Mutational specificity of gamma-radiation-induced guanine-thymine and thymine-guanine intrastrand cross-links in mammalian cells and translesion synthesis past the guanine-thymine lesion by human DNA polymerase eta

Comparative mutagenesis of gamma- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G --> T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5' and three bases 3' to the cross-link. The most prevalent semitargeted mutation was a C --> T transition immediately 5' to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of approximately 16%, and both included a dominant G --> T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5' to the lesion was increased and 3' to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called "A-rule". To determine if human polymerase eta (hpol eta) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol eta can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol eta was significantly different from that by yeast pol eta in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol eta opposite cross-linked T was 3-5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol eta was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol eta was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol eta may play a role in correct bypass of the cross-links.     

Structural and thermodynamic features of spiroiminodihydantoin damaged DNA duplexes

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.     

Interaction of nitroaromatic radiosensitizers with irradiated polyadenylic acid as measured by an indirect immunochemical assay with specificity for the 8,5'-cycloadenosine moiety

The relative reactivity of a series of nitroaromatic radiosensitizers toward the C(5') radical intermediate leading to 8,5'-cycloadenosine formation in deoxygenated solutions of irradiated polyadenylic acid (poly A) was assessed using standard competition kinetic analysis. Formation of 8,5'-cycloadenosine was assayed by an indirect, competitive, enzyme-linked immunosorbent assay (ELISA) described in an earlier report. In the absence of oxygen, the nitroaromatics inhibit 8,5'-cyclonucleoside formation in a way which generally increases with radiosensitizer electron affinity. Although hydroxyl radical scavenging by the nitroaromatics may account for a relatively small decrease in 8,5'-cyclonucleoside formation, the data suggest that oxidation of the C(5') radical intermediate is the more plausible explanation for the decreased yield of the 8,5'-cyclonucleoside with increasing nitroaromatic electron affinity.     

Human DNA polymerase kappa bypasses and extends beyond thymine glycols during translesion synthesis in vitro,preferentially incorporating correct nucleotides

Human polymerase kappa (polkappa), the product of the human POLK (DINB1) gene, is a member of the Y superfamily of DNA polymerases that support replicative bypass of chemically modified DNA bases (Ohmori, H., Friedberg, E. C., Fuchs, R. P., Goodman, M. F., Hanaoka, F., Hinkle, D., Kunkel, T. A., Lawrence, C. W., Livneh, Z., Nohmi, T., Prakash, L., Prakash, S., Todo, T., Walker, G. C., Wang, Z., and Woodgate, R. (2001) Mol. Cell 8, 7-8; Gerlach, V. L., Aravind, L., Gotway, G., Schultz, R. A., Koonin, E. V., and Friedberg, E. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11922-11927). Polkappa is shown here to bypass 5,6-dihydro-5,6-dihydroxythymine (thymine glycol) generated in two different DNA substrate preparations. Polkappa inserts the correct base adenine opposite thymine glycol in preference to the other three bases. Additionally, the enzyme correctly extends beyond the site of the thymine glycol lesion when presented with adenine opposite thymine glycol at the primer terminus. However, steady state kinetic analysis of nucleotides incorporated opposite thymine glycol demonstrates different misincorporation rates for guanine with each of the two DNA substrates. The two substrates differ only in the relative proportions of thymine glycol stereoisomers, suggesting that polkappa distinguishes among stereoisomers and exhibits reduced discrimination between purines when incorporating a base opposite a 5R thymine glycol stereoisomer. When extending beyond the site of the lesion, the misincorporation rate of polkappa for each of the three incorrect nucleotides (adenine, guanine, and thymine) is dramatically increased. Our findings suggest a role for polkappa in both nonmutagenic and mutagenic bypass of oxidative damage.     

Absolute configuration-dependent epoxide formation from isoflavan-4-ol stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain KF707

Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2' and C3' on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.     

