Differential effects of high fat diet and diet-induced obesity on skeletal acquisition in female C57BL/6J vs. FVB/NJ Mice.

The effects of obesity on bone metabolism are complex, and may be mediated by consumption of a high fat diet and/or by obesity-induced metabolic dysregulation. To test the hypothesis that both high fat (HF) diet and diet-induced metabolic disease independently decrease skeletal acquisition, we compared effects of HF diet on bone mass and microarchitecture in two mouse strains: diet-induced obesity (DIO)-susceptible C57BL/6J (B6) and DIO-resistant FVB/NJ (FVB). At 3 wks of age we weaned 120 female FVB and B6 mice onto normal (N, 10% Kcal/fat) or HF diet (45% Kcal/fat) and euthanized them at 6, 12 and 20 weeks of age (N = 10/grp). Outcomes included body mass; percent fat and whole-body bone mineral density (WBBMD, g/cm2) via DXA; cortical and trabecular bone architecture at the midshaft and distal femur via µCT; and marrow adiposity via histomorphometry. In FVB HF, body mass, percent body fat, WBBMD and marrow adiposity did not differ vs. N, but trabecular bone mass was lower at 6 wks of age only (p < 0.05), cortical bone geometric properties were lower at 12 wks only, and bone strength was lower at 20 wks of age only in HF vs. N (p < 0.05). In contrast, B6 HF had higher body mass, percent body fat, and leptin vs. N. B6 HF also had higher WBBMD (p < 0.05) at 9 and 12 wks of age but lower distal femur trabecular bone mass at 12 wks of age, and lower body mass-adjusted cortical bone properties at 20 wks of age compared to N (p < 0.05). Marrow adiposity was also markedly higher in B6 HF vs. N. Overall, HF diet negatively affected bone mass in both strains, but was more deleterious to trabecular bone microarchitecture and marrow adiposity in B6 than in FVB mice. These data suggest that in addition to fat consumption itself, the metabolic response to high fat diet independently alters skeletal acquisition in obesity.

Solution structure and backbone dynamics of the human DNA ligase IIIalpha BRCT domain.

BRCT (BRCA1 carboxyl terminus) domains are found in a number of DNA repair enzymes and cell cycle regulators and are believed to mediate important protein-protein interactions. The DNA ligase IIIalpha BRCT domain partners with the distal BRCT domain of the DNA repair protein XRCC1 (X1BRCTb) in the DNA base excision repair (BER) pathway. To elucidate the mechanisms by which these two domains can interact, we have determined the solution structure of human ligase IIIalpha BRCT (L3[86], residues 837-922). The structure of L3[86] consists of a beta2beta1beta3beta4 parallel sheet with a two-alpha-helix bundle packed against one face of the sheet. This fold is conserved in several proteins having a wide range of activities, including X1BRCTb [Zhang, X. D., et al. (1998) EMBO J. 17, 6404-6411]. L3[86] exists as a dimer in solution, but an insufficient number of NOE restraints precluded the determination of the homodimer structure. However, 13C isotope-filtered and hydrogen-deuterium exchange experiments indicate that the N-terminus, alpha1, the alpha1-beta2 loop, and the three residues following alpha2 are involved in forming the dimer interface, as similarly observed in the structure of X1BRCTb. NOE and dynamic data indicate that several residues (837-844) in the N-terminal region appear to interconvert between helix and random coil conformations. Further studies of other BRCT domains and of their complexes are needed to address how these proteins interact with one another, and to shed light on how mutations can lead to disruption of function and ultimately disease.

Psychiatric Darwinism = survival of the fittest + extinction of the unfit.

This article is a critical analysis of the American Health Security Act of 1993. Although AHSA was soundly defeated when first proposed, parts of it have been enacted into law in 1996, with the prospect of further piece-meal enactments in the future. It includes matters of fundamental importance to American mental health practitioners, to vulnerable citizens with psychiatric disorders, to their families, and to their few champions in medicine and law. Utilitarianism is the unstated philosophical substructure of AHSA and its legislative progeny, i.e., whatever cuts medical costs and saves money is good. The author delineates AHSA's mental health entitlements and limitations of in-patient, out-patient, and other patient care. She enumerates a dozen major imperfections and dangers of this mental health law, especially its medical utilitarianism emphasizing outcomes and quality of life. Dr. Cosman argues that medical cost, outcome, quality of life, and managed competition threaten the essential liberties and the lives of older persons, persons who are chronically ill, fatally ill, and most particularly those who are mentally impaired. She concludes that if limited money, medicine and time are invested only in inevitable medical success, then America's medicine by its medical law will be Medical Darwinism encouraging survival of the fittest by requiring extinction of the unfit.

