Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40-400 microM AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0. 1-4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.     

A viral genome containing an unstable aflatoxin B1-N7-guanine DNA adduct situated at a unique site.

A problem that has hindered the study of the biological properties of certain DNA adducts, such as those that form at the N7 atoms of purines, is their extreme chemical lability. Conditions are described for the construction of a single-stranded genome containing the chemically and thermally labile 8,9-dihydro-8- (N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct, the major DNA adduct of the potent liver carcinogen aflatoxin B1 (AFB1). A 13mer oligonucleotide, d(CCTCTTCGAACTC), was allowed to react with the exo-8,9-epoxide of AFB1 to form an oligonucleotide containing a single AFB1-N7-Gua (at the underlined guanine). This modified 13mer was 5'-phosphorylated and ligated into a gap in an M13 bacteriophage genome generated by annealing a 53mer uracil-containing scaffold to M13mp7L2 linearized by EcoRI. Following ligation, the scaffold was enzymatically removed with uracil DNA glycosylase and exonuclease III. The entire genome construction was complete within 3 h and was carried out at 16 degrees C, pH 6.6, conditions determined to be optimal for AFB1-N7-Gua stability. Characterization procedures indicated that the AFB1-N7-Gua genome was approximately 95% pure with a small (5%) contamination by unmodified genome. This construction scheme should be applicable to other chemically or thermally unstable DNA adducts.     

Mispairing of the 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 adduct with deoxyadenosine results in extrusion of the mismatched dA toward the major groove

The G --> T transversion is the dominant mutation induced by the cationic trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B(1) adduct. The structure of d(ACATC(AFB)GATCT).d(AGATAGATGT), in which the cationic adduct was mismatched with deoxyadenosine, was refined using molecular dynamics calculations restrained by NOE data and dihedral restraints obtained from NMR spectroscopy. Restrained molecular dynamics calculations refined structures with pairwise rmsd <1 A and a sixth root R1x factor between the refined structure and NOE data of 10.5 x 10-2. The mismatched duplex existed in a single conformation at neutral pH. The aflatoxin moiety intercalated above the 5' face of the modified (AFB)G. The mismatched dA was in the anti conformation about the glycosyl bond. It extruded toward the major groove and did not participate in hydrogen bonding with (AFB)G. The structure was compared with that of d(ACATCGATCT).d(AGATAGATGT) containing the corresponding unmodified G.A mismatch and with d(ACATC(AFB)GATCT).d(AGATCGATGT) containing the aflatoxin lesion in the correctly paired (AFB)G.C context. The correctly paired oligodeoxynucleotide exhibited Watson-Crick-type geometry at the (AFB)G.C pair. It melted at higher temperature than the mismatched (AFB)G.A duplex. The unmodified mismatched G.A duplex exhibited spectral line broadening at neutral pH, suggesting a mixture of conformations. It exhibited a lower melting temperature than did the mismatched (AFB)G.A duplex. These differences correlated with replication bypass experiments performed in vitro utilizing DNA polymerase I exo- [Johnston, D. S., and Stone, M. P. (2000) Chem. Res. Toxicol. 13, 1158-1164]. Those experiments showed that correct insertion of dC opposite (AFB)G blocked replication by the enzyme, whereas incorrect insertion of dA opposite (AFB)G allowed full-length replication of the adducted template strand.     

Studies of the recognition sequence of phi X174 gene A protein. Cleavage site of phi X gene A protein in St-1 RFI DNA.

It is already known that phi X gene A protein converts besides phi X RFI DNA also the RFI DNAs of the single-stranded bacteriophages G4, St-1, alpha 3 and phi K into RFII DNA. We have extended this observations for bacteriophages G14 and U3. Restriction enzyme analysis placed the phi X gene A protein cleavage site in St-1 RF DNA in the HinfI restriction DNA fragment F10 and in the overlapping HaeIII restriction DNA fragment Z7. The exact position and the nucleotide sequence at the 3'-OH end of the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment F10 obtained by gelelectrophoresis in denaturing conditions. A stretch of 85 nucleotides of St-1 DNA around the position of the phi X gene A protein cleavage site was established by DNA sequence analysis of the restriction DNA fragment Z7F1. Comparison of this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of phi X gene A protein in phi X174 RF DNA and G4 RF DNA revealed an identical sequence of only 10 nucleotides. The results suggest that the recognition sequence of the phi X174 gene A protein lies within these 10 nucleotides.     

Protection of DNA sequences by triplex-bridge formation.