Fluorescence postlabeling assay of cis-thymidine glycol monophosphate in X-irradiated calf-thymus DNA

DNA damage was induced by irradiating calf-thymus DNA with a GE Maxitron-250 as an X-ray source. The use of nitrous oxide as a scavenger of solvated electrons in the irradiation process, resulted in essentially a monoreactant system of the biologically important hydroxyl radical. A novel approach combining the enzymatic digestion of the irradiated DNA to nucleoside 5' monophosphates and fluorescence postlabeling was applied to detect a specific modified nucleotide induced by ionizing radiation, namely the 5,6-dihydroxy-5,6-dihydrothymine lesion. This modification, often referred to as the glycol lesion, is polar and is generated mainly as the cis stereoisomers. In order to demonstrate the detection of this lesion in DNA by fluorescence labeling, the lesion was first produced chemically in a DNA model compound d(CGTA). The modified oligomers were isolated intact by HPLC and characterized by NMR as cis stereoisomers of glycol derivatives of d(CGTA). The major isomer of the modified d(CGTA) was enzymatically digested to yield 5' monophosphates. The digest was chromatographed by HPLC to enrich the modified nucleotide. The fraction containing the modified nucleotide was labeled with dansyl chloride. The fluorescent labeled nucleotide was chromatographed by HPLC. The same overall procedure was applied to DNA X-irradiated in aqueous solution. With a conventional fluorescence detector, HPLC analysis of the fluorescence labeled nucleotides detected 1 modified nucleotide/10(6) normal nucleotides from 100 micrograms DNA. The two cis glycol lesions were detected in the irradiated DNA by co-chromatography with fluorescent labeled markers. The initial assay of the modified oligomer demonstrated that the same stereoisomer of cis glycol was induced as a major modified nucleotide by both chemical oxidation and ionizing radiation.     

Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues.

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.     

Direct superoxide anion scavenging by a disodium disuccinate astaxanthin derivative: Relative efficacy of individual stereoisomers versus the statistical mixture of stereoisomers by electron paramagnetic resonance imaging

Carotenoids are a related group of greater than 600 natural compounds, irrespective of geometric- and stereoisomers, with demonstrated antioxidant efficacy. The carotenoids are broadly divided into "carotenes," or non-oxygen substituted hydrocarbon carotenoids, and "xanthophylls," oxygen-substituted carotenoids. The natural compounds are excellent singlet oxygen quenchers as well as lipid peroxidation chain-breakers; this dual antioxidant capacity is generally attributed to the activity of the polyene chain, and increases with the number of conjugated double bonds along the polyene chain length. However, the poor aqueous solubility of most carotenes and the vast majority of xanthophylls limits their use as aqueous-phase singlet oxygen quenchers and direct radical scavengers. A variety of introduction vehicles (e.g., organic solvents, cyclodextrins) have been used to introduce the insoluble carotenoids into aqueous test systems. Hawaii Biotech, Inc. (HBI) successfully synthesized a novel carotenoid derivative, the disodium disuccinate derivative of astaxanthin (3,3(')-dihydroxy-beta,beta-carotene-4,4(')-dione) in all-trans (all-E) form. The novel derivative is a water-dispersible symmetric chiral molecule with two chiral centers, yielding four stereoisomeric forms: 3R,3(')R and 3S,3(')S (enantiomers), and the diastereomeric meso forms (3R,3(')S and 3(')R,3S). The individual stereoisomers were synthesized at high purity (>90% by HPLC) and compared directly for efficacy with the statistical mixture of stereoisomers obtained from the synthesis from the commercial source of astaxanthin (1:2:1 ratio of 3S,3(')S, meso, and 3R,3(')R, respectively). Direct scavenging of superoxide anion was evaluated in a standard in vitro isolated human neutrophil assay by electron paramagnetic resonance (EPR) imaging, employing the spin-trap DEPMPO. Each novel derivative was tested in pure aqueous formulation and in ethanolic formulation shown to completely disaggregate the compounds in solution. In each case, the ethanolic formulation was a more potent scavenging vehicle. No significant differences in scavenging efficiency were noted among the individual stereoisomers and the statistical mixture of stereoisomers, suggesting that the polyene chain alone was responsible for superoxide scavenging. Dose-ranging revealed that the statistical mixture of stereoisomers of the novel derivative, at millimolar (mM) concentrations, could nearly completely eliminate the superoxide anion signal generated in the activated human neutrophil assay. All ethanolic formulations of the novel derivatives exhibited increased scavenging efficiency over equimolar concentrations of non-esterified astaxanthin delivered in a dimethyl sulfoxide (DMSO) vehicle. These novel compounds will likely find utility in applications requiring aqueous delivery of a highly potent direct radical scavenger.     

