Synthesis and spectroscopic characterization of site-specific 2-amino-1-methyl-6-phenylimidazo

The aim of the present study is to determine the chemical structure and conformation of DNA adducts formed by incubation of the bioactive form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-acetoxy-PhIP, with a single-stranded 11mer oligodeoxyribonucleotide. Using conditions optimized to give the C8-dG-PhIP adduct as the major product, sufficient material was synthesized for NMR solution structure determination. The NMR data indicate that in duplex DNA this adduct exists in equilibrium between two different conformational states. In the main conformer, the covalently bound PhIP molecule intercalates in the helix, whilst in the minor conformation the PhIP ligand is probably solvent exposed. In addition to the C8-dG-PhIP adduct, at least eight polar adducts are found after reaction of N-acetoxy-PhIP with the oligonucleotide. Three of these were purified for further characterization and shown to exhibit lowest energy UV absorption bands in the range 342–347 nm, confirming the presence of PhIP or PhIP derivative. Accurate mass determination of two of the polar adducts by negative ion MALDI-TOF MS revealed ions consistent with a spirobisguanidino-PhIP derivative and a ring-opened adduct. The third adduct, which has the same mass as the C8-dG-PhIP oligonucleotide adduct, may contain PhIP bound to the N² position of guanine.     

Recognition and incision of site-specifically modified C8 guanine adducts formed by 2-aminofluorene, N-acetyl-2-aminofluorene and 1-nitropyrene by UvrABC nuclease

Nucleotide excision repair plays a crucial role in removing many types of DNA adducts formed by UV light and chemical carcinogens. We have examined the interactions of Escherichia coli UvrABC nuclease proteins with three site-specific C8 guanine adducts formed by the carcinogens 2-aminofluorene (AF), N-acetyl-2-acetylaminofluorene (AAF) and 1-nitropyrene (1-NP) in a 50mer oligonucleotide. Similar to the AF and AAF adducts, the 1-NP-induced DNA adduct contains an aminopyrene (AP) moiety covalently linked to the C8 position of guanine. The dissociation constants for UvrA binding to AF–, AAF– and AP–DNA adducts, determined by gel mobility shift assay, are 33 ± 9, 8 ± 2 and 23 ± 9 nM, respectively, indicating that the AAF adduct is recognized much more efficiently than the other two. Incision by UvrABC nuclease showed that AAF–DNA was cleaved ~2-fold more efficiently than AF– or AP–DNA (AAF > AF ≈ AP), even though AP has the largest molecular size in this group. However, an opened DNA structure of six bases around the adduct increased the incision efficiency for AF–DNA (but not for AP–DNA), making it equivalent to that for AAF–DNA. These results are consistent with a model in which DNA damage recognition by the E.coli nucleotide excision repair system consists of two sequential steps. It includes recognition of helical distortion in duplex DNA followed by recognition of the type of nucleotide chemical modification in a single-stranded region. The difference in incision efficiency between AF– and AAF–DNA adducts in normal DNA sequence, therefore, is a consequence of their difference in inducing structural distortions in DNA. The results of this study are discussed in the light of NMR solution structures of these DNA adducts.     

Crosslinking reactions of 4-amino-6-oxo-2-vinylpyrimidine with guanine derivatives and structural analysis of the adducts

DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo-2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine. The AOVP CFO exhibited selective crosslinking reactivity with guanine and thymine in DNA, and with guanine in RNA. These crosslinking reactions with guanine were accelerated in the presence of CoCl₂, NiCl₂, ZnCl₂ and MnCl₂. In addition, we demonstrated that the AOVP CFO was reactive toward 8-oxoguanine opposite AOVP in the duplex DNA. The structural analysis of each guanine and 8-oxoguanine adduct in the duplex DNA was investigated by high-resolution NMR. The results suggested that AOVP reacts at the N2 amine in guanine and at the N1 or N2 amines in 8-oxoguanine in the duplex DNA. This study demonstrated the first direct determination of the adduct structure in duplex DNA without enzyme digestion.     

Solution structure of the covalent sterigmatocystin-DNA adduct.

