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Reactive oxygen species (ROS), resulting from endogenous metabolism and/or environmental exposure, can induce damage to the 2-deoxyribose moiety in DNA. Specifically, a hydrogen atom from each of the five carbon atoms in 2-deoxyribose can be abstracted by hydroxyl radical, and improper chemical repair of the ensuing radicals formed at the C1, C3, and C4 positions can lead to the stereochemical inversion at these sites to yield epimeric 2-deoxyribose lesions. Although ROS-induced single-nucleobase lesions have been well studied, the biological consequences of the C3-epimeric lesions of 2-deoxynucleosides, i.e., 2-deoxyxylonucleosides (dxN), have not been comprehensively investigated. Herein, we assessed the impact of dxN lesions on the efficiency and fidelity of DNA replication in Escherichia coli cells by conducting a competitive replication and adduct bypass assay with single-stranded M13 phage containing a site-specifically incorporated dxN. Our results revealed that, of the four dxN lesions, only dxG constituted a strong impediment to DNA replication, and intriguingly, dxT and dxC conferred replication bypass efficiencies higher than those of the unmodified counterparts. In addition, the three SOS-induced DNA polymerases (Pol II, Pol IV, and Pol V) did not play any appreciable role in bypassing these lesions. Among the four dxNs, only dxA directed a moderate frequency of dCMP misincorporation. These results provided important insights into the impact of the C3-epimeric lesions on DNA replication in E. coli cells.

Urea lesions in DNA arise from thymine glycol (Tg) or 8-oxo-dG; their genotoxicity is thought to arise in part due to their potential to accommodate the insertion of all four dNTPs during error-prone replication. Replication bypass with human DNA polymerase η (hPol η) confirmed that all four dNTPs were inserted opposite urea lesions but with purines exhibiting greater incorporation efficiency. X-ray crystal structures of ternary replication bypass complexes in the presence of Mg 2+ ions with incoming dNTP analogs dAMPnPP, dCMPnPP, dGMPnPP, and dTMPnPP bound opposite urea lesions (hPol η·DNA·dNMPnPP complexes) revealed all were accommodated by hPol η. In each, the Watson-Crick face of the dNMPnPP was paired with the urea lesion, exploiting the ability of the amine and carbonyl groups of the urea to act as H-bond donors or acceptors, respectively. With incoming dAMPnPP or dGMPnPP, the distance between the imino nitrogen of urea and the N9 atoms of incoming dNMPnPP approximated the canonical distance of 9 Å in B-DNA. With incoming dCMPnPP or dTMPnPP, the corresponding distance of about 7 Å was less ideal. Improved base-stacking interactions were also observed with incoming purines vs pyrimidines. Nevertheless, in each instance, the α-phosphate of incoming dNMPnPPs was close to the 3'-hydroxyl group of the primer terminus, consistent with the catalysis of nucleotidyl transfer and the observation that all four nucleotides could be inserted opposite urea lesions. Preferential insertion of purines by hPol η may explain, in part, why the urea-directed spectrum of mutations arising from Tg vs 8-oxo-dG lesions differs.

The secondary metabolites of polypropanoids, methyleugenol (MEG), and estragole (EG), found in many herbs and spices, are commonly used as food flavoring agents and as ingredients in cosmetics. MEG and EG have been reported to cause hepatocarcinogenicity in rodents, human livers, and lung cells. The formation of N 2 -dA DNA adducts is primarily attributed to the carcinogenicity of these compounds. Therefore, these compounds have been classified as "possible human carcinogens" by the International Agency for Research on Cancer and "reasonably anticipated to be a human carcinogen" by the National Toxicology Program. Herein, we report the synthesis of the N 6 -EG-dG modified oligonucleotides to study the mutagenicity of these DNA adducts. Our studies show that N 2 -MEG-dG and N 2 -EG-dG modified oligonucleotides to study the mutagenicity of these DNA adducts. Our studies show that N 2 -MEG-dG and N 2 -EG-dG could be bypassed by human translesion synthesis (TLS) polymerases hpolκ and hpolη in an error-free manner. The steady-state kinetics of dCTP incorporation by hpolκ across N 2 -MEG-dG and N 2 -EG-dG adducts show that the catalytic efficiencies ( k cat / K m ) were ∼2.5- and ∼4.4-fold higher, respectively, compared to the unmodified dG template. A full-length primer extension assay demonstrates that hpolκ exhibits better catalytic efficiency than hpolη. Molecular modeling and dynamics studies capturing pre-insertion, insertion, and post-insertion steps reveal the structural features associated with the efficient bypass of the N 2 -MEG-dG adduct by hpolκ and indicate the reorientation of the adduct in the active site allowing the successful insertion of the incoming nucleotide. Together, these results suggest that though hpolκ and hpolη perform error-free TLS across MEG and EG during DNA replication, the observed carcinogenicity of these adducts could be attributed to the involvement of other low fidelity polymerases.