We have demonstrated that the DNA sequence between two triplex-forming polypurine.polypyrimidine (Pu.Py) tracts was protected from DNA modifying enzymes upon formation of triplex DNA structures with an oligodeoxyribonucleotide in which two triplex-forming Pu or Py tracts were placed at the termini (triplex-bridge formation). In model experiments, when two triplex structures were formed between double-stranded DNA with the sequence (AG)17-(N)18-(T)34, and an oligodeoxyribonucleotide, (T)34-(N)18-(GA)17, not only the Pu.Py tracts but also the 18 bp non-Pu.Py sequence in the duplex DNA between the tracts was protected from restriction enzymes, HpaII methylase and DNase I. This protection occurred only when both of the Pu.Py tracts were involved as triplexes. The length of the tracts could be as short as 21 bp, while the difference in length between the non-Pu.Py sequences on the duplex and the oligodeoxyribonucleotide should be within 10 nucleotides. The efficiency of protection was enhanced in the presence of a cationic detergent, cetyltrimethylammonium bromide, during triplex formation. Protection was also observed with another type of the triplex bridge formed between (G)34 and (T)34 tracts with an oligodeoxyribonucleotide, (T)34-(N)20-(G)34. These findings suggest that the protection of specific DNA sequences from enzymes by triplex-bridge formation can be applied to any DNA sequence by placing it between two triplex-forming sequences.     

Mapping the binding site of aflatoxin B1 in DNA: systematic analysis of the reactivity of aflatoxin B1 with guanines in different DNA sequences

The mutagenic and carcinogenic chemical aflatoxin B1 (AFB1) reacts almost exclusively at the N(7)-position of guanine following activation to its reactive form, the 8,9-epoxide (AFB1 oxide). In general N(7)-guanine adducts yield DNA strand breaks when heated in base, a property that serves as the basis for the Maxam-Gilbert DNA sequencing reaction specific for guanine. Using DNA sequencing methods, other workers have shown that AFB1 oxide gives strand breaks at positions of guanines; however, the guanine bands varied in intensity. This phenomenon has been used to infer that AFB1 oxide prefers to react with guanines in some sequence contexts more than in others and has been referred to as "sequence specificity of binding". Herein, data on the reaction of AFB1 oxide with several synthetic DNA polymers with different sequences are presented, and (following hydrolysis) adduct levels are determined by high-pressure liquid chromatography. These results reveal that for AFB1 oxide (1) the N(7)-guanine adduct is the major adduct found in all of the DNA polymers, (2) adduct levels vary in different sequences, and, thus, sequence specificity is also observed by this more direct method, and (3) the intensity of bands in DNA sequencing gels is likely to reflect adduct levels formed at the N(7)-position of guanine. Knowing this, a reinvestigation of the reactivity of guanines in different DNA sequences using DNA sequencing methods was undertaken. The reactivities of 190 guanines were determined quantitatively and considered in a pentanucleotide context, 5'-WXGYZ-3', where the central, underlined G represents the reactive guanine and W, X, Y, and Z can be any of the nucleotide bases. Methods are developed to determine that the X (5'-side) base and the Y (3'-side) base are most influential in determining guanine reactivity. The influence of the bases in the 5'-position (X) is 5'-G (1.0) greater than C (0.8) greater than A (0.3) greater than T (0.2), while the influence of the bases in the 3'-position (Y) is 3'-G (1.0) greater than T (0.8) greater than C (0.4) greater than A (0.3). These rules in conjunction with molecular modeling studies (to be published elsewhere) were used to assess the binding sites that might be utilized by AFB1 oxide in its reaction with DNA.     

Protonated pyrimidine-purine-purine triplex.

We have studied a protonated pyrimidine-purine-purine (Py-Pu-Pu) triplex, which is formed between the d(C)nd(G)n duplex and the d(AG)m oligonucleotide as the third strand and carries the CG*A+ protonated base-triads. We have observed such an intermolecular complex between a plasmid carrying the d(C)18 d(G)18 insert and the d(AG)5 oligonucleotide without bivalent cations in 200 mM of Na+ at pH4.0. Bivalent cations additionally stabilize the complex. We propose the structures for nearly isomorphous base-triads TA*A, CG*G and CG*A+. To identify the H-DNA-like structure, which includes the triplex between d(C)n d(G)n duplex and the AG-strand, we have cloned in a superhelical plasmid the insert: G10TTAA(AG)5. The data on photofootprinting and chemical modification with diethyl pyrocarbonate, potassium permanganate and dimethyl sulfate demonstrate that the H-like structure with triplex carrying CG*G and CG*A+ base triads is actually formed under acid conditions. In the course of this study we have come across unexpected results on probing of Py-Pu-Pu triplexes by dimethyl sulfate (DMS): the protection effect is observed not only for guanines entering the duplex but also for guanines in the third strand lying in the major groove. We have demonstrated this effect not only for the case the novel protonated Py-Pu-Pu triplex but also for the traditional non-protonated Py-Pu-Pu intramolecular triplex (H*-DNA) formed by the d(C)37 d(G)37 insert in supercoiled plasmid in the presence of Mg2+ ions.     