Genetic requirement for mutagenesis of the G[8,5-Me]T cross-link in Escherichia coli: DNA polymerases IV and V compete for error-prone bypass

γ-Radiation generates a variety of complex lesions in DNA, including the G[8,5-Me]T intrastrand cross-link in which C8 of guanine is covalently linked to the 5-methyl group of the 3'-thymine. We have investigated the toxicity and mutagenesis of this lesion by replicating a G[8,5-Me]T-modified plasmid in Escherichia coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these DNA polymerases. In the single-polymerase knockout strains, viability was the lowest in a pol V-deficient strain, which suggests that pol V is most efficient in bypassing this lesion. Most mutations were single-base substitutions or deletions, though a small population of mutants carrying two point mutations at or near the G[8,5-Me]T cross-link was also detected. Mutations in the progeny occurred at the cross-linked bases as well as at bases near the lesion site, but the mutational spectrum varied on the basis of the identity of the DNA polymerase that was knocked out. Mutation frequency was the lowest in a strain that lacked the three SOS DNA polymerases. We determined that pol V is required for most targeted G → T transversions, whereas pol IV is required for the targeted T deletions. Our results suggest that pol V and pol IV compete to carry out error-prone bypass of the G[8,5-Me]T cross-link.     

Sequence effects on translesion synthesis of an aminofluorene-DNA adduct: conformational, thermodynamic, and primer extension kinetic studies

The DNA sequence effect is an important structural factor for determining the extent and nature of carcinogen-induced mutational and repair outcomes. In this study, we used two 16-mer template sequences, TG*A [d(5'-CTTCTTG*ACCTCATTC-3')] and CG*A [d(5'-CTTCTCG*ACCTCATTC-3')], to study the impact of the 5'-flanking nucleotide (T vs C) on aminofluorene (AF)-induced stacked (S)/major groove (B)/wedge (W) conformational heterogeneity during a simulated translesion synthesis. In addition, we probed the sequence effect on nucleotide insertion efficiencies catalyzed by the Klenow fragment (exonuclease-deficient) of DNA polymerase I. Our (19)F NMR/ICD/DSC results showed that AF in the CG*A duplex sequence adopts a greater population of S-conformer than the TG*A sequence. We found that the S conformer of CG*A thermodynamically favors insertion of A over C at the lesion site (n). Significant stalling occurred at both the prelesion (n - 1) and lesion (n) sites; however, the effect was more persistent for the S conformer of CG*A than TG*A at the lesion site (n). Kinetics show that relative nucleotide insertion frequencies (f(ins)) were greater for TG*A than the S conformer of CG*A for either dCTP or dATP at the lesion site (n), and the insertion rate was significantly reduced at immediate upstream base pairs (n, n + 1). Taken together, the results provide insight into how the mutagenic AF could exhibit an S/B/W equilibrium in the active site of a polymerase, causing different mutations. This work represents a novel structure-function relationship in which adduct structure is directly linked to nucleotide insertion efficiency in a conformation-specific manner during translesion DNA synthesis.     

The stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence

The trans-4-hydroxynonenal (HNE)-derived exocyclic 1, N(2)-dG adduct with (6S,8R,11S) stereochemistry forms interstrand N(2)-dG-N(2)-dG cross-links in the 5'-CpG-3' DNA sequence context, but the corresponding adduct possessing (6R,8S,11R) stereochemistry does not. Both exist primarily as diastereomeric cyclic hemiacetals when placed into duplex DNA [Huang, H., Wang, H., Qi, N., Kozekova, A., Rizzo, C. J., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 10898-10906]. To explore the structural basis for this difference, the HNE-derived diastereomeric (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were examined with respect to conformation when incorporated into 5'-d(GCTAGC XAGTCC)-3' x 5'-d(GGACTCGCTAGC)-3', containing the 5'-CpX-3' sequence [X = (6S,8R,11S)- or (6R,8S,11R)-HNE-dG]. At neutral pH, both adducts exhibited minimal structural perturbations to the DNA duplex that were localized to the site of the adduction at X(7) x C(18) and its neighboring base pair, A(8) x T(17). Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were located within the minor groove of the duplex. However, the respective orientations of the two cyclic hemiacetals within the minor groove were dependent upon (6S) versus (6R) stereochemistry. The (6S,8R,11S) cyclic hemiacetal was oriented in the 5'-direction, while the (6R,8S,11R) cyclic hemiacetal was oriented in the 3'-direction. These cyclic hemiacetals effectively mask the reactive aldehydes necessary for initiation of interstrand cross-link formation. From the refined structures of the two cyclic hemiacetals, the conformations of the corresponding diastereomeric aldehydes were predicted, using molecular mechanics calculations. Potential energy minimizations of the duplexes containing the two diastereomeric aldehydes predicted that the (6S,8R,11S) aldehyde was oriented in the 5'-direction while the (6R,8S,11R) aldehyde was oriented in the 3'-direction. These stereochemical differences in orientation suggest a kinetic basis that explains, in part, why the (6S,8R,11S) stereoisomer forms interchain cross-links in the 5'-CpG-3' sequence whereas the (6R,8S,11R) stereoisomer does not.     

Translesion DNA synthesis by yeast DNA polymerase eta on templates containing N2-guanine adducts of 1,3-butadiene metabolites

Yeast DNA polymerase eta can replicate through cis-syn cyclobutane pyrimidine dimers and 8-oxoguanine lesions with the same efficiency and accuracy as replication of an undamaged template. Previously, it has been shown that Escherichia coli DNA polymerases I, II, and III are incapable of bypassing DNA substrates containing N(2)-guanine adducts of stereoisomeric 1,3-butadiene metabolites. Here we showed that yeast polymerase eta replicates DNA containing the monoadducts (S)-butadiene monoepoxide and (S,S)-butadiene diolepoxide N(2)-guanines albeit at an approximately 200-300-fold lower efficiency relative to the control guanine. Interestingly, nucleotide incorporation opposite the (R)-butadiene monoepoxide and the (R,R)-butadiene diolepoxide N(2)-guanines was approximately 10-fold less efficient than incorporation opposite their S stereoisomers. Polymerase eta preferentially incorporates the correct nucleotide opposite and downstream of all four adducts, except that it shows high misincorporation frequencies for elongation of C paired with (R)-butadiene monoepoxide N(2)-guanine. Additionally, polymerase eta does not bypass the (R,R)- and (S,S)-butadiene diolepoxide N(2)-guanine-N(2)-guanine intra- strand cross-links, and replication is completely blocked just prior to the lesion. Collectively, these data suggest that polymerase eta can tolerate the geometric distortions in DNA conferred by the N(2)-guanine butadiene monoadducts but not the intrastrand cross-links.     

Prenylated flavonoids from Moghania philippinensis

Five prenylated flavonoids, 8-(1,1-dimethylallyl)genistein (1), 5,7,3',4'-tetrahydroxy-2',5'-di(3-methylbut-2-enyl)isoflavone (2), 5,7,3'-trihydroxy-2'-(3-methylbut-2-enyl)-4',5'-(3,3-dimethylpyrano)isoflavone (3), (2R)-5,2',4'-trihydroxy-8,5'-di(3-methylbut-2-enyl)-6,7-(3,3-dimethylpyrano)flavanone (4a) and (2S)-5, 2', 4'-trihydroxy-8,5'-di(3-methylbut-2-enyl)-6,7-(3,3-dimethylpyrano)flavanone (4b), were isolated from the roots of Moghania philippinensis. The structures of these compounds were determined on the basis of spectroscopic and chemical means.     