Cisplatin (CP) and oxaliplatin (OX), platinum-based drugs used widely in chemotherapy, form adducts on intrastrand guanines (5'GG) in genomic DNA. DNA damage recognition proteins, transcription factors, mismatch repair proteins, and DNA polymerases discriminate between CP- and OX-GG DNA adducts, which could partly account for differences in the efficacy, toxicity, and mutagenicity of CP and OX. In addition, differential recognition of CP- and OX-GG adducts is highly dependent on the sequence context of the Pt-GG adduct. In particular, DNA binding protein domain HMGB1a binds to CP-GG DNA adducts with up to 53-fold greater affinity than to OX-GG adducts in the TGGA sequence context but shows much smaller differences in binding in the AGGC or TGGT sequence contexts. Here, simulations of the HMGB1a-Pt-DNA complex in the three sequence contexts revealed a higher number of interface contacts for the CP-DNA complex in the TGGA sequence context than in the OX-DNA complex. However, the number of interface contacts was similar in the TGGT and AGGC sequence contexts. The higher number of interface contacts in the CP-TGGA sequence context corresponded to a larger roll of the Pt-GG base pair step. Furthermore, geometric analysis of stacking of phenylalanine 37 in HMGB1a (Phe37) with the platinated guanines revealed more favorable stacking modes correlated with a larger roll of the Pt-GG base pair step in the TGGA sequence context. These data are consistent with our previous molecular dynamics simulations showing that the CP-TGGA complex was able to sample larger roll angles than the OX-TGGA complex or either CP- or OX-DNA complexes in the AGGC or TGGT sequences. We infer that the high binding affinity of HMGB1a for CP-TGGA is due to the greater flexibility of CP-TGGA compared to OX-TGGA and other Pt-DNA adducts. This increased flexibility is reflected in the ability of CP-TGGA to sample larger roll angles, which allows for a higher number of interface contacts between the Pt-DNA adduct and HMGB1a. [ABSTRACT FROM AUTHOR]

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 19F 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 (fins) 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. [ABSTRACT FROM AUTHOR]

The nucleotide excision repair (NER) machinery, the primary defense against cancer-causing bulky DNA lesions, is surprisingly inefficient in recognizing certain mutagenic DNA adducts and other forms of DNA damage. However, the biochemical basis of resistance to repair remains poorly understood. To address this problem, we have investigated a series of intercalated DNA-adenine lesions derived from carcinogenic polycyclic aromatic hydrocarbon (PAH) diol epoxide metabolites that differ in their response to the mammalian NER apparatus. These stereoisomeric PAH-derived adenine lesions represent ideal model systems for elucidating the effects of structural, dynamic, and thermodynamic properties that determine the recognition of these bulky DNA lesions by NER factors. The objective of this work was to gain a systematic understanding of the relation between aromatic ring topology and adduct stereochemistry with existing experimental NER efficiencies and known thermodynamic stabilities of the damaged DNA duplexes. For this purpose, we performed 100 ns molecular dynamics studies of the lesions embedded in identical double-stranded 11-mer sequences. Our studies show that, depending on topology and stereochemistry, stabilizing PAH-DNA base van der Waals stacking interactions can compensate for destabilizing distortions caused by these lesions that can, in turn, cause resistance to NER. The results suggest that the balance between helix stabilizing and destabilizing interactions between the adduct and nearby DNA residues can account for the variability of NER efficiencies observed in this class of PAH-DNA lesions. [ABSTRACT FROM AUTHOR]

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of umnodifled DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop. [ABSTRACT FROM AUTHOR]

Previous structural studies of the cyclopropapyrroloindole (CPI) antitumor antibiotics have shown that these ligands bind covalently edge-on into the minor groove of double-stranded DNA. Reversible covalent modification of the DNA via N3 of adenine occurs in a sequence-specific fashion. Early nuclear magnetic resonance and molecular modeling studies with both mono- and bis-alkylating ligands indicated that the ligands fit tightly within the minor groove, causing little distortion of the helix. In this study, we propose a new binding model for several of the CPI-based analogues, in which the aromatic secondary rings form π-stacked complexes within the minor groove. One of the adducts, formed with adozelesin and the d(ATTAAT)2 sequence, also demonstrates the ability of these ligands to manipulate the DNA of the binding site, resulting in a Hoogsteen base-paired adduct. Although this type of base pairing has been previously observed with the bisfunctional CPI analogue bizelesin, this is the first time that such an observation has been made with a monoalkylating nondimeric analogue. Together, these results provide a new model for the design of CPI-based antitumor antibiotics, which also has a significant bearing on other structurally related and structurally unrelated minor groove-binding ligands. They indicate the dynamic nature of ligand-DNA interactions, demonstrating both DNA conformational flexibility and the ability of two DNA-bound ligands to interact to form stable covalent modified complexes. [ABSTRACT FROM AUTHOR]