Development of aflatoxin B(1)-lysine adduct monoclonal antibody for human exposure studies

Mouse monoclonal antibodies were developed against a synthetic aflatoxin B(1) (AFB)-lysine-cationized bovine serum albumin conjugate. The isotype of one of these antibodies, IIA4B3, has been classified as immunoglobulin G1(lambda). The affinity and specificity of IIA4B3 were further characterized by a competitive radioimmunoassay. The affinities of IIA4B3 for AFB and its associated adducts and metabolites are ranked as follows: AFB-lysine > 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB > AFB = 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB > aflatoxin M(1) > aflatoxin Q(1). IIA4B3 had about a 10-fold higher affinity for binding to AFB-lysine adduct than to AFB when (3)H-AFB-lysine was used as the tracer. The concentration for 50% inhibition for AFB-lysine was 0.610 pmol; that for AFB was 6.85 pmol. IIA4B3 had affinities at least sevenfold and twofold higher than those of 2B11, a previously developed antibody against parent AFB, for the major aflatoxin-DNA adducts 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB and 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB, respectively. An analytical method based on a competitive radioimmunoassay with IIA4B3 and (3)H-AFB-lysine was validated with a limit of detection of 10 fmol of AFB-lysine adduct. The method has been applied to the measurement of AFB-albumin adduct levels in human serum samples collected from the residents of areas at high risk for liver cancer.     

Triple helix DNA oligomer melting measured by fluorescence polarization anisotropy

A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 mer single strand sequence onto the complementary purinic sequence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10-90 degrees C. The T(m) of the triplex, 37 degrees C, was well separated from the onset of duplex melting. When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the T(m) of the triplex was shifted to approximately 32 degrees C and overlapped the melting of the duplex. We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and triplex samples to determine the hydrodynamic parameters in the temperature range 10-40 degrees C. The fluorescence lifetime of EB in the samples of double and triple stranded DNA is the same (21.3 +/- 0.5 ns) at 20 degrees C, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal triplex, and nicked triplex samples were 10.7 +/- 0.2, 12.2 +/- 0.2, and 12.0 +/- 0.2 A. FPA measurements on normal triplex DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the triplex carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal triplex sample. The torsional rigidity of the triplex forms was found to be higher than that of the duplex form.     

Detoxification of aflatoxin B1 by manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624

Aflatoxin B(1) (AFB(1) ) is a potent mycotoxin with mutagenic, carcinogenic, teratogenic, hepatotoxic, and immunosuppressive properties. In order to develop a bioremediation system for AFB(1) -contaminated foods by white-rot fungi or ligninolytic enzymes, AFB(1) was treated with manganese peroxidase (MnP) from the white-rot fungus Phanerochaete sordida YK-624. AFB(1) was eliminated by MnP. The maximum elimination (86.0%) of AFB(1) was observed after 48 h in a reaction mixture containing 5 nkat of MnP. The addition of Tween 80 enhanced AFB(1) elimination. The elimination of AFB(1) by MnP considerably reduced its mutagenic activity in an umu test, and the treatment of AFB(1) by 20 nkat MnP reduced the mutagenic activity by 69.2%. (1) H-NMR and HR-ESI-MS analysis suggested that AFB(1) is first oxidized to AFB(1) -8,9-epoxide by MnP and then hydrolyzed to AFB(1) -8,9-dihydrodiol. This is the first report that MnP can effectively remove the mutagenic activity of AFB(1) by converting it into AFB(1) -8,9-dihydrodiol.     