Sterigmatocystin and aflatoxin are potent mutagens that contaminate foodstuffs stored under conditions that permit fungal growth. These food mycotoxins can be metabolically activated to their epoxides, which subsequently form covalent adducts with DNA and can eventually induce tumor development. We have generated the sterigmatocystin-d(A1-A2-T3-G4-C5-A6-T7-T8) covalent adduct (two sterigmatocystins per duplex) by reacting sterigmatocystin-1,2-epoxide with the self-complementary d(A-A-T-G-C-A-T-T) duplex and determined its solution structure by the combined application of two-dimensional NMR experiments and molecular dynamics calculations. The self-complementary duplex retains its 2-fold symmetry following covalent adduct formation of sterigmatocystin at the N7 position of G4 residues on each strand of the duplex. The H8 proton of [ST]G4 exchanges rapidly with water and resonates at 9.58 ppm due to the presence of the positive charge on the guanine ring following adduct formation. We have assigned the exchangeable and nonexchangeable proton resonances of sterigmatocystin and the duplex in the covalent adduct and identified the intermolecular proton-proton NOEs that define the orientation and mode of binding of the mutagen to duplex DNA. The analysis was aided by intermolecular NOEs between the sterigmatocystin protons with both the major groove and minor groove protons of the DNA. The molecular dynamics calculations were aided by 180 intramolecular nucleic acid constraints, 16 intramolecular sterigmatocystin constraints, and 56 intermolecular distance constraints between sterigmatocystin and the nucleic acid protons in the adduct. The sterigmatocystin chromophore intercalates between the [ST]G4.C5 and T3.A6 base pairs and stacks predominantly over the modified guanine ring in the adduct duplex. The overall conformation of the DNA remains right-handed on adduct formation with unwinding of the helix, as well as widening of the minor groove. Parallel NMR studies on the sterigmatocystin-d(A1-A2-A3-G4-C5-T6-T7-T8) covalent adduct (two sterigmatocystins per duplex) provide supportive evidence that the mutagen covalently adducts the N7 position of G4 and its chromophore intercalates to the 5' side of the guanine and stacks over it. The present NMR-molecular dynamics studies that define a detailed structure for the sterigmatocystin-DNA adduct support key structural conclusions proposed previously on the basis of a qualitative analysis of NMR parameters for the adduct formed by the related food mutagen aflatoxin B1 and DNA [Gopalakrishnan, S., Harris, T. M., & Stone, M. P. (1990) Biochemistry 29, 10438-10448].     

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.     

Antiproliferative and antimalarial anthraquinones of Scutia myrtina from the Madagascar forest

Bioassay-guided fractionation of an ethanol extract of a Madagascar collection of the bark of Scutia myrtina led to the isolation of three new anthrone–anthraquinones, scutianthraquinones A, B and C (1–3), one new bisanthrone–anthraquinone, scutianthraquinone D (4), and the known anthraquinone, aloesaponarin I (5). The structures of all compounds were determined using a combination of 1D and 2D NMR experiments, including COSY, TOCSY, HSQC, HMBC, and ROESY sequences, and mass spectrometry. All the isolated compounds were tested against the A2780 human ovarian cancer cell line for antiproliferative activities, and against the chloroquine-resistant Plasmodium falciparum strains Dd2 and FCM29 for antiplasmodial activities. Compounds 1, 2 and 4 showed weak antiproliferative activities against the A2780 ovarian cancer cell line, while compounds 1–4 exhibited moderate antiplasmodial activities against P. falciparum Dd2 and compounds 1, 2, and 4 exhibited moderate antiplasmodial activities against P. falciparum FCM29.     

Crystal structure of trioxacarcin A covalently bound to DNA

We report a crystal structure that shows an antibiotic that extracts a nucleobase from a DNA molecule ‘caught in the act’ after forming a covalent bond but before departing with the base. The structure of trioxacarcin A covalently bound to double-stranded d(AACCGGTT) was determined to 1.78 Å resolution by MAD phasing employing brominated oligonucleotides. The DNA–drug complex has a unique structure that combines alkylation (at the N7 position of a guanine), intercalation (on the 3′-side of the alkylated guanine), and base flip-out. An antibiotic-induced flipping-out of a single, nonterminal nucleobase from a DNA duplex was observed for the first time in a crystal structure.     