Glycation of biopolymers by glucose-derived α-oxo-aldehydes such as methylglyoxal (MG) is believed to play a major role in the complex pathologies associated with diabetes and metabolic disease. In contrast to the extensive literature detailing the formation and physiological consequences of protein glycation, there is little information about the corresponding phenomenon for DNA. To assess the potential contribution of DNA glycation to genetic instability, we prepared shuttle vectors containing defined levels of the DNA glycation adduct N²-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) and transfected them into isogenic human fibroblasts that differed solely in the capacity to conduct nucleotide excision repair (NER). In the NER-compromised fibroblasts, the induced mutation frequencies increased up to 18-fold relative to background over a range of ∼10-1400 CEdG adducts/105 dG, whereas the same substrates transfected into NER-competent cells induced a response that was 5-fold over background at the highest adduct density. The positive linear correlation (R² = 0.998) of mutation frequency with increasing CEdG level in NER-defective cells suggested that NER was the primary if not exclusive mechanism for repair of this adduct in human fibroblasts. Consistent with predictions from biochemical studies using CEdG-substituted oligonucleotides, guanine transversions were the predominant mutation resulting from replication of MG-modified plasmids. At high CEdG levels, significant increases in the number of AT → GC transitions were observed exclusively in NER-competent cells (P < 0.0001). This suggested the involvement of an NER-dependent mutagenic process in response to critical levels of DNA damage, possibly mediated by error-prone Y-family polymerases. [ABSTRACT FROM AUTHOR]

Nucleotide excision repair (NER) is the main pathway used for the repair of bulky DNA adducts such as those caused by UV light exposure and the chemotherapeutic drug cisplatin. The xeroderma pigmentosum group C (XPC)—Rad23B complex is involved in the recognition of these bulky DNA adducts and initiates the global genomic nucleotide excision repair pathway (GG-NER). Photo-cross-linking experiments revealed that the human XPC—Rad23B complex makes direct contact with both the cisplatindamaged DNA strand and the complementary undamaged strand of a duplex DNA substrate. Coupling photo-cross-linking with denaturation and immunoprecipitation of protein—DNA complexes, we identified the XPC subunit in complex with damaged DNA. While the interaction of the XPC subunit with DNA was direct, studies revealed that although Rad23B was found in complex with DNA, the Rad23B—DNA interaction was largely indirect via its interaction with XPC. Using Site specific cross-linking, we determined that the XPC—Rad23B complex is preferentially cross-linked to the damaged DNA when the photo- reactive FAP-dCMP (exo-N(-(2-[N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]aminoethyl_-2'-deoxycytidine 5'-monophosphate) analogue is located to the 5' side of the cisplatin—DNA adduct. When the FAP-dCMP analogue is located to the 3' side of the adduct, no difference in binding was detected between undamaged and damaged DNA, Collectively, these data suggest a model in which XPC—DNA interactions drive the damage recognition process contacting both the damaged and undamaged DNA strand. Preferential cross-linking 5' of the cisplatin-damaged Site suggests that the XPC—Rad23B complex displays orientation specific binding to eventually impart directionality to the downstream binding and incision events relative to the site of DNA damage. [ABSTRACT FROM AUTHOR]

DNA damage by adduct formation is a critical step for the initiation of carcinogenesis. Aromatic amines are strong inducers of environmental carcinogenesis. Their DNA adducls are known to exist irs an equilibrium between the major groove (B) and base-displaced stacked (S) conformations. However, the factors governing such heterogeneity remain unclear. Here we conducted extensive calorimetry/NMR/CD studies on the model DNA lesions caused by fluorinated 2-aminfluorene (FAF) and 4-aminobiphenyl (FABP) in order to gain thermodynamic and kinetic insights into the S/B conformational equilibrium. We demonstrate that there are large differences in enthalpy-entropy compensations for FABP and 19FAF. The small and flexible FABP exclusively adopts the less perturbed B conformer with small enthalpy (ΔΔ-2.7 kcal/mol)/entropy (ΔΔS-0.7 eu) change. In contrast, FAF stacks better and exists as a mixture of B and S conformers with large enthalpy (ΔΔH-13.4 kcal/mol)/entropy (ΔS-34.2 eu) compensation. van't Hoff analysis of dynamic 9F NMR data indicated ΔHB⃡S = 4.1 kcal/mol in favor of the B conformer and ΔSB⃡S = 15.6 cal mol-1 K-1 in favor of the intercalated S conformer. These findings demonstrate that the favorable entropy of the S conformer over B conformer determines the S/B population ratios at physiological temperatures. [ABSTRACT FROM AUTHOR]

