens, including benzo[a]pyrene (BP). A tumorigenic metabolite of BP, (+)-anti-benzo-[a]pyrene diol epoxide, attacks DNA to form the major 10S (+)-trans-anti-[BP]-N2-dG adduct, which has been shown to be mutagenic in a number of prokaryotic and eukaryotic systems. The 10S (+)-trans-anti-[BP]-N2-dG adduct can cause all three base substitution mutations, and the SOS response in Escherichia coli increases bypass of bulky adducts, suggesting that Y-family DNA polymerases are involved in the bypass of such lesions. Dpo4 belongs to the DinB branch of the Y-family, which also includes E. coli pol IV and eukaryotic pol κ. We carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in nucleotide incorporation opposite the bulky 10S (+)-trans-anti-[BP]-N2-dG adduct by Dpo4. Dpo4 is able to bypass the 10S (+)-trans-anti-[BP]-N2-dG adduct, albeit to a lesser extent than unmodified guanine, and the Vmax values for insertion of all four nucleotides opposite the adduct by Dpo4 are similar. Computational studies suggest that 10S (+)-trans-anti-[BP]-N2-dG can be accommodated in the active site of Dpo4 in either the anti or syn conformation due to the limited protein-DNA contacts and the open nature of both the minor and major groove sides of the nascent base pair, which can contribute to the promiscuous nucleotide incorporation opposite this lesion.

[1]  R. Woodgate,et al.  Snapshots of replication through an abasic lesion; structural basis for base substitutions and frameshifts. , 2004, Molecular cell.

[2]  Jun Yin,et al.  A role for DNA polymerase V in G --> T mutations from the major benzo[a]pyrene N2-dG adduct when studied in a 5'-TGT sequence in E. coli. , 2004, DNA repair.

[3]  D. Jerina,et al.  Crystal structure of a benzo[a]pyrene diol epoxide adduct in a ternary complex with a DNA polymerase. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[4]  T. Kunkel,et al.  The Efficiency and Specificity of Apurinic/Apyrimidinic Site Bypass by Human DNA Polymerase η and Sulfolobus solfataricus Dpo4* , 2003, Journal of Biological Chemistry.

[5]  R. Woodgate,et al.  Replication of a cis–syn thymine dimer at atomic resolution , 2003, Nature.

[6]  S. Broyde,et al.  Extending the understanding of mutagenicity: structural insights into primer-extension past a benzo[a]pyrene diol epoxide-DNA adduct. , 2003, Journal of molecular biology.

[7]  Yanbin Zhang,et al.  Effects of base sequence context on translesion synthesis past a bulky (+)-trans-anti-B[a]P-N2-dG lesion catalyzed by the Y-family polymerase pol kappa. , 2003, Biochemistry.

[8]  T. Kunkel,et al.  Functions of eukaryotic DNA polymerases. , 2003, Science of aging knowledge environment : SAGE KE.

[9]  Wei Yang Damage repair DNA polymerases Y. , 2003, Current opinion in structural biology.

[10]  D. Volk,et al.  Solution structure of a cis-opened (10R)-N6-deoxyadenosine adduct of (9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a DNA duplex. , 2003, Biochemistry.

[11]  E. G. Frank,et al.  polι-dependent lesion bypass in vitro , 2002 .

[12]  R. Fuchs,et al.  How DNA lesions are turned into mutations within cells? , 2002, Oncogene.

[13]  E. G. Frank,et al.  Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota. , 2002, Nucleic acids research.

[14]  R. Woodgate,et al.  Localization of DNA polymerases η and ι to the replication machinery is tightly co‐ordinated in human cells , 2002, The EMBO journal.

[15]  Y. Shinkai,et al.  Polκ protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  D. Jerina,et al.  Benzo[a]pyrene diol epoxide-deoxyguanosine adducts are accurately bypassed by yeast DNA polymerase zeta in vitro. , 2002, Mutation research.

[17]  S. Broyde,et al.  Toward understanding the mutagenicity of an environmental carcinogen: structural insights into nucleotide incorporation preferences. , 2002, Journal of molecular biology.

[18]  Yanbin Zhang,et al.  trans-Lesion Synthesis Past Bulky Benzo[a]pyrene Diol Epoxide N 2-dG and N 6-dA Lesions Catalyzed by DNA Bypass Polymerases* , 2002, The Journal of Biological Chemistry.

[19]  R. Fuchs,et al.  Lesion bypass in yeast cells: Pol η participates in a multi‐DNA polymerase process , 2002, The EMBO journal.

[20]  T. Kunkel,et al.  Low Fidelity DNA Synthesis by a Y Family DNA Polymerase Due to Misalignment in the Active Site* , 2002, The Journal of Biological Chemistry.

