We have synthesized, separated, and purified =10 mg of a deoxyundecanucleotide duplex containing a single centrally positioned covalent adduct between (+)-antibenzo[a]pyrene (BP) diol epoxide and the exocyclic amino group of guanosine. Excellent proton NMR spectra are observed for the (+)-trans-anti-BP diol epoxide-N2-dG adduct positioned opposite dC and flanked by G-C pairs in theWe have determined the solution structure centered about the BP covalent adduct site in the (BP)G-C ll-mer duplex by incorporating intramolecular and intermolecular proton-proton distance bounds deduced from the NMR data sets as constraints in energy minimization computations. The BP ring is positioned in the minor groove and directed toward the 5' end of the modified strand. One face of the BP ring of (BP)G6 is stacked over the G18 and A19 sugarphosphate backbone on the partner strand and the other face is exposed to solvent. A minimally perturbed B-DNA helix is observed for the d[T4-C5-(BP)G6-C7-T8]Jd[A15-G16-C17-G18-A19] segment centered about the adduct site with WatsonCrick alignment for both the (BP)G6-C17 pair and flanking GC pairs. A widening of the minor groove at the adduct site is detected that accommodates the BP ring whose long axis makes an angle of =45°with the average direction of the DNA helix axis. Our study holds future promise for the characterization of other stereoisomerically pure adducts of BP diol epoxides with DNA to elucidate the molecular basis of structure-activity relationships associated with the stereoisomerdependent spectrum of mutational and carcinogenic activities.Benzo[a]pyrene (BP), a ubiquitous environmental pollutant, is metabolized in mammalian cells to highly reactive, mutagenic, and tumorigenic diol epoxide derivatives (the field of carcinogen-DNA adducts is reviewed in refs.
This paper reports on the solution structure of the (+)-cis-anti-[BP]dG adduct positioned opposite dC in a DNA oligomer duplex which provides the first experimentally based solution structure of an intercalative complex of a polycyclic aromatic hydrocarbon covalently bound to the N2 of deoxyguanosine. The combined NMR-energy minimization computation studies were undertaken on the (+)-cis-anti-[BP]dG adduct embedded in the same d(C5-[BP]G6-C7).d(G16-C17-G18) trinucleotide segment of the complementary 11-mer duplex studied previously with the stereoisomeric trans adducts. The exchangeable and nonexchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY data sets as restraints in energy minimization computations. The benzo[a]pyrene ring of [BP]dG6 is intercalated between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs in a right-handed DNA helix. The benzylic ring is in the minor groove while the pyrenyl ring sacks with flanking dC5 and dC7 bases on the same strand. The deoxyguanosine ring of [BP]dG6 is not Watson-Crick base paired but displaced into the minor groove with its plane parallel to the helix axis and stacks over the sugar ring of dC5. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove by the intercalated benzo[a]pyrene ring. This intercalative structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex exhibits several unusually shifted proton resonances which can be readily accounted for by the ring current contributions of the deoxyguanosine and pyrenyl rings of the [BP]dG6 adduct. Several phosphorus resonances are shifted to low and high field of the unperturbed phosphorus spectral region and have been assigned to internucleotide phosphates centered about the [BP]dG6 modification site. These studies define the changes in the helix at the central trinucleotide segment needed to generate the intercalation site for the covalently bound (+)-cis-anti-[BP]dG adduct.(ABSTRACT TRUNCATED AT 400 WORDS)
Benzo[a]pyrene (BP) is an environmental genotoxin, which, following metabolic activation to 7,8-diol 9,10-epoxide (BPDE) derivatives, forms covalent adducts with cellular DNA. A major fraction of adducts are derived from the binding of N2 of guanine to the C10 position of BPDE. The mutagenic and carcinogenic potentials of these adducts are strongly dependent on the chirality at the four asymmetric benzylic carbon atoms. We report below on the combined NMR-energy minimization refinement characterization of the solution conformation of (-)-trans-anti-[BP]G positioned opposite C and flanked by G.C base pairs in the d(C1-C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17- G18-A19-T20-G21-G22) duplex. Two-dimensional NMR techniques were applied to assign the exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid in the modified duplex. These results establish Watson-Crick base pair alignment at the [BP]G6.C17 modification site, as well as the flanking C5.G18 and C7.G16 pairs within a regular right-handed helix. The solution structure of the (-)-trans-anti-[BP]G.C 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOE buildup curves as constraints in energy minimization computations. The BP ring spans both strands of the duplex in the minor groove and is directed toward the 3'-end of the modified strand in the refined structure. One face of the BP ring of [BP]G6 stacks over the C17 residue across from it on the partner strand while the other face is exposed to solvent.(ABSTRACT TRUNCATED AT 250 WORDS)
Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.
7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG•C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain–DNA phosphate contacts translocate by one nucleotide step, while the thumb domain–DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain–phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.
by benzo[a]pyrene diol epoxide (BPDE), a potent and ubiquitous mutagen that induces mainly G ⅐ C3T ⅐ A transversions and frameshift deletions. We found that human nucleotide excision repair processes the predominant (؉)-trans-BPDE-N 2 -dG adduct 15 times less efficiently than a standard acetylaminofluorene-C 8 -dG lesion in the same sequence. No difference was observed between (؉)-trans-and (؊)-trans-BPDE-N 2 -dG, but excision was enhanced about 10-fold by changing the adduct configurations to either (؉)-cis-or (؊)-cis-BPDE-N 2 -dG. Conversely, excision of (؉)-cis-and (؊)-cis-but not (؉)-trans-BPDE-N 2 -dG was reduced about 10-fold when the complementary cytosine was replaced by adenine, and excision of these BPDE lesions was essentially abolished when the complementary deoxyribonucleotide was missing. Thus, a set of chemically identical BPDE adducts yielded a greater-than-100-fold range of repair rates, demonstrating that nucleotide excision repair activity is entirely dictated by local DNA conformation. In particular, this unique comparison between structurally highly defined substrates shows that fast excision of BPDE-N 2 -dG lesions is correlated with displacement of both the modified guanine and its partner base in the complementary strand from their normal intrahelical positions. The very slow excision of carcinogen-DNA adducts located opposite deletion sites reveals a cellular strategy that minimizes the fixation of frameshifts after mutagenic translesion synthesis.Mammalian nucleotide excision repair promotes genomic stability by removing UV radiation products and a wide range of chemical carcinogen-DNA adducts (14,20,37,42,52). This multisubunit DNA repair system operates by cleavage of damaged strands on either side of the targeted lesion (28, 35) followed by excision of oligonucleotide segments 24 to 32 residues in length (25,31). In subsequent reactions, double-helical integrity and the correct nucleotide sequence are reestablished by DNA repair synthesis and DNA ligation (1, 40).The mechanism by which mammalian nucleotide excision repair enzymes discriminate damaged sites as substrates for dual DNA incision is not understood (14,24,26,42), but several reports have demonstrated that excision activity is highly nonuniform in the context of mammalian chromosomes. For example, bulky UV radiation products are excised at variable rates, with cyclobutane pyrimidine dimers being processed considerably more slowly than the less frequently occurring pyrimidine(6-4)pyrimidone lesion (30). Generally, active genes are repaired faster than inactive loci, and the template strand of RNA polymerase II-transcribed genes is repaired faster than the coding strand (2,3,20,29,46). Yet another level of heterogeneity emerged when DNA excision repair rates were compared between closely related genomic sites. Along the nontranscribed strand of the human hypoxanthine phosphoribosyltransferase gene, for example, excision repair of guanine adducts formed by benzo[a]pyrene diol epoxide (BPDE) varies by more than 1 order of magni...
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