DNA sustains a wide variety of damage, such as the formation of abasic sites, pyrimidine dimers, alkylation adducts, or oxidative lesions, upon exposure to UV radiation, alkylating agents, or oxidative conditions. Since these forms of damage may be acutely toxic or mutagenic and potentially carcinogenic, it is of interest to gain insight into how their structures impact biochemical processing of DNA, such as synthesis, transcription, and repair. Lesion-specific molecular probes have been used to study polymerase-mediated translesion DNA synthesis of abasic sites and TI dimers, while other probes have been developed for specifically investigating the alkylation adduct O6-Bn-G and the oxidative lesion 8-oxo-G. In this review, recent examples of lesion-specific molecular probes are surveyed; their specificities of incorporation opposite target lesions compared to unmodified nucleotides are discussed, and limitations of their applications under physiologically relevant conditions are assessed. [ABSTRACT FROM AUTHOR]

Oxidative stress can induce the formation of reactive electrophiles, such as DNA peroxidation products, e.g., base propenals, and lipid peroxidation products, e.g., malondialdehyde. Base propenals and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-β-D-erythro-pentofuranosyl) pyrimido[1,2-α]purin-10(3H)-one (M1dG). When paired opposite cytosine in duplex DNA at physiological pH, M1dG) undergoes ring opening to form N2-3-oxo-1-propenyl)-dG(N2-OPdG). Previous work has shown that M1dG is mutagenic in bacteria and mammalian cells and that its mutagenicity in Escherichia coli is depending on induction of the SOS response, indicating a role for translesion DNA polymerases in the bypass of M1dG. To probe the mechanism by which translesion polymerases bypass M1dG, kinetic and structural studies were conducted wit a model Y-family DNA polymerase, Dpo4 from Sulfolobus. The level of steady-state incorporation of dNTPs opposite M1dG was reduced 260-2900-fold and exhibited a preference for dATP incorporation. Liquid chromatography-tandem mass spectrometry analysis of the full-length extension products revealed a spectrum of products arising principally by incorporation of dC or dA opposite M1dG followed by partial or full-length extension. A greater proportion of -1 deletions were observed when dT was positioned 5' of M1dG. Two crystal structures were determined, including a "type II" frameshift deletion cmplex and another complex with Dpo4 bound to a dC∙M1dG pair located in the postinsetion context. Importantly, M1dG was in the ring-closed state in both structures, and in the structures with dC opposite M1dG, the dC residue moved out of the Dpo4 active site, into the minor groove. The results are consistent with the reported mutagenicity of M1dG and illustrate how the lesion may affect replication events. [ABSTRACT FROM AUTHOR]

The equine estrogen equilin (EQ) and equilenin (EN) are the active components in the widely prescribed hormone replacement therapy formulation Premarin. Metabolic activation of EQ and EN generates the catechol 4-hydroxyequilenin (4-OHEN) that autoxidizes to the reactive o-quinone form in aerated aqueous solution. The o-quinones react predominantly with C, and to a lesser extent with A and G, to form premutagenic cyclic covalent DNA adducts in vitro. To obtain insight into the structural properties of these biologically important DNA lesions, we have synthesized site-specifically modified oligonucleotides containing the stereoisomeric 1'S,2'R,3'R-4-OHEN-C3 and 1'R,2'S,3'S-4-OHEN-C4 adducts derived from the reaction of 4-OHEN with the C in the oligonucleotide 5'-GGTAGCGATGG in aqueous solution. A combined NMR and computational approach was utilized to determine the conformational characteristics of the two major 4-OHEN-C3 and 4-OHEN-C4 stereoisomeric adducts formed in this oligonucleotide hybridized with its complementary strand. In both cases, the modified C adopts an anti glycosidic bond conformation; the equilenin distal ring protrudes into the minor groove while its two proximal hydroxyl are exposed on the major groove side of the DNA duplex The bulky 4-OHEN-C adducts distorts the duplex within the central GC*G portion, but Watson-Crick pairing is maintained adjacent to C* in both stereoisomeric adducts. For the 4-OHEN-C3 adduct, the equilenin rings are oriented toward the 5'-end of the modified strand, while in 4-OHEN-C4 the equilenin is 3'-directed. Correspondingly, the distortions of the double-helical structures are more pronounced on the 5'- or the 3'-side of the lesion, respectively. These differences in stereoisomeric adduct conformations may party a role in the processing of these lesions in cellular environments. [ABSTRACT FROM AUTHOR]

