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ABSTRACT: Polycyclic aromatic hydrocarbons (PAHs) are environmental carcinogens whose metabolites can react with DNA to form bulky DNA
adducts. We focus on the well-studied planar bay-region PAH benzo[a]pyrene (B[a]P) and on the twisted, fjord-region PAHs dibenzo[a,l]pyrene (DB[a,l]P), benzo[g]chrysene (B[g]C), and benzo[c]phenanthrene (B[c]Ph). The unusually potent tumorigenicity of the fjord-region carcinogens, particularly DB[a,l]P, is noted. DNA adducts derived from the selected prototypes are then described. Mutagenic properties of these lesions are
briefly considered as anchors for their connection to cancer initiation. We next describe structural characteristics of the
bulky adducts as determined in solution by NMR methods and computational treatments. Structure–function relationships are
subsequently discussed. We connect solution structures to observed relative susceptibilities to nucleotide excision repair
(NER) with human HeLa cell extracts, as NER is the fundamental defense against bulky adducts in humans. Processing of the
bulky lesions by DNA polymerases in connection with lesion mutagenicity is also considered. Finally, we offer perspectives
concerning adduct structures in relation to cancer prevention and treatment, and the need for deeper understanding of the
processing of structurally different lesions on cellular and systems biology levels.
03/2011: pages 181-207;
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ABSTRACT: Bypass across DNA lesions by specialized polymerases is essential for maintenance of genomic stability. Human DNA polymerase iota (poliota) is a bypass polymerase of the Y family. Crystal structures of poliota suggest that Hoogsteen base pairing is employed to bypass minor groove DNA lesions, placing them on the spacious major groove side of the enzyme. Primer extension studies have shown that poliota is also capable of error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF). We present molecular dynamics simulations and free energy calculations suggesting that Watson-Crick base pairing could be employed in poliota for bypass of dG-AAF. In poliota with Hoogsteen-paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for error-free incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick-paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Thus our results suggest that poliota would utilize the same spacious region for lesion bypass of both major and minor groove adducts. Therefore, purine adducts with bulk on the minor groove side would use Hoogsteen pairing, while adducts with the bulky lesion on the major groove side would utilize Watson-Crick base pairing as indicated by our MD simulations for dG-AAF. This suggests the possibility of an expanded role for poliota in lesion bypass.
Biochemistry 01/2009; 48(1):7-18. · 3.42 Impact Factor
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ABSTRACT: Equilin and equilenin, components of the hormone replacement therapy drug Premarin, can be metabolized to the catechol 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dC, dA, and dG to form unusual stable cyclic adducts, which have been found in human breast tumor tissue. Four stereoisomeric adducts have been identified for each base. These 12 Premarin-derived adducts provide a unique opportunity for analyzing effects of stereochemistry and base damage on DNA structure and consequently its function. Our computational studies have shown that these adducts, with obstructed Watson-Crick hydrogen-bond edges and near-perpendicular ring systems, have limited conformational flexibility and near-mirror-image conformations in stereoisomer pairs. The dC and dA adducts can adopt major- and minor-groove positions in the double helix, but the dG adducts are positioned only in the major groove. In all cases, opposite orientations of the equilenin rings with respect to the 5' --> 3' direction of the damaged strand are found in stereoisomer pairs derived from the same base, and no Watson-Crick pairing is possible. However, detailed structural properties in DNA duplexes are distinct for each stereoisomer of each damaged base. These differences may underlie observed differential stereoisomer and base-dependent mutagenicities and repair susceptibilities of these adducts.
Chemical Research in Toxicology 05/2008; 21(5):1064-73. · 3.78 Impact Factor
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ABSTRACT: The equine estrogens, equilin and equilenin, are major components of the drug Premarin, the most widely used formula for hormone replacement therapy. The derivative 4-hydroxyequilenin (4-OHEN), a major phase I metabolite of equilin and equilenin, autoxidizes to potent cytotoxic quinoids that can react in vitro and in vivo with cytosine and adenine in DNA. Unique cyclic adducts containing the same bicyclo[3.3.1]nonane-type connection ring are produced. Each base adduct has four stereoisomers. In order to elucidate the structural effects of A versus C modification, we have carried out molecular dynamics simulations of the stereoisomeric 4-OHEN-A adducts in DNA 11-mer duplexes and compared results with an earlier study of the C adducts (Ding, S., Shapiro, R., Geacintov, N.E., and Broyde, S. (2005) Equilenin-Derived DNA Adducts to Cytosine in DNA Duplexes: Structures and Thermodynamics, Biochemistry 44, 14565-14576). Similar stereochemical principles govern the orientations in DNA duplexes of the 4-OHEN-A adducts as for the analogous C adducts, with opposite orientations of the equilenin rings in stereoisomeric pairs of adducts characterized by near-mirror image circular dichroism (CD) spectra. However, the larger purine adducts have unique structural properties in the duplexes that distinguish their characteristics from those of the pyrimidine adducts. Significant differences are observed in terms of hydrogen bonding, stacking, bending, groove dimensions, solvent exposure, and hydrophobic interactions; also, each of the four stereoisomeric 4-OHEN-A adducts exhibit distinct structural features. Each base adduct and stereoisomer distorts the structure of the DNA duplex differently. These characteristics may manifest themselves in terms of differential nucleotide excision repair susceptibilities and mutagenic activities of the 4-OHEN-A and C adducts.