Conformation and Configuration at the Central Amine Nitrogen of a Nucleotide Adduct of the Carcinogen 2-(Acetylamino)flourene as Studied by 13C and 15N NMR Spectroscopy

Abstract

The conformation and configuration at the central nitrogen of the adduct 8-(N-fluoren-2-ylamino)-2′-deoxyguanosine 5′-monophosphate has been investigated by high-field ¹³C and ¹⁵N NMR spectroscopy. One-bond nitrogen-hydrogen coupling constants and ¹³C chemical shifts for the adduct as well as for the model compounds diphenylamine, 4-nitrodiphenylamine and 2-aminofluorene have been measured in nonaqueous solutions. The data indicate a near planar configuration at the amine nitrogen that links the guanine and fluorene rings of the adduct. The orientations about the guanyl-nitrogen and fluorenyl-nitrogen bonds place the two ring systems in either perpendicular (Type A) or helical (Type B) conformations. It is suggested, based on structural similarities to diarylamines, that the C-N-C bond angle of the adduct is greater than 120° in order to reduce unfavorable steric interactions between the two ring systems. Space-filling molecular models of the adduct in duplex DNA show that the aminofluorene moiety can be oriented into both Type A and Type B conformations within the major groove. The configuration at nitrogen of diphenylamine, 4-nitrodiphenylamine and 2-aminofluorene has also been examined.     

Copper generated reactive oxygen leads to formation of lysine-DNA adducts

This work describes the addition of a lysine derivative to guanine base in a nucleoside, an oligonucleotide, and to a large DNA that occurs via oxidation by copper generated reactive oxygen species. Nucleophiles present during oxidation leads to the formation of adducts. In this work, 2′-deoxyguanosine is oxidized by copper generated reactive oxygen species in the presence of a lysine derivative, Nα-acetyl-lysine methyl ester. Under these conditions the guanidinohydantoin-lysine adduct is observed in a relative yield of 27% when compared to other guanine oxidation products. MS² strongly supports that lysine is added to the 5-position during the formation of guanidinohydantoin-lysine. A fourteen-nucleotide DNA duplex was oxidized under similar conditions. Digestion showed formation of the same guanidinohydantoin-lysine nucleoside. The reaction was then examined on a 392-nucleotide DNA substrate. Oxidation in the presence of the lysine ester showed adduct formation as stops in a primer extension assay. Adducts predominately formed at a 5′-GGG at position 415. Six of the seven sites that showed reaction greater than 3-fold above background were guanine sites. We conclude from this study that copper can catalyze the formation of DNA-protein adducts and may form in cells with elevated copper and oxidative stress.     

Alkylation of guanine in DNA by S23906-1, a novel potent antitumor compound derived from the plant alkaloid acronycine

The discovery of a new DNA-targeted antitumor agent is a challenging enterprise, and the elucidation of its mechanism of action is an essential first step in investigating the structural and biological consequences of DNA modification and to guide the rational design of analogues. Here, we have dissected the mode of action of the newly discovered antitumor agent S23906-1. Gel retardation experiments reveal that the diacetate compound S23906-1 and its monoacetate analogue S28687 form highly stable covalent adducts with DNA. The covalent adducts formed between S23906-1 and a 7-bp hairpin oligonucleotide duplex were identified by spectrometry. In contrast, the inactive compound S23907, lacking the two acetate groups of S23906-1, fails to yield covalent DNA adducts, indicating that the C1-C2 functionality is the DNA reactive moiety. DNase I footprinting and DNA alkylation experiments indicate that S23906-1 reacts primarily with guanine residues. A 30-mer oligonucleotide containing only G.C bp forms highly stable complexes with S23906-1 and S28687, whereas the equivalent A.T oligonucleotide is not a good substrate for these two drugs. The use of an oligonucleotide duplex containing inosines instead of guanosines identifies the guanine 2-amino group exposed in the minor groove of DNA as the potential reactive site. The reactivity of S23906-1 toward the guanine-N2 group was independently confirmed by fluorescence spectroscopy. Covalent DNA adducts were also identified in the genomic DNA of B16 melanoma cells exposed to S23906-1, and the specific accumulation of the drug in the nucleus of the cells was visualized by confocal microscopy. The elucidation of the mechanism of action of this highly potent anticancer agent opens a new field for future drug design.     

Solution structure of a guanine-N7-linked complex of the mitomycin C metabolite 2,7-diaminomitosene and DNA. Basis of sequence selectivity