Non-invasive strategies for the analysis of endogenous DNA damage are of interest for the purpose of monitoring genomic exposure to biologically produced chemicals. We have focused our research on the biological processing of DNA adducts and how this may impact the observed products in biological matrixes. Preliminary research has revealed that pyrimidopurinone DNA adducts are subject to enzymatic oxidation in vitro and in vivo and that base adducts are better substrates for oxidation than the corresponding 2'-deoxynucleosides. We tested the possibility that structurally similar exocyclic base adducts may be good candidates for enzymatic oxidation in vitro. We investigated the in vitro oxidation of several endogenously occurring etheno adducts [1,N²-ε-guanine (1,N²-ε-Gua), N²,3-ε-Gua, heptanone- 1,N²-ε-Gua, 1,N6-ε-adenine (1,N6-ε-Ade), and 3,N4-ε-cytosine (3,N4-ε-Cyt)] and their corresponding 2'-deoxynucleosides. Both 1,N²-ε-Gua and heptanone-1,N²-ε-Gua were substrates for enzymatic oxidation in rat liver cytosol; heteronuclear NMR experiments revealed that oxidation occurred on the imidazole ring of each substrate. In contrast, the partially or fully saturated pyrimidopurinone analogues [i.e., 5,6-dihydro-M1G and 1,N²-propanoguanine (PGua)] and their 2'-deoxynucleoside derivatives were not oxidized. The 2'-deoxynucleoside adducts, 1,N²-ε-dG and 1,N6-ε-dA, underwent glycolytic cleavage in rat liver cytosol. Together, these data suggest that multiple exocyclic adducts undergo oxidation and glycolytic cleavage in vitro in rat liver cytosol, in some instances in succession. These multiple pathways of biotransformation produce an array of products. Thus, the biotransfonnation of exocyclic adducts may lead to an additional class of biomarkers suitable for use in animal and human studies. [ABSTRACT FROM AUTHOR]

Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped fiavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 Å resolution X-ray crystal structure of choline oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. We propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures. [ABSTRACT FROM AUTHOR]

The naturally occurring pyrrolo[2,1-c][1,4]benzodiazepine (PBD) monomers such as sibiromycin, anthramycin, and tomaymycin form stable covalent adducts with duplex DNA at purine–guanine–purine sites. A correlative relationship between DNA-binding affinity, as measured by enhanced thermal denaturation temperature of calf thymus DNA (Tm), and cytotoxicity is well documented for these naturally occurring compounds and a range of synthetic analogues with sibiromycin having the highest ΔTm value (16.3 °C), reflecting favorable hydrogen-bonding interactions between the molecule and DNA bases. We report here that, surprisingly, the structurally simple synthetic C2-(2-naphthyl)-substituted pyrrolo[2,1-c][1,4]benzodiazepine monomer (5) has a ΔTm value (15.8 °C) similar to that of sibiromycin and significantly higher than the values for either anthramycin (13.0 °C) or tomaymycin (2.6 °C). 5 also has similar cytotoxic potency to sibiromycin which is widely regarded as the most potent naturally occurring PBD monomer. To investigate this, we have used NMR in conjunction with molecular dynamics to study the 2:1 adduct formed between 5 and the DNA duplex d(AATCTTTAAAGATT)2. In contrast to the hydrogen-bonding interactions which predominate in the case of sibiromycin and anthramycin adducts, we have shown that the high binding affinity of 5 is due predominantly to hydrophobic (van der Waals) interactions. The high-resolution 2D NOESY, TOCSY, and COSY data obtained have also allowed unequivocal determination of the orientation of the PBD molecule (A-ring toward 3′-end of covalently bound strand), the stereochemistry at the C11 position of the PBD (C11S), and the conformation of the C2-naphthyl ring which extends along the floor of the minor groove thus optimizing hydrophobic interactions with DNA. These results provide opportunities for future drug design in terms of extending planar hydrophobic groups at the C2 position of PBDs to maximize binding affinity. [ABSTRACT FROM AUTHOR]

