Reaction of trans-2-chlorovinylarsine oxide with polydeoxynucleotides

Trans-2-chlorovinylarsine oxide (in DCl/acetone-d6) was added to various polydeoxynucleotides. The arsenical did react with poly[dG].poly[dC], releasing guanine, and resulting in a partial apurinic duplex.     

Reaction of trans-2-Chlorovinylarsine Oxide with Polydeoxynucleotides

ABSTRACT

Trans-2-chlorovinylarsine oxide (in DCl/acetone-d₆) was added to various polydeoxynucleotides. The arsenical did react with poly[dG]poly[dC], releasing guanine, and resulting in a partial apurinic duplex.     

Dependence of conformations of benzo[a]pyrene diol epoxide-DNA adducts derived from stereoisomers of different tumorigenicities on base sequence

The conformations of covalent adducts derived from the binding of the highly tumorigenic stereoisomer (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyren e [(+)-anti-BPDE] and its nontumorigenic (-)-anti-BPDE isomer with poly[(dG).(dC)], poly[(dG-dC).(dG-dC)], poly[(dT-dC).(dG-dA)], and poly[(dA-dC).(dG-dT)] were investigated by employing UV absorbance and linear dichroism methods. The degrees of orientation of the BPDE residues (bound covalently to N2 of deoxyguanosine), relative to the DNA bases, are most pronounced in the alternating and nonalternating (dG).(dC) polymers and decrease in polymers with neighboring dA.dT base pairs. The tumorigenic (+)-anti-BPDE isomer gives rise predominantly to external (solvent-exposed) site II adducts, while the (-)-enantiomer gives rise predominantly to site I adducts with significant carcinogen-nucleoside interactions. In the mixed (dA-dC).(dG-dT) and (dT-dC).(dG-dA) copolymers, the (+)-anti-BPDE isomer also binds predominantly to N2 of deoxyguanosine, but the adducts are weakly oriented with respect to the DNA bases. The incidence of site II adducts is considerably reduced as compared to the (dG).(dC) and (dG-dC).(dG-dC) polymers, and there is a greater proportion of site I adducts; the presence of a significant proportion of unordered adduct forms is also suggested from the diffuseness and broadness of the absorption spectra in the dA.dT base pair containing polymers. The preference of formation of site II adducts in dG-rich sequences in the case of the biologically highly active (+)-anti-BPDE isomer is discussed in terms of the known binding and mutation spectra.     

Comparative laser spectroscopic study of DNA and polynucleotide adducts from the (+)-anti-diol epoxide of benzo[a]pyrene

A recently developed methodology [Jankowiak, R., Lu, P., Small, G. J., and Geacintov, N. E. (1990) Chem. Res. Toxicol. 3, 39-46], which combines fluorescence line narrowing spectroscopy at 4.2 K with non-line-narrowed (S2----S0 laser excitation) fluorescence spectroscopy at 77 K and fluorescence quenching, is used to characterize adducts formed from (+)-anti-BPDE and the alternating copolymers poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), the nonalternating poly (dG).poly(dC), single-strand poly(dG), and the oligonucleotide d(ATATGTATA). Detailed comparisons of the fluorescence spectra and quenching (with acrylamide) of the properties of the adducts with those of (+)-anti-BPDE-DNA adducts are made. Fluorescence spectra of the trans and cis isomers of the adduct formed from guanosine monophosphate and the adducts of d(ATATGTATA) are used to assign the stereochemistry of the two major DNA adducts as trans-N2-dG moieties which occupy two different DNA sites. Evidence for the existence of minor cis-type guanine adducts is provided. Finally, a fourth type of DNA adduct (minor) is identified and assigned as trans-N6-dA.     