Intercalation of the (1S,2R,3S,4R)-N6-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence

The (1S,2R,3S,4R)-N(6)-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, results from trans opening of (1R,2S,3S,4R)-1,2-epoxy-1,2,3, 4-tetrahydrobenz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Two conformations of this adduct exist, in slow exchange on the NMR time scale. A structure for the major conformation, which represents approximately 80% of the population, is presented. In this conformation, an anti glycosidic torsion angle is observed for all nucleotides, including S,R,S,RA6. The refined structure is a right-handed duplex, with the benz[a]anthracene moiety intercalated on the 3'-face of the modified base pair, from the major groove. It is located between S,R,S,RA6.T17 and A7.T16. Intercalation is on the opposite face of the modified S,R,S,RA6.T17 base pair as compared to the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2, 3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct, which intercalated 5' to the modified R,S,R,SA6.T17 base pair [Li, Z. , Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981]. The spectroscopic data do not allow refinement of the minor conformation, but suggest that the adenyl moiety in the modified nucleoti111S,R, S,RA6 adopts a syn glycosidic torsion angle. Thus, the minor conformation may create greater distortion of the DNA duplex. The results are discussed in the context of site-specific mutagenesis studies which reveal that the S,R,S,RA6 lesion is less mutagenic than the R,S,R,SA6 lesion.     

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.     

Minor groove orientation for the (1S,2R,3S,4R)-N2- [1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in the N-ras codon 12 sequence

The conformation of the trans-anti-(1S,2R,3S,4R)-N(2)-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in d(G(1)G(2)C(3)A(4)G(5)X(6)T(7)G(8)G(9)T(10)G(11)).d(C(12)A(13)C(14)C(15)A(16)C(17)C(18)T(19)G(20)C(21)C(22)), bearing codon 12 of the human N-ras protooncogene (underlined), was determined. This adduct had S stereochemistry at the benzylic carbon. Its occurrence in DNA is a consequence of trans opening by the deoxyguanosine amino group of (1R,2S,3S,4R)-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracenyl-3,4-diol. The resonance frequencies, relative to the unmodified DNA, of the X(6) H1' and H6 protons were shifted downfield, whereas those of the C(18) and T(19) H1', H2', H2' ', and H3' deoxyribose protons were shifted upfield. The imino and amino resonances exhibited the expected sequential connectivities, suggesting no interruption of Watson-Crick pairing. A total of 426 interproton distances, including nine uniquely assigned BA-DNA distances, were used in the restrained molecular dynamics calculations. The refined structure showed that the benz[a]anthracene moiety bound in the minor groove, in the 5'-direction from the modified site. This was similar to the (+)-trans-anti-benzo[a]pyrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon [Cosman, M., De Los Santos, C., Fiala, R., Hingerty, B. E., Singh, S. B., Ibanez, V., Margulis, L. A., Live, D., Geacintov, N. E., Broyde, S., and Patel, D. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918]. It differed from the (-)-trans-anti-benzo[c]phenanthrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon, which intercalated in the 5'-direction [Lin, C. H., Huang, X., Kolbanovskii, A., Hingerty, B. E., Amin, S., Broyde, S., Geacintov, N. E., and Patel, D. J. (2001) J. Mol. Biol. 306, 1059-1080]. The results provided insight into how PAH molecular topology modulates adduct structure in duplex DNA.     

Inhibition of Werner syndrome helicase activity by benzo[c]phenanthrene diol epoxide dA adducts in DNA is both strand-and stereoisomer-dependent