[21]  R. Woodgate,et al.  Structure-based interpretation of missense mutations in Y-family DNA polymerases and their implications for polymerase function and lesion bypass. , 2002, DNA repair.

[22]  A. Grollman,et al.  Translesion synthesis by human DNA polymerase kappa on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). , 2002, Biochemistry.

[23]  L. Blanco,et al.  Human DNA polymerase mu (Pol mu) exhibits an unusual replication slippage ability at AAF lesion. , 2002, Nucleic acids research.

[24]  D. Jerina,et al.  Preferential Misincorporation of Purine Nucleotides by Human DNA Polymerase η Opposite Benzo[a]pyrene 7,8-Diol 9,10-Epoxide Deoxyguanosine Adducts* , 2002, The Journal of Biological Chemistry.

[25]  L. Romano,et al.  Effects of benzo[a]pyrene adduct stereochemistry on downstream DNA replication in vitro: evidence for different adduct conformations within the active site of DNA polymerase I (Klenow fragment). , 2002, Biochemistry.

[26]  J. Wagner,et al.  Genetics of mutagenesis in E. coli: various combinations of translesion polymerases (Pol II, IV and V) deal with lesion/sequence context diversity. , 2002, DNA repair.

[27]  D. Jerina,et al.  Efficiency and Accuracy of SOS-induced DNA Polymerases Replicating Benzo[a]pyrene-7,8-diol 9,10-Epoxide A and G Adducts* , 2002, The Journal of Biological Chemistry.

[28]  R. Woodgate,et al.  The “tale” of UmuD and its role in SOS mutagenesis † , 2002, BioEssays : news and reviews in molecular, cellular and developmental biology.

[29]  E. Kool Active site tightness and substrate fit in DNA replication. , 2002, Annual review of biochemistry.

[30]  M. Goodman Error-prone repair DNA polymerases in prokaryotes and eukaryotes. , 2002, Annual review of biochemistry.

[31]  R. Woodgate,et al.  Unique misinsertion specificity of polι may decrease the mutagenic potential of deaminated cytosines , 2001, The EMBO journal.

[32]  R. Woodgate,et al.  Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic poleta. , 2001, Nucleic acids research.

[33]  R. Woodgate,et al.  Crystal Structure of a Y-Family DNA Polymerase in Action A Mechanism for Error-Prone and Lesion-Bypass Replication , 2001, Cell.

[34]  T. Kunkel,et al.  The Y-family of DNA polymerases. , 2001, Molecular cell.

[35]  S. Amin,et al.  Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I. , 2001, Biochemistry.

[36]  S. Broyde,et al.  Evading the proofreading machinery of a replicative DNA polymerase: induction of a mutation by an environmental carcinogen. , 2001, Journal of molecular biology.

[37]  Thomas A. Steitz,et al.  Structure of the Replicating Complex of a Pol α Family DNA Polymerase , 2001, Cell.

[38]  L. Dzantiev,et al.  Effects of benzo[a]pyrene DNA adducts on Escherichia coli DNA polymerase I (Klenow fragment) primer-template interactions: evidence for inhibition of the catalytically active ternary complex formation. , 2001, Biochemistry.

[39]  J. Wagner,et al.  All three SOS‐inducible DNA polymerases (Pol II, Pol IV and Pol V) are involved in induced mutagenesis , 2000, The EMBO journal.

[40]  Fenghua Yuan,et al.  Error-free and error-prone lesion bypass by human DNA polymerase κ in vitro , 2000 .

[41]  M. Goodman,et al.  The expanding polymerase universe , 2000, Nature Reviews Molecular Cell Biology.

[42]  R. Fuchs,et al.  The processing of a Benzo(a)pyrene adduct into a frameshift or a base substitution mutation requires a different set of genes in Escherichia coli , 2000, Molecular microbiology.

[43]  E. Kool,et al.  Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases. , 2000, Biochemistry.

[44]  T. Kunkel,et al.  Minor groove interactions at the DNA polymerase beta active site modulate single-base deletion error rates. , 2000, The Journal of biological chemistry.

[45]  S. Broyde,et al.  Conformational determinants of structures in stereoisomeric cis-opened anti-benzo[a]pyrene diol epoxide adducts to adenine in DNA. , 2000, Chemical research in toxicology.

[46]  L. Romano,et al.  In vitro replication of primer-templates containing benzo[a]pyrene adducts by exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment): effect of sequence context on lesion bypass. , 2000, Biochemistry.

[47]  K. Guckian,et al.  Mimicking the Structure and Function of DNA: Insights into DNA Stability and Replication. , 2000, Angewandte Chemie.