Bulge migration of the malondialdehyde OPdG DNA adduct when placed opposite a two-base deletion in the (CpG)3 frameshift hotspot of the Salmonella typhimurium hisD3052 gene

The OPdG adduct N (2)-(3-oxo-1-propenyl)dG, formed in DNA exposed to malondialdehyde, was introduced into 5'-d(ATCGC XCGGCATG)-3'.5'-d(CATGCCGCGAT)-3' at pH 7 (X = OPdG). The OPdG adduct is the base-catalyzed rearrangement product of the M 1dG adduct, 3-(beta- d-ribofuranosyl)pyrimido[1,2- a]purin-10(3 H)-one. This duplex, named the OPdG-2BD oligodeoxynucleotide, was derived from a frameshift hotspot of the Salmonella typhimuium hisD3052 gene and contained a two-base deletion in the complementary strand. NMR spectroscopy revealed that the OPdG-2BD oligodeoxynucleotide underwent rapid bulge migration. This hindered its conversion to the M 1dG-2BD duplex, in which the bulge was localized and consisted of the M 1dG adduct and the 3'-neighbor dC [ Schnetz-Boutaud, N. C. , Saleh, S. , Marnett, L. J. , and Stone, M. P. ( 2001) Biochemistry 40, 15638- 15649 ]. The spectroscopic data suggested that bulge migration transiently positioned OPdG opposite dC in the complementary strand, hindering formation of the M 1dG-2BD duplex, or alternatively, reverting rapidly formed intermediates in the OPdG to M 1dG reaction pathway when dC was placed opposite from OPdG. The approach of initially formed M 1dG-2BD or OPdG-2BD duplexes to an equilibrium mixture of the M 1dG-2BD and OPdG-2BD duplexes was monitored as a function of time, using NMR spectroscopy. Both samples attained equilibrium in approximately 140 days at pH 7 and 25 degrees C.     

Preferential alkylation by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of guanines with guanines as neighboring bases in DNA

The base sequence of DNA has been shown to influence the kinds and amounts of alkylation of purine bases by N-methyl-N-nitrosourea [W. T. Briscoe and L-E. Cotter, Chem. Biol. Interact. 56, 321 (1985)]. In the present study, the alkylation of DNA polymers of defined sequence by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) has been investigated. The assay involved treating poly (dG).poly(dC), poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT), poly(dA-dG).poly(dC-dT), and calf thymus DNA with BCNU, followed by hydrolysis to release the modified purine bases and separation and quantitation of these by HPLC. Analysis of the results revealed that there was a 24-fold increase of 7-(beta-hydroxyethyl)guanine (HOEtG) in poly(dG).poly(dC) relative to poly(dA-dG).poly(dC-dT). There was also a 3-fold increase in HOEtG in poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and calf thymus DNA relative to poly(dA-dG).poly(dC-dT). A 2- to 4-fold increase of 7(beta-aminoethyl)guanine (AmEtG) was observed in poly(dG-dC).poly(dG-dC) relative to the other polymers tested. This study has determined that guanines in certain base sequences in polydeoxyribonucleotides are more susceptible to BCNU alkylation at the N-7 position than guanines in other sequences.     

1,N2-Etheno-2'-deoxyguanosine adopts the syn conformation about the glycosyl bond when mismatched with deoxyadenosine

The oligodeoxynucleotide 5'-CGCATXGAATCC-3'·5'-GGATTCAATGCG-3' containing 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdG) opposite deoxyadenosine (named the 1,N(2)-εdG·dA duplex) models the mismatched adenine product associated with error-prone bypass of 1,N(2)-εdG by the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) and by Escherichia coli polymerases pol I exo(-) and pol II exo(-). At pH 5.2, the T(m) of this duplex was increased by 3 °C as compared to the duplex in which the 1,N(2)-εdG lesion is opposite dC, and it was increased by 2 °C compared to the duplex in which guanine is opposite dA (the dG·dA duplex). A strong NOE between the 1,N(2)-εdG imidazole proton and the anomeric proton of the attached deoxyribose, accompanied by strong NOEs to the minor groove A(20) H2 proton and the mismatched A(19) H2 proton from the complementary strand, establish that 1,N(2)-εdG rotated about the glycosyl bond from the anti to the syn conformation. The etheno moiety was placed into the major groove. This resulted in NOEs between the etheno protons and T(5) CH(3). A strong NOE between A(20) H2 and A(19) H2 protons established that A(19), opposite to 1,N(2)-εdG, adopted the anti conformation and was directed toward the helix. The downfield shifts of the A(19) amino protons suggested protonation of dA. Thus, the protonated 1,N(2)-εdG·dA base pair was stabilized by hydrogen bonds between 1,N(2)-εdG N1 and A(19) N1H(+) and between 1,N(2)-εdG O(9) and A(19)N(6)H. The broad imino proton resonances for the 5'- and 3'-flanking bases suggested that both neighboring base pairs were perturbed. The increased stability of the 1,N(2)-εdG·dA base pair, compared to that of the 1,N(2)-εdG·dC base pair, correlated with the mismatch adenine product observed during the bypass of 1,N(2)-εdG by the Dpo4 polymerase, suggesting that stabilization of this mismatch may be significant with regard to the biological processing of 1,N(2)-εdG.     

Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro.

The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6-C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BP-DNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of 1,4-dioxo-2-butene-derived adducts of 2'-deoxyadenosine and 2'-deoxycytidine in oxidized DNA

Oxidation of deoxyribose in DNA produces a variety of electrophilic residues that are capable of reacting with nucleobases to form adducts such as M(1)dG, the pyrimidopurinone adduct of dG. We now report that deoxyribose oxidation in DNA leads to the formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA. We previously demonstrated that these adducts arise in reactions of nucleosides and DNA with trans-1,4-dioxo-2-butene, the beta-elimination product of the 2-phosphoryl-1,4-dioxobutane residue arising from 5'-oxidation of deoxyribose in DNA, and with cis-1,4-dioxo-2-butene, a metabolite of furan. Treatment of DNA with enediyne antibiotics capable of oxidizing the 5'-position of deoxyribose (calicheamicin and neocarzinostatin) led to a concentration-dependent formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA, while the antibiotic bleomycin, which is capable of performing only 4-oxidation of deoxyribose, did not give rise to the adducts. The nonspecific DNA oxidant, gamma-radiation, also produced the adducts that represented approximately 0.1% of the 2-phosphoryl-1,4-dioxobutane residues formed during the irradiation. These results suggest that the oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA could represent endogenous DNA lesions arising from oxidative stresses that also give rise to other DNA adducts.     

Inhibition of deoxyribonucleic acid polymerases of human leukemic leukocytes by 2',3'-dideoxythymidine triphosphate

The effects of 2',3'-dideoxythymidine 5'-triphosphate (d₂TTP) on the activities of DNA polymerases α and β isolated from leukocytes of a patient with acute myelogenous leukemia have been examined. DNA polymerase β was more sensitive than DNA polymerase a to d₂TTP inhibition; the concentration of d₂TTP required for a 50 per cent inhibition of enzyme activity was 20-fold lower for DNA polymerase β than for DNA polymerase α in assays with activated DNA template. A similar difference in sensitivity was observed also when synthetic primer templates such as (dT) ~₁₅· (dA)_n or (dT) ~ ₁₅· (A)_n were used. However, with (dG) ~₁₅ · (dC)_n primer template, neither DNA polymerase α nor DNA polymerase β activity was inhibited by d₂TTP. Kinetic analysis with activated DNA template showed that the d₂TTP inhibition was competitive with dTTP but noncompetitive with dGTP; the values of the K_m for dTTP and K_m for d₂TTP for the DNA polymerase α were approximately the same, while for DNA polymerase β the K_m was twenty times more than K_i. Neither DNA polymerase α nor DNA polymerase β had any exonuclease activity and, therefore, the resistance of DNA polymerase α to d₂TTP inhibition was not due to exonuclease activity. The extent of d₂TTP inhibition was not altered significantly by changes in the concentration of either the template or the enzyme. Preincubation of the inhibitor with either the template or the enzyme was not necessary for inhibition. The compound was inhibitory even when added after the initiation of the reaction.     