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) η and κ, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6α- and 6β-diastereoisomers of dG-N2-6-estrogen or dA-N6-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pots has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6α or 6β-isomeric N2-(estradiol-6- yl)-2'-deoxyguanosine (dG-N2-6-E2) into oligodeoxynucleotides. Using such site-specific modified bligomer as a template, the specificity and frequency of miscoding by dG-N2-6α-E2 or dG-N2-6β-E2 were explored using pol η and a truncated form of pol κ (pol κΔC). Translesion synthesis catalyzed by pol η bypassed both the 6α and 6β-isomers of dG-N2-6-E22-6-E2, with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol κΔC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol κΔC was more efficient than pol η by incorporating dCMP opposite both 6α- and 6β-isomeric dG-N2-6-E2 adducts. The miscoding events occurred more frequently with pot η, but not with pol κΔC. Pol η promoted incorporation of dAMP and dTMP at both the 6α- and 6β-isomeric adducts, generating G → T transversions and G Δ A transitions. One- and two-base deletions were also formed. The 6α-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6β-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6α-isomeric dG-N2-6-E2 is likely to be similar to that of the 6β-isomeric adduct. [ABSTRACT FROM AUTHOR]

The Y-family DNA polymerase Dpo4, from the archaeon bacterium Sulfolobus solfataricus, is a member of the DinB family, which also contains human Pol κ. It has a spacious active site that can accommodate two templating bases simultaneously, with one of them skipped by the incoming dNTP. Assays of single dNTP insertion opposite a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), reveal that an incoming dATP is significantly preferred over the other three dNTPs in the TG1*G2 sequence context. Molecular modeling and dynamics simulations were carried out to interpret this experimental observation on a molecular level. Modeling studies suggest that the significant preference for dATP insertion observed experimentally can result from two possible dATP incorporation modes. The dATP can be inserted opposite the T on the 5' side of the adduct G1*, using an unusual 5'-slippage pattern, in which the unadducted G2, rather than G1*, is skipped, to produce a -1 deletion. In addition, the dATP can be misincorporated opposite the adduct. The 5'-slippage pattern may be generally facilitated in cases where the base 3' to the lesion is the same as the adducted base. [ABSTRACT FROM AUTHOR]

Proteins that discriminate between cisplatin-DNA adducts and oxaliplatin-DNA adducts are thought to be responsible for the differences in tumor range, toxicity, and mutagenicity of these two important chemotherapeutic agents. However, the structural basis for differential protein recognition of these adducts has not been determined and could be important for the design of more effective platinum anticancer agents. We have determined high-resolution NMR structures for cisplatin-GG and undamaged DNA dodecamers in the AGGC sequence context and have compared these structures with the oxaliplatin-GG structure in the same sequence context determined previously in our laboratory. This structural study allows the first direct comparison of cisplatin-GG DNA and oxaliplatin-GG DNA solution structures referenced to undamaged DNA in the same sequence context. Non-hydrogen atom rmsds of 0.81 and 1.21 were determined for the 15 lowest-energy structures for cisplatin-GG DNA and undamaged DNA, respectively, indicating good structural convergence. The theoretical NOESY spectra obtained by back-calculation from the final average structures showed excellent agreement with the experimental data, indicating that the final structures are consistent with the NMR data. Several significant conformational differences were observed between the cisplatin-GG adduct and the oxaliplatin-GG adduct, including buckle at the 5′ G6·C19 base pair, opening at the 3′ G7·C18 base pair, twist at the A5G6·T20C19 base pair step, slide, twist, and roll at the G6G7·C19C18 base pair step, slide at the G7C8·C18G17 base pair step, G6G7 dihedral angle, and overall bend angle. We hypothesize that these conformational differences may be related to the ability of various DNA repair proteins, DNA binding proteins, and DNA polymerases to discriminate between cisplatin-GG and oxaliplatin-GG adducts. [ABSTRACT FROM AUTHOR]