Biochemistry 02/2007; 46(1):182-91. · 3.42 Impact Factor
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ABSTRACT: 5-Guanidino-4-nitroimidazole (NI), derived from guanine oxidation by reactive oxygen and nitrogen species, contains an unusual flexible ring-opened structure, with nitro and guanidino groups which possess multiple hydrogen bonding capabilities. In vitro primer extension experiments with bacterial and mammalian polymerases show that NI incorporates C as well as A and G opposite the lesion, depending on the polymerase. To elucidate structural and thermodynamic properties of the mutagenic NI lesion, we have investigated the structure of the modified base itself and the NI-containing nucleoside with high-level quantum mechanical calculations and have employed molecular modeling and molecular dynamics simulations in solution for the lesion in B-DNA duplexes, with four partner bases opposite the NI. Our results show that NI adopts a planar structure at the damaged base level. However, in the nucleoside and in DNA duplexes, steric hindrance between the guanidino group and its linked sugar causes NI to be nonplanar. The NI lesion can adopt both syn and anti conformations on the DNA duplex level, with the guanidino group positioned in the DNA major and minor grooves, respectively; the specific preference depends on the partner base. On the basis of hydrogen bonding and stacking interactions, groove dimensions, and bending, we find that the least distorted NI-modified duplex contains partner C, consistent with observed incorporation of C opposite NI. However, hydrogen bonding interactions between NI and partner G or A are also found, which would be compatible with the observed mismatches.
Biochemistry 06/2006; 45(21):6644-55. · 3.42 Impact Factor
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ABSTRACT: Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics (MD) simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP adduct enhances incorporation of these mismatches. Finally, bulky C8-dG adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N2-dG adducts, which can reside on the minor groove side, appear to cause less hindrance when in this position.
Nucleic Acids Research 02/2006; 34(11):3326-37. · 8.03 Impact Factor
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ABSTRACT: The drug Premarin is the most widely used formula for hormone replacement therapy. However, long-term exposure to estrogens from the Premarin drug increases the risk of breast cancer. Equilin and equilenin, major components of Premarin, are predominantly metabolized to 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form unusual stable cyclic bulky adducts, with four stereoisomers identified for each base adduct. The 4-OHEN-dC adducts are most predominant. They are mutagenic in vitro and have been found in human tumor tissue. We have carried out molecular modeling and molecular dynamics simulations to investigate structures and thermodynamics of the four 4-OHEN-dC stereoisomeric adducts in DNA duplexes. Our results show that the structure of each stereoisomer adduct in duplex DNA is specifically governed by its unique stereochemistry. The bulky adducts, with an obstructed Watson-Crick edge and an equilenin ring system near perpendicular to the damaged cytosine, are located in the B-DNA major or minor groove, with the modified cytosine in the syn or anti conformation, respectively. The DNA duplex structures are distorted, in terms of Watson-Crick pairing at and near the lesion, stacking interactions, and groove dimensions. Stereochemistry determines the orientation of the equilenin rings with respect to the 5'- to 3'-direction of the modified strand, as well as the positioning of the equilenin moiety's methyl and hydroxyl groups for each stereoisomer. The unusual structures and the stereochemical effects underlie their biological processing as miscoding DNA lesions whose mutagenic properties may contribute to breast cancer.
Biochemistry 12/2005; 44(44):14565-76. · 3.42 Impact Factor
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ABSTRACT: Oxidation of guanine or 8-oxo-7,8-dihydroguanine can produce spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G --> C and G --> T transversion mutations. Therefore, they are of interest as potential endogenous cancer causing lesions. However, their structural properties in DNA duplexes remain to be elucidated. We have employed computational methods to study the Sp lesions in 11-mer DNA duplexes with A, C, G, and T partners. Molecular dynamics simulations have been carried out to obtain ensembles of structures, and the trajectories were employed to analyze the structures and compute free energies. The structural and thermodynamic analyses reveal that the Sp stereoisomers energetically favor positioning in the B-DNA major groove, with minor groove conformers also low energy in some cases, depending on the partner base. The R and S stereoisomers adopt opposite orientations with respect to the 5' to 3' direction of the modified strand. Both syn and anti glycosidic bond conformations are energetically feasible, with partner base and stereochemistry determining the preference. The lesions adversely impact base stacking and Watson-Crick hydrogen bonding interactions in the duplex, and cause groove widening. The chemical nature of the partner base determines specific hydrogen bonding and stacking properties of the damaged duplexes. The structural characteristics may relate to observed mutagenic properties of the Sp stereoisomers, including possible stereoisomer-dependent differences.