Replication bypass of N2-deoxyguanosine interstrand cross-links by human DNA polymerases η and ι

DNA-interstrand cross-links (ICLs) can be repaired by biochemical pathways requiring DNA polymerases that are capable of translesion DNA synthesis (TLS). The anticipated function of TLS polymerases in these pathways is to insert nucleotides opposite and beyond the linkage site. The outcome of these reactions can be either error-free or mutagenic. TLS-dependent repair of ICLs formed between the exocyclic nitrogens of deoxyguanosines (N(2)-dG) can result in low-frequency base substitutions, predominantly G to T transversions. Previously, we demonstrated in vitro that error-free bypass of a model acrolein-mediated N(2)-dG ICL can be accomplished by human polymerase (pol) κ, while Rev1 can contribute to this bypass by inserting dC opposite the cross-linked dG. The current study characterized two additional human DNA polymerases, pol η and pol ι, with respect to their potential contributions to either error-free or mutagenic bypass of these lesions. In the presence of individual dNTPs, pol η could insert dA, dG, and dT opposite the cross-linked dG, but incorporation of dC was not apparent. Further primer extension was observed only from the dC and dG 3' termini, and the amounts of products were low relative to the matched undamaged substrate. Analyses of bypass products beyond the adducted site revealed that dG was present opposite the cross-linked dG in the majority of extended primers, and short deletions were frequently detected. When pol ι was tested for its ability to replicate past this ICL, the correct dC was preferentially incorporated, but no further extension was observed. Under the steady-state conditions, the efficiency of dC incorporation was reduced ~500-fold relative to the undamaged dG. Thus, in addition to pol κ-catalyzed error-free bypass of N(2)-dG ICLs, an alternative, albeit low-efficiency, mechanism may exist. In this pathway, either Rev1 or pol ι could insert dC opposite the lesion, while pol η could perform the subsequent extension.     

Interaction between A 3-nitrobenzothiazolo (3,2-a) quinolinium antitumour drug and deoxyribonucleic acid

The interaction of 3-nitrobenzothiazolo (3,2-a) quinolinium (NBQ) perchlorate with DNA was studied by u.v.-visible and fluorescence spectrophotometry as well as by hydrodynamic methods. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts, and the fluorescence was quenched. Binding parameters, determined from spectrophotometric measurements by Scatchard analysis according to an excluded-site model, indicated a binding constant of 2.4 × 10⁵ M^?1 for calf thymus DNA at ionic strength 0.01. The interaction was markedly suppressed by increasing the salt concentration. Binding to the GC-rich DNA of Micrococcus lysodeikticus was weaker than the binding to calf thymus DNA at ionic strength 0.01. NBQ increased the viscosity of sonicated rod-like DNA fragments, producing a calculated increment in length of 2.4 Å/bound drug molecule. It removed and reversed the supercoiling of closed circular duplex plasmid pBR322 DNA by virtue of a helix-unwinding angle estimated as approximately 13°/bound ligand molecule. We conclude that the binding of NBQ to DNA occurs by a mechanism of intercalation, which probably accounts for its reported antitumor activity.     

Combined effects of okadaic acid and cadmium on lipid peroxidation and DNA bases modifications (m5dC and 8-(OH)-dG) in Caco-2 cells