The equine estrogens, equilin and equilenin, are major components of the drug Premarin, the most widely used formula for hormone replacement therapy. The derivative 4-hydroxyequilenin (4-OHEN), a major phase I metabolite of equilin and equilenin, autoxidizes to potent cytotoxic quinoids that can react in vitro and in vivo with cytosine and adenine in DNA. Unique cyclic adducts containing the same bicyclo[3.3.1]nonane-type connection ring are produced. Each base adduct has four stereoisomers. In order to elucidate the structural effects of A versus C modification, we have carried out molecular dynamics simulations of the stereoisomeric 4-OHEN-A adducts in DNA 11-mer duplexes and compared results with an earlier study of the C adducts (Ding, S., Shapiro, R., Geacintov, N.E., and Broyde, S. (2005) Equilenin-Derived DNA Adducts to Cytosine in DNA Duplexes: Structures and Thermodynamics, Biochemistry 44, 14565-14576). Similar stereochemical principles govern the orientations in DNA duplexes of the 4-OHEN-A adducts as for the analogous C adducts, with opposite orientations of the equilenin rings in stereoisomeric pairs of adducts characterized by near-mirror image circular dichroism (CD) spectra. However, the larger purine adducts have unique structural properties in the duplexes that distinguish their characteristics from those of the pyrimidine adducts. Significant differences are observed in terms of hydrogen bonding, stacking, bending, groove dimensions, solvent exposure, and hydrophobic interactions; also, each of the four stereoisomeric 4-OHEN-A adducts exhibit distinct structural features. Each base adduct and stereoisomer distorts the structure of the DNA duplex differently. These characteristics may manifest themselves in terms of differential nucleotide excision repair susceptibilities and mutagenic activities of the 4-OHEN-A and C adducts. [ABSTRACT FROM AUTHOR]

The electrophilic lipid oxidation product 4-hydroxy-2-nonenal (HNE) reacts with proteins to form covalent adducts, and this damage has been implicated in pathologies associated with oxidative stress. HNE adduction of blood proteins, such as human serum albumin (HSA), yields adducts that may serve as markers of oxidative stress in vivo. We used liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the P-Mod algorithm to map the sites of 10 adducts formed by reaction of HNE with HSA in vitro. The detected adducts included Michael adducts formed at histidine and lysine residues. The selectivity of HNE in competing adduction reactions was evaluated by analysis of kinetics for HNE Michael adduction at six targeted HSA histidine residues. Reaction kinetics were analyzed by selected reaction monitoring in LC-MS-MS using stable isotope tagging with phenyl isocyanate. Rate constants ranged over 4 orders of magnitude, with the order of reactivity being H242 > H510 > H67 > H367 > H247 ≈ K233. The most reactive target, H242, is located in a fatty acid- and drug binding cavity in subdomain IIa of HSA and appears to be a hot-spot for HNE modification. Analysis of adduction kinetics together with HSA structure and target residue pKa values suggest that location in the hydrophobic binding cavity and low predicted pKa of H242 account for its high reactivity toward HNE. H242 adducts may be preferred products of adduction by lipophilic electrophiles and may comprise a family of biomarkers for oxidative stress. [ABSTRACT FROM AUTHOR]

DNA methylation is an important cellular mechanism for controlling gene expression. Whereas the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons, have been widely studied, little is known about their influence on DNA methylation. We have constructed site-specifically modified 1 8-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts derived from a benzo [a]pyrene-derived diol epoxide [(+)- and (- )- r7 ,t8-dihydroxy-19, 1 0-epoxy-7 ,8,9,- io∼tetrahydrobenzo[a]pyrene, or B[a]PDEI bound to the exocyclic amino group of guanine. The adducts, either (+)- or (-)∼trans∼anti-B[a]PN∼dG (G*), positioned either at the 5'-side or the 3'-side deoxyguanosine residue in the recognition sequence of EcoRil restriction-modification enzymes (5'-...CCA/ TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on complex formation and the catalytic activity of the EcoRTI DNA methyltransferase (M.EcoRll) and EcoRil restriction endonuclease (R.EcoRll) were investigated. The M.EcoRII catalyzes the transfer of a methyl group to the C5 position of the 3'-side cytosine of each strand of the recognition sequence, whereas R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRll to the oligodeoxynucleotide duplexes and the catalytic cleavage were completely abolished when G* was positioned at the 3'-side dG position (5'∼...CCTGG*...). When G* was at the 5'-side dO position, binding was moderately diminished, but cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5-30 but the catalytic activity was either abolished or reduced 4-80-fold when the adducts were located at either position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes. These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression of genes that promote tumor development. [ABSTRACT FROM AUTHOR]

The study of the interactions of double-stranded (ds) DNA with the dirhodium carboxylate compounds Rh2(O2CCH3)4(H20)2 (Rid), [Rh2(O2CCH3)2(CH3CN)6I(B1∼4)2 (Rh2), and Rh2(O2CCF3)4 (Rh3) supports the presence of covalently linked DNA adducts, including stable DNA interstrand cross-links. The present biochemical study refutes earlier claims that no reaction between dirhodium compounds and dsDNA occurs. The reversal behavior of these interstrand cross-links in 5 M urea at 95 °C (for different heating times) implies the presence of various coordination modes involving ax/ax, ax/eq, and eq/eq DNA interactions with the dirhodium core. The reaction rates of the dirhodium compounds with dsDNA were determined spectroscopically and are in the order Rh! <