2,7-Diaminomitosene (2,7-DAM), the major metabolite of the antitumor antibiotic mitomycin C, forms DNA adducts in tumor cells. 2,7-DAM was reacted with the deoxyoligonucleotide d(GTGGTATACCAC) under reductive alkylation conditions. The resulting DNA adduct was characterized as d(G-T-G-[M]G-T-A-T-A-C-C-A-C) (5), where [M]G stands for a covalently modified guanine, linked at its N7-position to C10 of the mitosene. The adducted oligonucleotide complements with itself, retaining 2-fold symmetry in the 2:1 drug-duplex complex, and provides well-resolved NMR spectra, amenable for structure determination. Adduction at the N7-position of G4 ([M]G, 4) is characterized by a downfield shift of the G4(H8) proton and separate resonances for G4(NH(2)) protons. We assigned the exchangeable and nonexchangeable proton resonances of the mitosene and the deoxyoligonucleotide in adduct duplex 5 and identified intermolecular proton-proton NOEs necessary for structural characterization. Molecular dynamics computations guided by 126 intramolecular and 48 intermolecular distance restraints were performed to define the solution structure of the 2,7-DAM-DNA complex 5. A total of 12 structures were computed which exhibited pairwise rmsd values in the 0.54-1.42 A range. The 2,7-DAM molecule is anchored in the major groove of DNA by its C10 covalently linked to G4(N7) and is oriented 3' to the adducted guanine. The presence of 2,7-DAM in the major groove does not alter the overall B-DNA helical structure. Alignment in the major groove is a novel feature of the complexation of 2,7-DAM with DNA; other known major groove alkylators such as aflatoxin, possessing aromatic structural elements, form intercalated complexes. Thermal stability properties of the 2,7-DAM-DNA complex 5 were characteristic of nonintercalating guanine-N7 alkylating agents. Marked sequence selectivity of the alkylation by 2,7-DAM was observed, using a series of oligonucleotides incorporating variations of the 5'-TGGN sequence as substrates. The selectivity correlated with the sequence specificity of the negative molecular electrostatic potential of the major groove, suggesting that the alkylation selectivity of 2,7-DAM is determined by sequence-specific variation of the reactivity of the DNA. The unusual, major groove-aligned structure of the adduct 5 may account for the low cytotoxicity of 2,7-DAM.     

Long-range oxidative damage in duplex DNA: the effect of bulged G in a G–C tract and tandem G/A mispairs

Two series of duplex DNA oligomers were prepared having an anthraquinone derivative (AQ) covalently linked at a 5′-terminus. Irradiation of the AQ at 350 nm leads to injection of an electron hole (radical cation) into the DNA. The radical cation migrates through the DNA causing reaction primarily at G_n sequences. In one series, GA tandem mispairs are inserted between GG steps to assess the effect of the mispair on the transport of the radical cation, reaction (damage) caused by the radical cation at the mispair, and repair of the resulting damage by formamidopyrimidine DNA glycosylase (Fpg). In the second series, a bulged guanine in a G₃C₂ sequence is interposed between the GG steps. These experiments reveal that neither G/A tandem mispairs nor bulged guanines are significant barriers to long-range charge migration in DNA. The radical cation does not cause reaction at guanines in the G/A tandem mispair. Reaction does occur at the bulged guanine, but it is repaired by Fpg.     

Distant Neighbor Base Sequence Context Effects in Human Nucleotide Excision Repair of a Benzo[a]pyrene-derived DNA Lesion

The effects of non-nearest base sequences, beyond the nucleotides flanking a DNA lesion on either side, on nucleotide excision repair (NER) in extracts from human cells were investigated. We constructed two duplexes containing the same minor groove-aligned 10S (+)-trans-anti-B[a]P-N²-dG (G?) DNA adduct, derived from the environmental carcinogen benzo[a]pyrene (B[a]P): 5′-C-C-A-T-C-G?-C-T-A-C-C-3′ (CG?C-I), and 5′-C-A-C3-A4-C5-G?-C-A-C-A-C-3′ (CG?C-II). We used polyacrylamide gel electrophoresis to compare the extent of DNA bending, and molecular dynamics simulations to analyze the structural characteristics of these two DNA duplexes. The NER efficiencies are 1.6(± 0.2)-fold greater in the case of the CG?C-II than the CG?C-I sequence context in 135-mer duplexes. Gel electrophoresis and self-ligation circularization experiments revealed that the CG?C-II duplex is more bent than the CG?C-I duplex, while molecular dynamics simulations showed that the unique -C3-A4-C5- segment in the CG?C-II duplex plays a key role. The presence of a minor groove-positioned guanine amino group, the Watson-Crick partner to C3, acts as a wedge; facilitated by a highly deformable local -C3-A4- base step, this amino group allows the B[a]P ring system to produce a more enlarged minor groove in CG?C-II than in CG?C-I, as well as a local untwisting and enlarged and flexible Roll only in the CG?C-II sequence. These structural properties fit well with our earlier findings that in the case of the family of minor groove 10S (+)-trans-anti-B[a]P-N²-dG lesions, flexible bends and enlarged minor groove widths constitute NER recognition signals, and extend our understanding of sequence context effects on NER to the neighbors that are distant to the lesion.     