Solution structure of the 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine C8-deoxyguanosine adduct in duplex DNA.

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of various meats. To enable structure/activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with N-acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP-N-methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiester-sugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is syn, and the displaced guanine base is directed toward the 3' end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.

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.

Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIalpha.

The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region. These regions, known as BRCT domains, have been found to mediate important protein-protein interactions. We produced the BRCT domain of DNA ligase IIIalpha (L3[86]) for biophysical and structural characterization. A glutathione S-transferase (GST) fusion with the L3[86] domain (residues 837-922 of ligase IIIalpha) was expressed in Escherichia coli and purified by glutathione affinity chromatography. The GST fusion protein was removed by thrombin digestion and further purification steps. Using this method, (15)N-labeled and (13)C/(15)N-double-labeled L3[86] proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy. To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied. Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation. However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3[86] or X1BRCTb proteins alone. This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIalpha activity.

Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding.

Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions.

An empirical relationship between rotational correlation time and solvent accessible surface area.

Structure-dynamics interrelationships are important in understanding protein function. We have explored the empirical relationship between rotational correlation times (tau(c) and the solvent accessible surface areas (SASA) of 75 proteins with known structures. The theoretical correlation between SASA and tau(c) through the equation SASA = K(r)tau(c) ((2/3)) is also considered. SASA was determined from the structure, tau(c) (calc) was determined from diffusion tensor calculations, and tau(c) (expt) was determined from NMR backbone(13) C or (15)N relaxation rate measurements. The theoretical and experimental values of tau(c) correlate with SASA with regression analyses values of K(r) as 1696 and 1896 m(2)s(-(2/3)), respectively, and with corresponding correlation coefficients of 0.92 and 0.70.

Solution structure of an oligodeoxynucleotide duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyadenosine, determined by NMR spectroscopy and restrained molecular dynamics.

The d(C-G-T-A-C-epsilon C-C-A-T-G-C).d(G-C-A-T-G-A-G-T-A-C-G) oligodeoxynucleotide duplex containing the 3, N4-etheno-2'-deoxycytidine adduct positioned opposite 2'-deoxyadenosine in the center of the helix has been analyzed by proton NMR spectroscopy and restrained molecular dynamics. The spectroscopic data establish a right-handed duplex, with sugar puckers in the C2'-endo/C3'-exo range, residues adopting an anti conformation around the glycosidic torsion angle and, with the exception of epsilon C.dA, Watson-Crick hydrogen bond alignment for all base pairs. Molecular dynamics simulations, restrained by the full relaxation matrix approach, produced a three-dimensional model with an NMR R-factor of 7%. The duplex structure shows no significant perturbation of the sugar-phosphate backbone, which remains in B-form. The exocyclic adduct and its partner dA are incorporated into the helix without producing a noticeable kink. The epsilon C.dA alignment adopts a staggered conformation with each residue displaced toward the 5'-terminus and intercalated between bases on the opposite strand, without increase of inter-phosphate distances. The partial intercalation of the epsilon C (anti).dA(anti) alignment allows stacking between the aromatic rings of epsilon C and dA and with base pairs adjacent to the lesion, suggesting an important role played by hydrophobic forces in the stabilization of the solution structure.

Site-specific adducts derived from the binding of anti-5-methylchrysene diol epoxide enantiomers to DNA: synthesis and characteristics.