Helicases are among the first enzymes to encounter DNA damage during DNA processing within the cell and thus are likely to be targets for the adverse effects of DNA lesions induced by environmental chemicals. Here we examined the effect of cis- and trans-opened 3,4-diol 1,2-epoxide (DE) DNA adducts of benzo[c]phenanthrene (BcPh) at N6 of adenine on helicase activity. These adducts are derived from the highly tumorigenic (-)-(1R,2S,3S,4R)-DE as well as its less carcinogenic (+)-(1S,2R,3R,4S)-DE enantiomer in both of which the benzylic 4-hydroxyl group and epoxide oxygen are trans. The hydrocarbon portions of these adducts intercalate into DNA on the 3' or the 5' side of the adducted deoxyadenosine for the 1S- and 1R-adducts, respectively. These adducts inhibited the human Werner (WRN) syndrome helicase activity in a strand-specific and stereospecific manner. In the strand along which WRN translocates, cis-opened adducts were significantly more effective inhibitors than trans-opened isomers, indicating that WRN unwinding is sensitive to adduct stereochemistry. WRN helicase activity was also inhibited but to a lesser extent by cis-opened BcPh DE adducts in the displaced strand independent of their direction of intercalation, whereas inhibition by the trans-opened stereoisomers in the displaced strand depended on their orientation, such that only adducts oriented toward the advancing helicase inhibited WRN activity. A BcPh DE adduct positioned in the helicase-translocating strand did not sequester WRN, nor affect the rate of ATP hydrolysis relative to an unadducted control. Although the Bloom (BLM) syndrome helicase was also inhibited by a cis-opened adduct in a strand-specific manner, this helicase was not as severely affected as WRN. Because BcPh DEs form substantial amounts of deoxyadenosine adducts at dA, their adverse effects on helicases could contribute to genetic damage and cell transformation induced by these DEs. Thus, the unwinding activity of RecQ helicases is sensitive to the strand, orientation, and stereochemistry of intercalated polycyclic aromatic hydrocarbon adducts.     

New sequential-assignment routes of nucleic acid NMR signals using a [5'-(13)C]-labeled DNA dodecamer

NMR signal assignments for DNA oligomers have been performed by the well-established sequential assignment procedures based on NOESY and COSY. The H4'/H5'/H5' resonance region is congested and difficult to analyze without the use of isotope-labeled DNA oligomers. Here a DNA dodecamer constructed with 2'-deoxy[5'-(13)C]ribonucleotides, 5'-d(*C*G*C*G*A*A*T*T*C*G*CG)-3' (*N = [5'-(13)C]Nucleotide), was prepared in an effort to analyze the H4'/H5'/H5' resonance region by 2D 1H-13C HMQC-NOESY. In the C5' and H1' resonance region, weak and strong cross peaks for C5'(i)-H1'(i) and C5'(i)-H1'(i-1), respectively, were found, thus enabling the sequential assignment within this region. A similar sequential assignment route was found between C5' and H2'. Proton pair distances evaluated from the canonical B-DNA as well as A-DNA indicated that these sequential-assignment routes on a 2D 1H-13C HMQC-NOESY spectrum work for most nucleic acid stem regions.     

Free-radical-induced formation of an 8,5''-cyclo-2''-deoxyguanosine moiety in deoxyribonucleic acid.

Isolation and identification of a novel .OH-induced product, namely an 8,5'-cyclo-2'-deoxyguanosine moiety, in DNA and 2'-deoxyguanosine are described. .OH radicals were generated in dilute aqueous solutions by gamma-irradiation. Analyses of 2'-deoxyguanosine and enzymic hydrolysates of DNA by gas chromatography-mass spectrometry (g.c.-m.s.) after trimethylsilylation showed the presence of 8,5-cyclo-2'-deoxyguanosine on the basis of its fragment ions. This product was isolated by h.p.l.c. Its u.v. and n.m.r. spectra taken were in agreement with the structure suggested by its mass spectrum. Exact masses of the typical ions from the mass spectrum of the trimethylsilyl derivative of this product were measured by high-resolution m.s. The values found were in excellent agreement with the theoretical mass derived from the suggested fragmentation patterns. Both (5'R)- and (5'S)-epimers of 8,5'-cyclo-2'-deoxyguanosine were observed. These two diastereomers were separated from each other by g.c. as well as by h.p.l.c. The assignment of the epimers was accomplished on the basis of the n.m.r. data. The formation of 8,5'-cyclo-2'-deoxyguanosine was suppressed by the presence of O2 in the solutions. The use of g.c.-m.s. with the selected-ion monitoring technique facilitated the detection of 8,5'-cyclo-2'-deoxyguanosine in DNA at radiation doses as low as 1 Gy. Its mechanism of formation probably involves hydrogen atom abstraction by .OH radicals from the C-5' of the 2'-deoxyguanosine moiety followed by intramolecular cyclization with the formation of a covalent bond between the C-5' and C-8 and subsequent oxidation of the resulting N-7-centred radical.