Influence of an exocyclic guanine adduct on the thermal stability, conformation, and melting thermodynamics of a DNA duplex.

As part of an overall program to characterize the impact of mutagenic lesions on the physiochemical properties of DNA, we report here the results of a comparative spectroscopic study on pairs of DNA duplexes both with and without an exocyclic guanine lesion. Specifically, we have studied a family of four 13-mer duplexes of the form d(CGCATGYGTACGC).d(GCGTACZCATGCG) in which Y is either the normal deoxyguanosine residue (G) or the exocyclic guanine adduct 1,N2-propanodeoxyguanosine (X), while Z is either deoxycytosine (C) or deoxyadenosine (A). Thus, the four duplexes studied, which can be designated by the identity of their central Y.Z base pair, are a Watson-Crick duplex (GC), a duplex with a central mismatch (GA), and two duplexes with exocyclic guanine lesions (X), that differ only by the base opposite the lesion (XC and XA). The data derived from our spectroscopic measurements on these four duplexes have allowed us to evaluate the influence of the exocyclic guanine lesion, as well as the base opposite the lesion, on the conformation, thermal stability, and melting energetics of the host DNA duplex. To be specific, our circular dichroism (CD) spectra show that the exocyclic guanine lesion induces alterations in the duplex structure, while our temperature-dependent optical measurements reveal that these lesion-induced structural alterations reduce the thermal stability, the transition enthalpy, and the transition free energy of the duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Sequence Dependence of Human Nucleotide Excision Repair Efficiencies of Benzo[a]pyrene-derived DNA Lesions: Insights into the Structural Factors that Favor Dual Incisions

Nucleotide excision repair (NER) is a vital cellular defense system against carcinogen-DNA adducts, which, if not repaired, can initiate cancer development. The structural features of bulky DNA lesions that account for differences in NER efficiencies in mammalian cells are not well understood. In vivo, the predominant DNA adduct derived from metabolically activated benzo[a]pyrene (BP), a prominent environmental carcinogen, is the 10S (+)-trans-anti-[BP]-N²-dG adduct (G*), which resides in the B-DNA minor groove 5′-oriented along the modified strand. We have compared the structural distortions in double-stranded DNA, imposed by this adduct, in the different sequence contexts 5′-…CGG*C…, 5′-…CG*GC…, 5′-…CIG*C… (I is 2′-deoxyinosine), and 5′-…CG*C…. On the basis of electrophoretic mobilities, all duplexes manifest moderate bends, except the 5′-…CGG*C…duplex, which exhibits an anomalous, slow mobility attributed to a pronounced flexible kink at the site of the lesion. This kink, resulting from steric hindrance between the 5′-flanking guanine amino group and the BP aromatic rings, both positioned in the minor groove, is abolished in the 5′-…CIG*C…duplex (the 2′-deoxyinosine group, I, lacks this amino group). In contrast, the sequence-isomeric 5′-…CG*GC…duplex exhibits only a moderate bend, but displays a remarkably increased opening rate at the 5′-flanking base pair of G*, indicating a significant destabilization of Watson-Crick hydrogen bonding. The NER dual incision product yields were compared for these different sequences embedded in otherwise identical 135-mer duplexes in cell-free human HeLa extracts. The yields of excision products varied by a factor of as much as ∼ 4 in the order 5′-...CG*GC…> 5′...CGG*C…≥ 5′...CIG*C…≥ 5′-…CG*C…. Overall, destabilized Watson-Crick hydrogen bonding, manifested in the 5′-...CG*GC...duplex, elicits the most significant NER response, while the flexible kink displayed in the sequence-isomeric 5′-...CGG*C...duplex represents a less significant signal in this series of substrates. These results demonstrate that the identical lesion can be repaired with markedly variable efficiency in different local sequence contexts that differentially alter the structural features of the DNA duplex around the lesion site.     

Alanine scan-guided synthesis and biological evaluation of analogues of culicinin D,a potent anticancer peptaibol

Culicinin D (1), a 10 amino acid peptaibol originally isolated from Culicinomyces clavisporus, exhibits potent activity against a range of cancer cell lines. Building on our previous work exploring the structure–activity relationship (SAR) of the unusual (2S,4S,6R)-AHMOD residue, a series of analogues of culicinin D were prepared to further investigate the SAR of these peptaibols. Alanine scanning of a potent and readily accessible analogue 23 revealed the effect of each residue on antiproliferative activity, and a small panel of analogues were prepared to explore the SAR of the non-natural amino acid residue (2S,4R)-AMD. Results from the alanine scan were used to design an expanded library of culicinin D analogues, leading to the discovery of cyclohexylalanine analogue 52, which exhibited better antiproliferative activity than the natural product 1.     