Xanthobacter sp. C20 contains a novel bioconversion pathway for limonene

Xanthobacter sp. C20 was isolated from sediment of the river Rhine using cyclohexane as sole source of carbon and energy. Xanthobacter sp. C20 converted both enantiomers of limonene quantitatively into limonene-8,9-epoxide, a not previously described bioconversion product of limonene. With (4R)-limonene, (4R,8R)-limonene-8, 9-epoxide was formed as the only reaction product, while (4S)-limonene was converted into a (78:22) mixture of (4S,8R)- and (4S,8S)-limonene-8,9-epoxide. Cytochrome P-450 was shown to be induced concomitantly with limonene bioconversion activity following growth of Xanthobacter sp. C20 on cyclohexane. Maximal limonene bioconversion rate was observed at an initial substrate concentration of 12 mM. The amount of limonene-8,9-epoxide formed, up to 0.8 g l(-1), was limited by a strong product inhibition.     

Cytotoxicity and mutagenicity of aflatoxin dichloride in normal and repair deficient diploid human fibroblasts

The cytotoxic and mutagenic effect of aflatoxin B1-dichloride (AFB1-Cl2), a direct-acting carcinogen which is a model for the proposed ultimate reactive metabolite of AFB1 (the 2,3-epoxide), was compared in normal, repair-proficient, diploid human fibroblasts and in complementation Group A xeroderma pigmentosum cells (XP12BE) which are virtually incapable of excision repair of DNA damage induced by ultraviolet radiation, the 7,8-diol-9,10-epoxide of benzo[alpha]pyrene, and several reactive aromatic amide derivatives. The XP cells were significantly more sensitive than normal to the cytotoxic and mutagenic effects of AFB1-Cl2, not only as a function of concentration administered but also of the number of AFB1-Cl2 residues initially bound to DNA. Cytotoxicity was determined from survival of colony-forming ability; resistance to 6-thioguanine was the genetic marker used for mutagenicity. We compared the rate of loss of AFB1-Cl2-DNA adducts from cells treated and held in the non-dividing state (confluent) over several days, as well as their ability to recover from the potentially mutagenic and/or cytotoxic effects of the agent. AFB1-Cl2 residues were lost from both strains of cells and both exhibited a gradual increase in survival. However, the rate of loss of adducts from the DNA in the normal cells was more rapid than in XP cells and they exhibited recovery from higher doses of AFB1-Cl2 than XP cells. The major primary DNA adduct formed in the human cells and in isolated DNA was a chemically unstable guanine derivative which could undergo a change in structure with time posttreatment to form a more stable secondary adduct. The cytotoxic effect of AFB1-Cl2 was highly correlated with the presence of either of these guanine adducts. Evidence suggests that the primary adduct is an N7-guanine adduct. The kinetics of the loss of this guanine and its transformation into the more stable secondary adduct resembled that reported recently for the major primary DNA adduct formed by the reaction of AFB1 at the N-7 position of guanine in the DNA of normal and XP cells and its transformation into the putative AFB1-ring opened triamino pyrimidyl structure.     

NMR characterisation of a triple stranded complex formed by homo-purine and homo-pyrimidine DNA strands at 1:1 molar ratio and acidic pH.

Homo-purine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC) oligomers have been designed such that they are complementary in parallel orientation. When mixed in a 1:1 molar ratio, the system adopts an antiparallel duplex at neutral pH with three mismatched base pairs. On lowering the pH below 5.5, a new complex is formed. The NMR results show the coexistence of a intermolecular pyrimidine.purine:pyrimidine DNA triplex and a single stranded oligopurine at this pH. The triplex is stabilized by five T.A:T, four C+.G:C and two mismatched triads, namely, C+.G-T and T.A-C. This triplex is further stabilized by a Hoogsteen C+.G base-pair on one end. Temperature dependence of the imino proton resonances reveals that the triplex dissociates directly into single strands around 55 degrees C, without duplex intermediates. Parallel duplexes are not formed under any of the conditions employed in this study.     

Trapping of DNA topoisomerase I on nick-containing DNA in cell free extracts of Saccharomyces cerevisiae