Okadaic acid (OA) is a marine toxin, a tumour promoter and an inducer of apoptosis. It mainly inhibits protein-phosphatases, protein synthesis and enhances lipid peroxidation. Cadmium (Cd) is known to be carcinogenic in animals and humans (group 1 according to the International Agency for Research on Cancer (IARC) classification). Cd also induces oxidative stress in living organisms. Since they are sometimes found simultaneously in mussels, we have evaluated in the present investigation, the lipid peroxidation, as malondialdehyde (MDA) production, in the variation of the ratios of 8-(OH)-dG/10⁵dG and m⁵dC/ (dC + m⁵dC) induced by OA and/or Cd in Caco-2 cells. When cells were treated exclusively by OA (15 ng/ml) or Cd (0.625 and 5μg/ml) for 24 h, protein synthesis was inhibited (by 42 ± 5%, 18 ± 13%, and 90 ± 4% respectively) while MDA production was 2235 ± 129, 1710 ± 20, and 11496 ± 1624 pmol/mg protein respectively. In addition, each toxicant induced modified bases in DNA; increases in oxidised bases and methylated dC. The combination of OA and cadmium was more cytotoxic and caused more DNA base modifications; the ratio m⁵dC/(m⁵dC+dC) was increased from 3 ± 0.15 to 9 ± 0.15 and the ratio 8-(OH)-dG/10⁵ dG also (from 36 ± 2 to 76 ± 6). The combination of OA and Cd also increased the level of MDA (16874 ± 2189 pmole/mg protein). The present results strongly suggest that DNA damage resulting from the oxidative stress induced by these two toxicants may significantly contribute to increasing their carcinogenicity via epigenetic processes. Received: 28 September 1999 / Accepted: 10 January 2000     

Inhibition of DNA primase by nucleoside triphosphates and their arabinofuranosyl analogs

DNA primase (EC 2.7.7.6) produces an RNA oligomer of approximately 10 bases, which is required by DNA polymerase alpha (EC 2.7.7.7) for the initiation of DNA synthesis. We partially purified DNA primase from acute lymphocytic leukemia cells from patients using several chromatography columns. Poly(dT) and poly(dC), but not poly(dA) or poly(dG), were good templates for ribonucleoside triphosphate (rNTP)-dependent DNA synthesis (i.e., DNA primase activity), and they were used in the study of the effect of natural and arabinofuranosyl nucleoside triphosphates on DNA primase activity. The Km for GTP in the poly(dC) primase assay was approximately 175 microM. All noncomplementary natural rNTPs and deoxyribonucleoside triphosphates (dNTPs) inhibited poly(dC) primase activity to a similar extent (Ki values of ATP and CTP were 610 and 517 microM, respectively). 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) and 9-beta-D-arabinofuranosyladenine 5'-triphosphate (araATP) were more potent inhibitors of poly(dC) primase activity than were CTP and ATP (Ki values were approximately 125 microM). araCTP, araATP, CTP, and ATP inhibited DNA primase activity in a manner competitive with GTP. The concentration required to inhibit poly(dC) DNA primase activity by 50% was determined for a number of arabinofuranosyl nucleoside triphosphate analogs, and the relative potency of inhibition of DNA primase activity was as follows: rNTP = dNTP = 5-aza-dCTP less than ara-5-azaCTP = araTTP = araATP = araCTP less than 2-fluoro-araATP = 2'-azido-2'-deoxy araCTP less than 2'-fluoro-araTTP = 2'-fluoro-5-iodo-araCTP = 2'-fluoro-5-methyl-araCTP. In the poly(dT) primase assay ATP did not follow classic Michaelis-Menten kinetics (ATP exhibited positive cooperativity with a Hill coefficient of 2.0). However, this assay was very sensitive to araCTP (apparent Ki of 25 microM). In summary, these experiments suggested that DNA primase is controlled by the levels of ribonucleoside triphosphates, and that the perturbation of these pools by any agent could lead to the inhibition of DNA primase and thereby inhibit DNA synthesis. Furthermore, aranucleoside triphosphate analogs directly inhibited DNA primase, and it is possible that this effect may contribute to the cytotoxicity of these compounds.     

Coupling products of nucleosides with the glyoxal adduct of deoxyguanosine

Glyoxal is a widely dispersed environmental mutagen that reacts with DNA and deoxyguanosine to give primarily the 1,N(2)-guanine adduct, i.e., 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purin-9-one. Kasai et al. have reported [Kasai, et al. (1998) Carcinogenesis 19, 1459-1465] additional minor reactions of glyoxal to give bis-nucleosides of unknown structure involving glyoxal conjugation of dG with dA, dC, and dG itself. Reaction conditions have been modified to give large increases in the yields of the adducts, which has permitted structural characterization utilizing chemical and spectroscopic techniques. The glyoxal conjugates of dG with dA and dC are imidazo[1,2-a]purines involving displacement of the 6-hydroxyl group of the dG conjugate by the exocyclic amino groups of dA and dC. The dG conjugate is a symmetrical fusion of two imidazo[1,2-a]purines in which both the 6- and the 7-hydroxyl groups of the dG conjugate have been replaced. The glyoxal conjugates are formed as pairs of diastereomers. The dC and dA have a trans orientation of substituents at C6 and C7; the adduct of dG has a cis orientation. The absolute configurations of the individual diastereomers have been tentatively assigned based on comparison of their CD spectra with configurationally assigned diastereomers of the crotonaldehyde adduct of deoxyguanosine.     