[48]  R. Fuchs,et al.  Lesions in DNA: hurdles for polymerases. , 2000, Trends in biochemical sciences.

[49]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[50]  S. Amin,et al.  Primer length dependence of binding of DNA polymerase I Klenow fragment to template-primer complexes containing site-specific bulky lesions. , 1999, Biochemistry.

[51]  R. Woodgate A plethora of lesion-replicating DNA polymerases. , 1999, Genes & development.

[52]  D. Phillips,et al.  Polycyclic aromatic hydrocarbons in the diet. , 1999, Mutation research.

[53]  T. Steitz DNA Polymerases: Structural Diversity and Common Mechanisms* , 1999, The Journal of Biological Chemistry.

[54]  S. Broyde,et al.  Stereochemical origin of opposite orientations in DNA adducts derived from enantiomeric anti-benzo[a]pyrene diol epoxides with different tumorigenic potentials. , 1999, Biochemistry.

[55]  M. Sawaya,et al.  An open and closed case for all polymerases. , 1999, Structure.

[56]  E. Kool,et al.  Minor Groove Interactions between Polymerase and DNA: More Essential to Replication than Watson-Crick Hydrogen Bonds? , 1999, Journal of the American Chemical Society.

[57]  P. Kollman,et al.  A modified version of the Cornell et al. force field with improved sugar pucker phases and helical repeat. , 1999, Journal of biomolecular structure & dynamics.

[58]  N. Geacintov,et al.  The major, N2-dG adduct of (+)-anti-B[a]PDE induces G-->A mutations in a 5'-AGA-3' sequence context. , 1999, Carcinogenesis.

[59]  Gabriel Waksman,et al.  Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation , 1998, The EMBO journal.

[60]  S. Doublié,et al.  The mechanism of action of T7 DNA polymerase. , 1998, Current opinion in structural biology.

[61]  G L Verdine,et al.  Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. , 1998, Science.

[62]  D. Jerina,et al.  Effect of single benzo[a]pyrene diol epoxide-deoxyguanosine adducts on the action of DNA polymerases in vitro. , 1998, International journal of oncology.

[63]  S. Amin,et al.  Mutagenic potential of stereoisomeric bay region (+)- and (-)-cis-anti-benzo[a]pyrene diol epoxide-N2-2'-deoxyguanosine adducts in Escherichia coli and simian kidney cells. , 1998, Biochemistry.

[64]  S. Harvey,et al.  The flying ice cube: Velocity rescaling in molecular dynamics leads to violation of energy equipartition , 1998, J. Comput. Chem..

[65]  S. Amin,et al.  Role of hydrophobic effects in the reaction of a polynuclear aromatic diol epoxide with oligodeoxynucleotides in aqueous solutions. , 1998, Chemical research in toxicology.

[66]  M. Tang,et al.  Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers , 1998, Oncogene.

[67]  S. Doublié,et al.  Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution , 1998, Nature.

[68]  James R. Kiefer,et al.  Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal , 1998, Nature.

[69]  E. Kool Replication of non‐hydrogen bonded bases by DNA polymerases: A mechanism for steric matching , 1998, Biopolymers.

[70]  D. Patel,et al.  Structural alignment of the (+)-trans-anti-benzo[a]pyrene-dG adduct positioned opposite dC at a DNA template-primer junction. , 1997, Biochemistry.

[71]  N. Geacintov,et al.  How stereochemistry affects mutagenesis by N2-deoxyguanosine adducts of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene: configuration of the adduct bond is more important than those of the hydroxyl groups. , 1997, Biochemistry.

[72]  Samuel H. Wilson,et al.  Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism. , 1997, Biochemistry.

[73]  N. Geacintov,et al.  The major, N2-dG adduct of (+)-anti-B[a]PDE shows a dramatically different mutagenic specificity (predominantly, G --> A) in a 5'-CGT-3' sequence context. , 1997, Biochemistry.

[74]  Mihaly Mezei,et al.  Optimal position of solute for simulations , 1997, J. Comput. Chem..

[75]  N. Geacintov,et al.  Sequence specific mutagenesis of the major (+)-anti-benzo[a]pyrene diol epoxide-DNA adduct at a mutational hot spot in vitro and in Escherichia coli cells. , 1997, Chemical research in toxicology.

[76]  D. Patel,et al.  NMR solution structures of stereoisometric covalent polycyclic aromatic carcinogen-DNA adduct: principles, patterns, and diversity. , 1997, Chemical research in toxicology.

[77]  G. Grimmer,et al.  Determination of urinary metabolites of polycyclic aromatic hydrocarbons (PAH) for the risk assessment of PAH-exposed workers , 1997, International archives of occupational and environmental health.