Estrogen replacement therapy (ERT), composed of equilenin, is associated with increased risk of breast, ovarian, and endometrial cancers. Several diastereoisomers of unique dC and dA DNA adducts were derived from 4-hydroxyequilenin (4-OHEN), a metabolite of equilenin, and have been detected in women receiving ERT. To explore the miscoding property of 4-OHEN-dC adduct, site-specifically modified oligodeoxynucleotides (Pk-1, Pk-2, Pk-3, and Pk-4) containing a single diastereoisomer of 4-OHEN-dC were prepared by a postsynthetic method. Among them, major 4-OHEN-dC-modified oligodeoxynucleotides (Pk-3 and Pk-4) were used to prepare the templates for primer extension reactions catalyzed by DNA polymerase (pol) α, pol n, and poi κ. Primer extension was retarded one base prior to the lesion and opposite the lesion; stronger blockage was observed with pol α, while with human pol n or pol κ, a fraction of the primers was extended past the lesion. Steady-state kinetic studies showed that both pol κ and poi n inserted dCMP and dAMP opposite the 4-OHEN-dC and extended past the lesion. Never or less-frequently, dGMP, the correct base, was inserted opposite the lesion. The relative bypass frequency past the 4-OHEN-dC lesion with pol n was at least 3 orders of magnitude higher than that for pol κ, as observed for primer extension reactions. The bypass frequency past the dA·4-OHEN-dC adduct in Pk-4 was 2 orders of magnitude more efficient than that past the adduct in Pk-3. Thus, 4-OHEN-dC is a highly miscoding lesion capable of generating C →T transitions and C →G transversions. The miscoding frequency and specificity of 4-OHEN-dC were strikingly influenced by the adduct stereochemistry and DNA polymerase used. [ABSTRACT FROM AUTHOR]

The sequence specificity and time course of covalent DNA adduct formation of the novel platinum-acridine conjugate [PtCl(en)(ACRAMTU)](NO3)2 [PT-ACRAMTU, 2; en = ethane-1,2-diamine, ACRAMTU = 1 -[2-(acridin-9-ylamino)ethyl]- 1 ,3-dimethylthiourea] have been investigated using restriction enzyme cleavage and transcription footprinting assays and compared to the damage produced by the clinical agent cis-diamminedichloroplatinum(II) (cisplatin, 1). The rate of DNA binding of 1 and 2 was also monitored by atomic emission spectrometry. Restriction enzymes were chosen that cleave the phosphodiester linkage at, or adjacent to, the predicted damage sites. While conjugate 2 selectively protected supercoiled plasmid from cleavage by EcoRI and DraI enzymes at their respective restriction sites, G↓AATTC and TTT↓AAA, 1 inhibited DNA hydrolysis by HindIII and PspOMI at A↓AGCTT and G↓GGCCC (arrows mark cleavage sites) more efficiently. Transcription footprinting using T7 RNA polymerase revealed major single-base damage sites for 2 at adenine in 5'-TA and 5'-GA sequences. In addition, the enzyme is efficiently stalled at guanine bases, primarily in the sequence 5'-CGA where the damaged nucleobase is flanked by two high-affinity intercalation sites of ACRAMTU. While 1 targets poly(G) sequences, the binding of 2 appears to be dominated by the groove and sequence recognition of the intercalator. The biochemical assays used confirm previous structural information extracted from mass spectra of DNA fragments modified by 2 isolated from enzymatic digests [Barry, C. G., et al. (2003) J. Am. Chem. Soc. 125, 9629-9637]. Possible DNA-binding mechanisms and biological consequences of the unprecedented modification of alternating TA sequences by 2, which occurred at a faster rate than binding to G, are discussed. [ABSTRACT FROM AUTHOR]

D-Arginine dehydrogenase (DADH) catalyzes the flavin-dependent oxidative deamination of D-arginine and other d-amino acids to the corresponding imino acids. The 1.07 Å atomic-resolution structure of DADH crystallized with D-leucine unexpectedly revealed a covalent N(5) flavin adduct, instead of the expected iminoleucine product in the active site. This acyl adduct has been successfully reproduced by photoreduction of DADH in the presence of 4-methyl-2-oxopentanoic acid (ketoleucine). The iminoleucine may be released readily because of weak interactions in the binding site, in contrast to iminoarginine, converted to ketoleucine, which reacts with activated FAD to form the covalently linked acyl adduct. [ABSTRACT FROM AUTHOR]