Studies on interactions of carbazole derivatives with DNA,cell image,and cytotoxicity

DNA-binding agents have been considered as an established opportunity for the development of anticancer drugs and DNA fluorescence probes. This work reported the synthesis of two novel carbazole derivatives (1 and 2) and investigated their DNA binding properties, living cell image, and cytotoxicity. The results demonstrated that both compounds presented the higher binding affinity to G-quadruplex than to duplex DNA by means of UV–Vis absorption titration and fluorescent intercalator displacement. Continuous variation analysis indicated that their binding stoichiometries of the compound/G-quadruplex were near 2 except the compound/bcl-2. Circular dichroism spectra showed that both compounds had no influence on the conformation of G-quadruplexes. Fluorescence titrations indicated that 2 had the potential to be a G-quadruplex selective fluorescent probe, while 1 could be used as a fluorescent probe for duplex DNA. Confocal fluorescence images indicated that both compounds could enter the living HepG2 cells, and 1 mainly located in nucleus whereas 2 mainly distributed in cytoplasm. DNase and RNase digest tests indicated that both compounds could enter into the nucleus and interact with duplex DNA, especially, 2 could interact with RNA and/or G-quadruplex DNA. They also exhibited an obvious antiproliferative activity to HepG2 by using MTT assays, with IC₅₀ values of 2.7 and 9.5?μM for 1 and 2, respectively.     

Structural insights by molecular dynamics simulations into specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C

The benzetheno exocyclic adduct of the cytosine (C) base (pBQ-C) is a product of reaction between DNA and a stable metabolite of the human carcinogen benzene, p-benzoquinone (pBQ). We reported previously that the pBQ-C-containing duplex is a substrate for the human AP endonuclease (APE1), an enzyme that cleaves an apurinic/apyrimidinic (AP) site from double stranded DNA. In this work, using molecular dynamics simulation (MD), we provided a structural explanation for the recognition of the pBQ-C adduct by APE1. Molecular modeling of the DNA duplex containing pBQ-C revealed significant displacement of this adduct toward the major groove with pronounced kinking of the DNA at the lesion site, which could serve as a structural element recognized by the APE1 enzyme. Using 3 ns MD it was shown that the position of the pBQ-C adduct is stabilized by two hydrogen bonds formed between the adduct and the active site amino acids Asp 189 and Ala 175. The pBQ-C/APE1 complex, generated by MD, has a similar hydrogen bond network between target phosphodiester bond at the pBQ-C site and key amino acids at the active site, as in the crystallographically determined APE1 complexed with an AP site-containing DNA duplex. The position of the adduct at the enzyme active site, together with the hydrogen bond network, suggests a similar reaction mechanism for phosphodiester bond cleavage of oligonucleotide containing pBQ-C as reported for the AP site.     

Strong structural effect of the position of a single acetylaminofluorene adduct within a mutation hot spot.

The NarI restriction enzyme recognition site, G1G2CG3CC, has been identified as a hotspot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF) on the basis of a forward mutation assay in plasmid pBR322 in the bacterium Escherichia coli. AAF binds primarily to the C-8 position of guanine residues, and the three guanines of the NarI site are similarly reactive. Despite this similar chemical reactivity, only binding of AAF to the G3 residue causes the -2 frameshift mutations. To study the mechanisms underlying the specificity of the mutagenic processing further, we monitored the structural changes induced by a single AAF adduct within the NarI site by means of CD spectroscopy and thermal denaturation. The NarI sequence was studied as part of the 12-mer ACCGGCGCCACA. The purification and characterization of the three isomers having a single AAF adduct covalently bound to one of the three guanines of this 12 mer are described. The analysis of the melting profiles of the duplexes formed when these three isomers are annealed with the oligonucleotide of complementary sequence shows the same destabilizing effect of the AAF adduct on the three DNA helices. It is also shown, from the CD spectra, that modification of guanine G1 or G2 by AAF does not induce major changes in the helical structure of DNA. On the other hand, modification of guanine G3 induces a change in the CD signal that suggests the formation of a local left handed structure within the 12-mer duplex. These results show the polymorphic nature of the DNA structure in the vicinity of an AAF adduct.