[78]  S. Spiegel,et al.  Fidelity of translesional synthesis past benzo[a]pyrene diol epoxide-2'-deoxyguanosine DNA adducts: marked effects of host cell, sequence context, and chirality. , 1996, Biochemistry.

[79]  M. Tang,et al.  Preferential Formation of Benzo[a]pyrene Adducts at Lung Cancer Mutational Hotspots in P53 , 1996, Science.

[80]  J. Kraut,et al.  Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity. , 1996, Biochemistry.

[81]  R. Weinberg,et al.  How cancer arises. , 1996, Scientific American.

[82]  Thomas A. Steitz,et al.  Structure of Taq polymerase with DNA at the polymerase active site , 1996, Nature.

[83]  D. Patel,et al.  Structural alignments of (+)- and (-)-trans-anti-benzo[a]pyrene-dG adducts positioned at a DNA template-primer junction. , 1995, Biochemistry.

[84]  N. Geacintov,et al.  The major, N2-Gua adduct of the (+)-anti-benzo[a]pyrene diol epoxide is capable of inducing G-->A and G-->C, in addition to G-->T, mutations. , 1995, Biochemistry.

[85]  Peter A. Kollman,et al.  AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules , 1995 .

[86]  P. Kollman,et al.  A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules , 1995 .

[87]  T. Krugh,et al.  Structural characterization of a (+)-trans-anti-benzo[a]pyrene-DNA adduct using NMR, restrained energy minimization, and molecular dynamics. , 1995, Biochemistry.

[88]  N. Geacintov,et al.  Synthesis and characterization of covalent adducts derived from the binding of benzo[a]pyrene diol expoxide to a -GGG- sequence in a deoxyoligonucleotide. , 1995, Carcinogenesis.

[89]  E. Loechler,et al.  Are base substitution and frameshift mutagenesis pathways interrelated? An analysis based upon studies of the frequencies and specificities of mutations induced by the (+)-anti diol epoxide of benzo[a]pyrene. , 1995, Mutation research.

[90]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

[91]  A. Grollman,et al.  Translesional synthesis on DNA templates containing 8-oxo-7,8-dihydrodeoxyadenosine. , 1993, Biochemistry.

[92]  E. Loechler,et al.  Mutational specificity of the (+)-anti-diol epoxide of benzo[a]pyrene in a supF gene of an Escherichia coli plasmid: DNA sequence context influences hotspots, mutagenic specificity and the extent of SOS enhancement of mutagenesis. , 1993, Carcinogenesis.

[93]  E. Loechler,et al.  Mutagenesis by the (+)-anti-diol epoxide of benzo[a]pyrene: what controls mutagenic specificity? , 1993, Biochemistry.

[94]  P. Liska,et al.  Trace enrichment and HPLC analysis of PAHs in edible oils and fat products, using liquid chromatography on electron acceptor stationary phases in connection with reverse phase and fluorescence detection , 1993 .

[95]  K. Peltonen,et al.  DNA polymerase action on benzo[a]pyrene-DNA adducts. , 1992, Carcinogenesis.

[96]  M. Benasutti,et al.  Mutagenesis by (+)-anti-B[a]P-N2-Gua, the major adduct of activated benzo[a]pyrene, when studied in an Escherichia coli plasmid using site-directed methods. , 1992, Carcinogenesis.

[97]  D. Patel,et al.  Solution conformation of the major adduct between the carcinogen (+)-anti-benzo[a]pyrene diol epoxide and DNA. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[98]  T. Kunkel,et al.  DNA replication fidelity. , 1992, The Journal of biological chemistry.

[99]  N. Geacintov,et al.  Spectroscopic characteristics and site I/site II classification of cis and trans benzo[a]pyrene diolepoxide enantiomer-guanosine adducts in oligonucleotides and polynucleotides. , 1991, Carcinogenesis.

[100]  J. Timm,et al.  Relevance of polycyclic aromatic hydrocarbons as environmental carcinogens , 1991 .

[101]  J. M. Roman,et al.  DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide. , 1989, Chemical research in toxicology.

[102]  R. H. Ritchie,et al.  Dielectric effects in biopolymers: The theory of ionic saturation revisited , 1985 .

[103]  M. L. Connolly Solvent-accessible surfaces of proteins and nucleic acids. , 1983, Science.

[104]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[105]  A. Conney,et al.  Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. , 1982, Cancer research.

[106]  K. Straub,et al.  Double-stranded DNA stereoselectively binds benzo(a)pyrene diol epoxides , 1979, Nature.

[107]  D. Jerina,et al.  Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides in newborn mice: exceptional activity of (+)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[108]  J. W. Cook,et al.  106. The isolation of a cancer-producing hydrocarbon from coal tar. Parts I, II, and III , 1933 .