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.

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.

Replication through a single DNA lesion may give rise to a panel of translesion synthesis (TLS) events, which comprise error-free TLS, base substitutions and frameshift mutations. In order to determine the genetic control of the various TLS events induced by a single lesion, we have chosen the major N2-dG adduct of (+)-anti-Benzo(a)pyrene diol epoxide [(+)-anti-BPDE] adduct located within a short run of guanines as a model lesion. Within this sequence context, in addition to the major event, i.e. error-free TLS, the adduct also induces base substitutions (mostly G --> T transversions) and -1 frameshift mutations. The pathway leading to G --> T base substitution mutagenesis appears to be SOS independent, suggesting that TLS is most probably performed by the replicative Pol III holoenzyme itself. In contrast, both error-free and frameshift TLS pathways are dependent upon SOS-encoded functions that belong to the pool of inducible DNA polymerases specialized in TLS (translesional DNA polymerases), namely umuDC (Pol V) and dinB (Pol IV). It is likely that, given the diversity of conformations that can be adopted by lesion-containing replication intermediates, cells use one or several translesional DNA polymerases to achieve TLS.

Accomodation of S-cis-tamoxifen-N(2)-guanine adduct within a bent and widened DNA minor groove.

The non-steroidal anti-estrogen tamoxifen [TAM] has been in clinical use over the last two decades as a potent adjunct chemotherapeutic agent for treatment of breast cancer. It has also been given prophylactically to women with a strong family history of breast cancer. However, tamoxifen treatment has also been associated with increased endometrial cancer, possibly resulting from the reaction of metabolically activated tamoxifen derivatives with cellular DNA. Such DNA adducts can be mutagenic and the activities of isomeric adducts may be conformation-dependent. We therefore investigated the high resolution NMR solution conformation of one covalent adduct (cis-isomer, S-epimer of [TAM]G) formed from the reaction of tamoxifen [TAM] to N(2)-of guanine in the d(C-[TAM]G-C).d(G-C-G) sequence context at the 11-mer oligonucleotide duplex level. Our NMR results establish that the S-cis [TAM]G lesion is accomodated within a widened minor groove without disruption of the Watson-Crick [TAM]G. C and flanking Watson-Crick G.C base-pairs. The helix axis of the bound DNA oligomer is bent by about 30 degrees and is directed away from the minor groove adduct site. The presence of such a bulky [TAM]G adduct with components of the TAM residue on both the 5'- and the 3'-side of the modified base could compromise the fidelity of the minor groove polymerase scanning machinery.

The decomposition of peroxynitrite to nitroxyl anion (NO-) and singlet oxygen in aqueous solution.

The mechanism of decomposition of peroxynitrite (OONO(-)) in aqueous sodium phosphate buffer solution at neutral pH was investigated. The OONO(-) was synthesized by directly reacting nitric oxide with superoxide anion at pH 13. The hypothesis was explored that OONO(-), after protonation at pH 7.0 to HOONO, decomposes into (1)O(2) and HNO according to a spin-conserved unimolecular mechanism. Small aliquots of the concentrated alkaline OONO(-) solution were added to a buffer solution (final pH 7.0-7.2), and the formation of (1)O(2) and NO(-) in high yields was observed. The (1)O(2) generated was trapped as the transannular peroxide (DPAO(2)) of 9, 10-diphenylanthracene (DPA) dissolved in carbon tetrachloride. The nitroxyl anion (NO-) formed from HNO (pKa 4.5) was trapped as nitrosylhemoglobin (HbNO) in an aqueous methemoglobin (MetHb) solution. In the presence of 25 mM sodium bicarbonate, which is known to accelerate the rate of decomposition of OONO(-), the amount of singlet oxygen trapped was reduced by a factor of approximately 2 whereas the yield of trapping of NO(-) by methemoglobin remained unaffected. Because NO(3)(-) is known to be the ultimate decomposition product of OONO(-), these results suggest that the nitrate anion is not formed by a direct isomerization of OONO(-), but by an indirect route originating from NO(-).

Mass spectrometric sequencing of site-specific carcinogen-modified oligodeoxyribonucleotides containing bulky benzo[a]pyrene diol epoxide-deoxyguanosyl adducts.

Site-specific carcinogen-modified oligonucleotides are often used in site-directed mutagenesis and other biological and biochemical studies of structure-function relationships. Postsynthetic analysis and confirmation of the sites of carcinogen binding in such oligonucleotides is an important step in the characterization of these site-specific carcinogen-DNA adducts. It is shown here that negative ion mode electrospray tandem mass spectrometry methods and collision-induced dissociation offer a rapid and convenient approach for the sequencing of products derived from the reaction of the carcinogenic and mutagenic metabolite of benzo[a]pyrene, the diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), with the 11-mer oligonucleotide d(CATGCGGCCTAC). The site of reaction of anti-BPDE with either one of the three dG residues in this oligonucleotide can be accurately established by comparing the mass/charge ratios of the observed collision-induced dissociation fragments with calculated values.

Role of hydrophobic effects in the reaction of a polynuclear aromatic diol epoxide with oligodeoxynucleotides in aqueous solutions.