Biochemistry 10/2005; 44(40):13342-53. · 3.42 Impact Factor
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ABSTRACT: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of the carcinogenic heterocyclic aromatic amines in the human diet, and the major mutagenic effect of dietary PhIP is G-->T transversions. The major PhIP-derived DNA adduct is to C8 of guanine. We have investigated this adduct in a PhIP-induced mutational hotspot 5'-GGGA-3' of the Apc tumor suppressor gene, frequently mutated in mammalian colon tumors. We have carried out a molecular dynamics study to elucidate on a structural level nucleotide incorporation and extension opposite this major adduct during replication. The PhIP adduct was modeled into the ternary complex closed conformation of DNA polymerase RB69, at incorporation and extension positions, with normal cytosine or mismatched partner adenine. RB69 polymerase is a member of the B family as are most replicative eukaryotic DNA polymerases such as DNA polymerase alpha. These systems were subjected to molecular dynamics simulations with AMBER. Our results show that the adduct can reside on the major groove side of the modified DNA template opposite an incoming dCTP or dATP. In the case of the normal partner, disturbance to the active site is observed at the incorporation step, but there is less perturbance in the extension simulation. In the case of the mismatched partner, a less disturbed active site is observed during the incorporation step, but extension appears to be more difficult. Disturbances include adverse impacts on Watson-Crick hydrogen bonding in the nascent base pair, on the distance between the alpha-phosphate of the incoming dNTP and the primer terminus 3'-OH, and on critical protein interactions with the dNTP. However, in all of these cases, a near reaction ready distance (within 3.5 angstroms) between the 3'-terminal oxygen of the primer and the Palpha of the incoming nucleotide triphosphate is sampled occasionally (0.4-23.5% of the time). Thus, error-free bypass or the induction of a G-->T transversion mutation could occur at times and contribute to an extent to the mutagenic effect of PhIP. Polymerase stalling would be the more common outcome and in vivo could lead to switch to an error-prone bypass polymerase.
Chemical Research in Toxicology 09/2005; 18(9):1347-63. · 3.78 Impact Factor
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ABSTRACT: Reactive oxygen species present in the cell generate DNA damage. One of the major oxidation products of guanine in DNA, 8-oxo-7,8-dihydroguanine, formed by loss of two electrons, is among the most extensively studied base lesions. The further removal of two electrons from this product can yield spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G --> T and G --> C transversions. Hence, they are of interest as examples of endogenous DNA damage that may initiate cancer. To interpret the mutagenic properties of the Sp lesions, an understanding of their structural properties is needed. To elucidate these structural effects, we have carried out computational investigations at the level of the Sp-modified base and nucleoside. At the base level, quantum mechanical geometry optimization studies have revealed exact mirror image symmetry of the R and S stereoisomers, with a near-perpendicular geometry of the two rings. At the nucleoside level, an extensive survey of the potential energy surface by molecular mechanics calculations using AMBER has provided three-dimensional potential energy maps. These maps reveal that the range and flexibility of the glycosidic torsion angles are significantly more restricted in both stereoisomeric adducts than in unmodified 2'-deoxyguanosine. The structural and energetic results suggest that the unusual geometric, steric, and hydrogen bonding properties of these lesions underlie their mutagenicity. In addition, stereoisomer-specific differences indicate the possibility that their processing by cellular replication and repair enzymes may be differentially affected by their absolute configuration.