Effect of divalent cations on the daunomycin-deoxyribonucleic acid complex

Studies have been carried out on the interaction of Cu²⁺ and Mg²⁺ with complexes formed between daunomycin and DNA. In the visible region, at pH 5·2, the addition of Cu²⁺ (10^?4M) to a denatured DNA-daunomycin complex causes a spectral shift from 505 nm to 540 and 582 nm. These peaks disappear at pH values above and below 5·2. Mg²⁺ (10^?1M), at pH 6·9, causes the appearance of peaks similar to those obtained with Cu²⁺, but this occurs with either native or denatured DNA—daunomycin complexes. Daunomycin shows a fluorescence emission peak at 555 nm. In the presence of Cu²⁺, there is a reduction in the fluorescence intensity of the DNA—daunomycin complex, at pH values above 6·0. Below this pH, Cu²⁺ has no effect. Mg²⁺, on the other hand, has a marked quenching effect at pH 7·8. As the pH drops below 7·0, this effect gradually disappears. Below pH 3·0, where DNA has no effect on fluorescence, both Cu²⁺ and Mg²⁺ are without effect.     

Structure and stability of duplex DNA containing the 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG(N2)-AAF) lesion: a bulky adduct that persists in cellular DNA

The carcinogenic environmental pollutant 2-nitrofluorene produces several DNA adducts including the minor 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene (dG(N(2))-AAF) lesion, which persists for long times in rat tissue DNA after discontinuation of carcinogen administration. Here, we present the solution structure of a dG(N(2))-AAF duplex as determined by NMR spectroscopy and restrained molecular dynamics. The data establish a regular right-handed conformation with Watson-Crick base pair alignments throughout the duplex. The AAF moiety resides in the minor grove of the helix with its long axis directed toward the 5'-end of the modified strand. Restrained molecular dynamics shows that the duplex structure adjusts to the AAF lesion, reducing its exposure to water molecules. Analysis of UV melting profiles shows that the presence of dG(N(2))-AAF increases the thermal and thermodynamic stability of duplex DNA, an effect that is driven by a favorable entropy. The structure and stability of the dG(N(2))-AAF duplex have important implications in understanding the recognition of bulky lesions by the DNA repair system.     

Conformational properties of equilenin-DNA adducts: stereoisomer and base effects

Equilin and equilenin, components of the hormone replacement therapy drug Premarin, can be metabolized to the catechol 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dC, dA, and dG to form unusual stable cyclic adducts, which have been found in human breast tumor tissue. Four stereoisomeric adducts have been identified for each base. These 12 Premarin-derived adducts provide a unique opportunity for analyzing effects of stereochemistry and base damage on DNA structure and consequently its function. Our computational studies have shown that these adducts, with obstructed Watson-Crick hydrogen-bond edges and near-perpendicular ring systems, have limited conformational flexibility and near-mirror-image conformations in stereoisomer pairs. The dC and dA adducts can adopt major- and minor-groove positions in the double helix, but the dG adducts are positioned only in the major groove. In all cases, opposite orientations of the equilenin rings with respect to the 5' --> 3' direction of the damaged strand are found in stereoisomer pairs derived from the same base, and no Watson-Crick pairing is possible. However, detailed structural properties in DNA duplexes are distinct for each stereoisomer of each damaged base. These differences may underlie observed differential stereoisomer and base-dependent mutagenicities and repair susceptibilities of these adducts.