The need for large-scale direct synthesis of stereochemically defined and site-specific benzo[alpha]pyrenediol epoxide-oligodeoxyribonucleotide adducts for detailed NMR and other biochemical and physicochemical studies has necessitated a better understanding of variables that lead to an enhancement of the reaction yields. It is shown that, in aqueous solution, the formation of noncovalent hydrophobic complexes between 7r, 8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[alpha] pyrene (BPDE) and the single-stranded oligonucleotide 5'-d(CCATCGCTACC) precedes the covalent binding reaction of BPDE with the single deoxyguanosine residue. The yield of covalent reaction products (involving reaction of BPDE at the C10 position with the exocyclic amino group of the dG residue) increases with increasing DNA concentration and tends toward saturation at oligonucleotide single-strand concentrations above approximately 3 mM. The addition of NaCl (0.3 M) also tends to enhance the adduct reaction yields. However, organic solvents such as tetrahydrofuran in the reaction mixtures (10-40%) decrease the stabilities of the noncovalent complexes, which in turn leads to reductions in the yields of covalent BPDE-dG oligonucleotide adducts. The efficiencies of formation of hydrophobic complexes were probed by fluorescence and UV absorption techniques using the BPDE tetrol hydrolysis product 7,8,9,10-tetrahydroxytetrahydrobenzo[alpha]pyrene as a model system.

Solution conformation of the (-)-trans-anti-[BP]dG adduct opposite a deletion site in a DNA duplex: intercalation of the covalently attached benzo[a]pyrene into the helix with base displacement of the modified deoxyguanosine into the minor groove.

A combined NMR-computational approach was employed to determine the solution structure of the (-)-trans-anti-[BP]dG adduct positioned opposite a -1 deletion site in the d(C1-C2-A3-T4-C5- [BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14-A15-G1 6-G17-A18-T19-G20-G21) sequence context. The (-)-trans-anti-[BP]dG moiety is derived from the binding of the (-)-anti-benzo[a]pyrene diol epoxide [(-)-anti-BPDE] to N2 of dG6 and has a 10R absolute configuration at the [BP]dG linkage site. The exchangeable and non-exchangeable 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 conformation has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space followed by restrained molecular dynamics calculations based on a NOE distance and intensity refinement protocol. Our structural studies establish that the aromatic BP ring system intercalates into the helix opposite the deletion site, while the modified deoxyguanosine residue is displaced into the minor groove with its face parallel to the helix axis. The intercalation site is wedge-shaped and the BP aromatic ring system stacks over intact flanking Watson-Crick dG.dC base pairs. The modified deoxyguanosine stacks over the minor groove face of the sugar ring of the 5'-flanking dC5 residue. The BP moiety is positioned with the benzylic ring oriented toward the minor groove and the distal pyrenyl aromatic ring directed toward the major groove. This conformation strikingly contrasts with the corresponding structure in the full duplex with the same 10R (-)-trans-anti-[BP]dG lesion positioned opposite a complementary dC residue [de los Santos et al. (1992) Biochemistry 31, 5245-5252); in this case the aromatic BP ring system is located in the minor groove, and there is no disruption of the [BP]dG.dC Watson-Crick base pairing alignment. The intercalation-base displacement features of the 10R (-)-trans-anti-[BP]dG adduct opposite a deletion site have features in common to those of the 10S (+)-trans-anti-[BP]dG adduct opposite a deletion site previously reported by Cosman et al. [(1994)(Biochemistry 33, 11507-11517], except that there is a nearly 180 degrees rotation of the BP residue about the axis of the helix at the base-displaced intercalation site and the modified deoxyguanosine is positioned in the opposite groove. In the 10S adduct, the benzylic ring is in the major groove and the aromatic ring systems point toward the minor groove. This work extends the theme of opposite orientations of adducts derived from chiral pairs of (+)- and (-)-anti-BPDE enantiomers; both 10S and 10R adducts can be positioned with opposite orientations either in the minor groove or at base displaced intercalation sites, depending on the presence or absence of the partner dC base in the complementary strand.

Interference of benzo[a]pyrene diol epoxide-deoxyguanosine adducts in a GC box with binding of the transcription factor Sp1.

Previous studies indicated that DNA adducts formed by the carcinogenic diol epoxide 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. Whether adducts that form in the normal binding site, the GC box sequence, increase the affinity of Sp1 for the modified GC-box was not determined. Starting with a 23-nt sequence that contains two natural GC box sequences, site-specifically modified oligonucleotides were prepared with a single(+)-BPDE-deoxyguanosine adduct at one of three positions: the center of each GC-box or in between the two boxes. Four modified oligonucleotides were studied, two derived from cis addition of BPDE to the exocyclic amino group and two from trans addition. For three of these site-specifically modified oligonucleotides, there was a diminution in Sp1 affinity, whereas Sp1 binding to the fourth modified oligonucleotide was abolished. Furthermore, random modification of the oligonucleotide to a level of about 1 BPDE adduct per fragment slightly decreased the affinity for Sp1, and no evidence was found for a subpopulation of molecules with high affinity. These findings suggest that BPDE modification of the GC box does not lead to an increased affinity for Sp1. This is consistent with a model in which a BPDE-induced bend in the DNA mimics the conformation of the normal GC box:Sp1 complex, leading to high-affinity binding of Sp1 to non-Gc box sites.