The aim of the present study was to identify proteins that bind nicked DNA intermediates formed in the course of base excision repair (BER) in cell free extracts of Saccharomyces cerevisiae. In mammalian cells, nicks in DNA are targets of proteins such as PARP-1 or XRCC1 that have no homologues in yeast. One of the most promising methodologies to trap proteins that interact with damaged DNA lies in using a photocrosslinking technique with photoactivable dNTP analogues such as exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) for enzymatic synthesis of DNA probes with a photoreactive dNMP residue at the 3'-margin of a nick. Using this approach, we identified a major covalent DNA-protein adduct between a nick-containing 34-mer DNA duplex and a protein of a molecular mass of around 100-kDa. Unexpectedly, the formation of the 100-kDa adduct did not require the incorporation of the photoreactive dNMP residue at the 3'-margin of the nick nor exposure to near UV-light. However, the formation of the 100-kDa adduct strictly required a nick or a short gap in the DNA probe. Furthermore, the 100-kDa adduct was not detected in yeast extracts lacking DNA topoisomerase I (Top1). To further establish the nature of crosslinked protein, yeast Top1 was tagged with a Myc-epitope. In this case, the mobility of the Top1-DNA adduct increased by 7- kDa. Therefore, our data speak in favor of Top1 trapping by nicked DNA. In support of this hypothesis, purified yeast Top1 was also crosslinked to nicked DNA structures. Undamaged, uracil- and abasic (AP) site-containing DNAs were unable to trap Top1 under the same assay conditions. Since nicked DNA structures are frequently formed in the course of BER, their covalent linkage to Top1 has the potential to interfere with BER in vivo.     

Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.     

Intercalation of aflatoxin B1 in two oligodeoxynucleotide adducts: comparative 1H NMR analysis of d(ATCAFBGAT).d(ATCGAT) and d(ATAFBGCAT)2

8,9-Dihydro-8-(N7-guanyl-[d(ATCGAT)])-9-hydroxyaflatoxin B1.d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1.8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1 were prepared by direct addition of afltoxin B1 8,9-epoxide to d(ATCGAT)2 and d(ATGCAT)2, respectively. In contrast to reaction of aflatoxin B1 8,9-epoxide with d(ATCGAT)2 which exhibits a limiting stoichiometry of 1:1 aflatoxin B1:d(ATCGAT)2 [Gopalakrishnan, S., Stone, M. P., & Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239], reaction of aflatoxin B1 8,9-epoxide with d(ATGCAT)2 exhibits a limiting stoichiometry of 2:1 aflatoxin B1:d(ATGCAT)2. 1H NOE experiments, nonselective 1H T1 relaxation measurements, and 1H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5'-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide 1H NOEs are observed between aflatoxin and the 5'-neighbor base pair and include both the major groove and the minor groove. The aflatoxin methoxy and cyclopentenone ring protons face into the minor groove; the furofuran ring protons face into the major groove. No NOE is observed between the imino proton of the modified base pair and the imino proton of the 5'-neighbor base pair; sequential NOEs between nucleotide base and deoxyribose protons are interrupted in both oligodeoxynucleotide strands on the 5'-side of the modified guanine. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons; chemical shift perturbations of the oligodeoxynucleotide protons are confined to the immediate vicinity of the adducted base pair. The imidazole proton of the modified guanine exchanges with water and is observed at 9.75 ppm. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B1 8,9-epoxide and B-DNA. Insertion of aflatoxin B1-8,9 epoxide above the 5'-face of guanine in d(ATCGAT)2 would prevent the binding of a second molecule of aflatoxin B1 8,9-epoxide. In contrast, two intercalation sites would be available with d(ATGCAT)2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities.

Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4-(spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications.     

Conformation of DNA modified at a d(GG) or a d(AG) site by the antitumor drug cis-diamminedichloroplatinum(II)

The purpose of this work was the comparison of the conformational changes induced in the double helix by the adducts formed at d(GG) and d(AG) sites in the reaction between the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA. Two duplexes (20-mer) containing either a single d(A*G*) or a single d(G*G) adduct were studied by means of gel electrophoresis and artificial nuclease and chemical probes. It is shown that the d(G*G*) and the d(A*G*) adducts bend DNA similarly, but at the nucleotide level they distort differently the double helix. We suggest that the weaker interactions between platinated A residues and the other nucleotides, as compared to the interactions between platinated G residues and the other nucleotides, are largely responsible for the differences in the distortions induced in DNA by the d(A*G) and d(G*G*) adducts. This suggestion is supported by the study of the distortions induced in duplexes by the d(G*G*) adducts, one of the platinated G residues being paired with a T residue.     

Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I

The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.     

Reaction of 2-Deoxy-2-C-(3-bromoacetoxypropyl)-α-D-arabinofuranosides with Oligonucleotide1

Abstract

Reaction of methyl 2-deoxy-2-C-(3-bromoacetoxypropyl)-α-D-arabinofuranosides, prepared from methyl 2,3-anhydro-α-D-ribofuranoside, with oligodeoxyribonucleotide (21mer) in acetonitrile-H₂O (pH 7) and subsequent treatment with piperidine resulted in the cleavage of the nucleotide chain at the position G, A, and C.