Biochemistry 04/2005; 44(16):6043-51. · 3.42 Impact Factor
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ABSTRACT: An extensive conformational analysis has been carried out for two diastereoisomeric pairs of model estrogen quinone-derived DNA adducts, N6-(2-hydroxyestron-6(alpha,beta)-yl)-2'-deoxyadenosine (2-OHE1-6(alpha,beta)-N6-dA) and N2-(2-hydroxyestron-6(alpha,beta)-yl)-2'-deoxyguanosine (2-OHE1-6(alpha,beta)-N2-dG), in a B-DNA duplex and at a primer-template junction in a pol alpha family DNA polymerase. In vitro primer extension studies in pol alpha [Terashima, I., et al. (1998) Biochemistry 37, 13807-13815] have shown that the adenine adducts can incorporate dT, together with a small proportion of the incorrect base dC opposite the lesion, and they block less strongly than the guanine adducts. We have carried out conformational searches with energy minimization for four DNA duplexes containing 2-OHE1-6alpha-N6-dA, 2-OHE1-6beta-N6-dA, 2-OHE1-6alpha-N2-dG, or 2-OHE1-6beta-N2-dG. Our searches revealed that the four-ring nonplanar 2-hydroxyestrone (2-OHE1) moiety strongly prefers to reside in the major groove of the adenine adducts or the minor groove of the guanine adducts in a B-DNA duplex, with stereochemistry-dependent orientational differences in each case. No low energy conformations involving intercalation of the 2-OHE1 moiety were located in the searches. This stems from the largely nonplanar, nonaromatic nature of the 2-OHE1 ring system and implies that the proclivity for such bulky, nonplanar adducts to reside at the DNA helix exterior is a plausible conformational feature of other structurally similar estrogen quinone-derived DNA adducts, independent of base sequence context. In addition, the adenine adduct isomers, located in the major groove, manifest serious disturbance to the Watson-Crick base pairs at and near the lesion site, suggesting repair susceptibility. Possible structures of these adducts in a pol alpha family polymerase were also investigated through molecular modeling. The results rationalized the experimental in vitro primer extension studies. In addition, poor accommodation of the beta-stereoisomers within the polymerase was noted, suggesting that these stereoisomers would be more prone to cause blockage. Stereochemistry-dependent differences in adduct orientation could be expected to produce different biochemical effects, as has been observed in adducts derived from polycyclic aromatic hydrocarbons.
Chemical Research in Toxicology 04/2004; 17(3):311-24. · 3.78 Impact Factor
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ABSTRACT: Exposure to estrogen through estrogen replacement therapy increases the risk of women developing cancer in hormone sensitive tissues. Premarin (Wyeth), which has been the most frequent choice for estrogen replacement therapy in the United States, contains the equine estrogens equilin and equilenin as major components. 4-Hydroxyequilenin (4-OHEN) is a phase I metabolite of both of these substances. This catechol estrogen autoxidizes to potent cytotoxic quinoids that can react with dG, dA, and dC to form unusual stereoisomeric cyclic adducts (Bolton, J. L., et al. (1998) Chem. Res. Toxicol. 11, 1113-1127). Like other bulky DNA adducts, these lesions may exhibit different susceptibilities to DNA repair and mutagenic potential, if not repaired in a structure-dependent manner. To ultimately gain insights into structure-function relationships, we computed conformations of stereoisomeric guanine, adenine, and cytosine base adducts using density functional theory. We find near mirror image conformations in stereoisomer adduct pairs for each modified base, suggesting opposite orientations with respect to the 5' --> 3' direction of the modified strand when the stereoisomer pairs are incorporated into duplex DNA. Such opposite orientations could cause stereoisomer pairs of lesions to respond differently to DNA replication and repair enzymes.
Chemical Research in Toxicology 06/2003; 16(6):695-707. · 3.78 Impact Factor
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ABSTRACT: An oligodeoxyribonucleotide 5' -d(CTCATGAPATTCC), in which GAP denotes N -(guanin-8-yl)-1-aminopyrene, the C8-guanine adduct of reductively activated 1-nitropyrene, was synthesized and characterized by polyacrylamide gel electrophoresis, absorption and fluorescence spectroscopy, circular dichroism, and thermal melting studies. Polyacrylamide gel electrophoresis showed slower mobility of the adducted oligonucleotide in single-stranded form compared to its unmodified counterpart, as expected. In duplex form, however (with a deoxycytidine opposite the adduct), the adducted 11mer migrated faster than the parent duplex. Absorption and fluorescence studies indicated significant interaction of the aminopyrene residue with the DNA bases in the modified 11mer. The spectroscopic data also suggested the presence of one or more conformers in which the aminopyrene residue is quasi-intercalative, as well as one(s) in which the aminopyrene is externally bound. Thermo-dynamic parameters for the helix-to-coil transitions for the 11mer duplex were determined. The difference in free energy (δδG°) between the unmodified and modified sequences was relatively small (∼1.2 kcal/mol). Circular dichroism spectra indicated the presence of essentially B-form DNA. The energy minimizations suggested that the most stable conformers shared a common feature: displacement of the modified guanine from the double helix. In the global minimum, the aminopyrene residue was inserted in the helix in the site of displaced guanine. In other low energy structures, the aminopyrene was also displaced towards the minor groove (in addition to guanine), or partly inserted and partly in the groove. More conventional structures were also encountered, with anti -guanine within the helix and aminopyrene in the major groove, or syn -guanine within the helix, and aminopyrene in the minor groove. Such structures were 12–20 kcal/mol less stable than the global minimum, however. The C8-guanine adduct of aminopyrene thus appears to perturb the B-DNA structure to a greater extent than do the adducts of less bulky amines such as aminofluorene and 4-aminobiphenyl.