The direct synthesis and characterization of site-specific adducts derived from the binding of (+)-1R,2S-dihydroxy-3S,4R-epoxide-1,2,3,4-tetrahydro-5-methylchrysene and the (-)-1S,2R,3R,4S-enantiomer [(+)- and (-)-5-MeCDE, respectively], to the N2-guanine residues in the oligonucleotide d(CCATCGCTACC) are described. The spectroscopic characteristics of the 5-MeCDE-modified oligonucleotides are discussed, and it is shown that their CD characteristics can be used to distinguish between the trans-addition products of the binding of the (+)- and (-)-enantiomers of 5-MeCDE (C4 position). The 11-mer duplexes with the normal complementary strands are destabilized by the site-specific, covalently bound 5-MeCDE residues: the melting points, Tm, are 5-10 degrees lower than in the case of the unmodified duplex. Stereoselective exonuclease enzyme digestion patterns of the single-stranded (+)- and (-)-trans-5-MeCDE-modified oligonucleotides (Mao et al, 1993, Biochemistry, 32, 11785-11793) were used to probe the orientations of the covalently bound 5-MeCDE residues relative to the modified guanine and the 5'-3' strand polarity; the aromatic residues are positioned either on the 5'-side [(+)-5-MeCDE], or the 3'-side [(-)-5-MeCDE adduct] of the modified guanine residues. The electrophoretic mobilities of the (+)-5-MeCDE-modified 11-mer duplexes in native polyacrylamide gels are slower than those of unmodified and modified duplexes containing the stereoisomeric (-)-5-MeCDE-N2-dG lesions. This indicates that the lesions derived from the tumorigenic (+)-5-MeCDE induce greater degrees of bending or local flexibility than the non-tumorigenic (-)-5- MeCDE enantiomer. These differences in the orientational and structural characteristics are similar to those observed with analogous DNA adducts derived from the tumorigenic (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the non-tumorigenic 7S,8R,9R,10S-enantiomer, respectively. The adducts derived from BPDE and 5-MeCDE enantiomers thus display similar characteristics that depend primarily on the PAH diol epoxide enantiomer stereochemistry. This direct synthesis approach can be used to generate milligram quantities of site-specific 5-MeCDE-modified oligonucleotides that are suitable for NMR studies (Cosman, et al., 1995, Biochemistry, 34, 6247-6260).

Solution conformation of the (-)-cis-anti-benzo[a]pyrenyl-dG adduct opposite dC in a DNA duplex: intercalation of the covalently attached BP ring into the helix with base displacement of the modified deoxyguanosine into the major groove.

This paper reports on the combined NMR-molecular mechanics computational studies of the solution structure of the (-)-cis-anti-[BP]dG adduct positioned opposite dC in the sequence context d(C1- C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14- A15-G16-C17-G18-A19-T20- G21-G22) duplex [designated (-)-cis-anti-[BP]dG.dC 11-mer duplex]. This adduct is derived from cis addition at C10 of (-)-anti-7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] to the N2 position of dG6 in this duplex sequence. The exchangeable and nonexchangeable protons of the benzo[a]pyrenyl moiety and nucleic acid of the major conformation were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. There was a general broadening of proton resonances for a three-nucleotide segment centered about the lesion site which resulted in a tentative assignment for the sugar protons of the C7 residue in the spectrum of the adduct duplex. The solution conformation of the major conformation of the (-)-cis-anti-[BP]dG.dC 11-mer duplex has been determined by incorporating DNA-DNA and intermolecular BP-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the covalently attached benzo[a]pyrenyl ring intercalates between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs. The modified deoxyguanosine [BP]-dG6 and its partner cytosine dC17 are looped out of the helix into the major groove. The purine ring of the [BP]dG6 residue is directed toward the 5'-end of the modified strand and stacks over the major groove edge of its 5'-side neighbor dC5 residue. The solution structure of the (-)-cis-anti-[BP]dG.dC 11-mer duplex is compared with those of the stereoisomeric (+)-trans-anti-[BP]dG [Cosman, M., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918], (-)-trans-anti-[BP]dG [de los Santos, C., et al. (1992) Biochemistry 31, 5245-5252], and (+)-cis-anti-[BP]dG [Cosman, M., et al. (1993a) Biochemistry 32, 4146-4155] adducts positioned opposite dC in the same duplex sequence context. A key finding is that the long axes of the intercalated benzo[a]pyrenyl rings in the solution structures of the (+)- and (-)- cis-anti-[BP]dG.dC 11-mer duplexes are oriented in opposite directions with the benzylic ring directed toward the minor groove in the (+)-cis isomer and toward the major groove in the (-)-cis isomer. In addition, a comparison is also made with the solution structure of the (+)-trans-anti-[BP]dG adduct opposite a deletion site [Cosman, M., et al. (1994a) Biochemistry 33, 11507-11517] since this adduct duplex displays several conformational features in common with the structure of the (-)-cis-anti-[BP]dG.dC 11-mer duplex. The structures of both duplex adducts exhibit intercalation of the covalently attached ligand into the helix and displacement of the modified deoxyguanosine into the major groove. Studies of the biological activities of stereochemically defined BP-DNA adducts and the comparison of the solution structure of the (-)-cis-anti-[BP]dG.dC 11-mer duplex with its stereoisomeric counterparts should lead to new insights into the relationships between defined helical distortions and mutagenic specificity and activity.

Structural alignments of (+)- and (-)-trans-anti-benzo[a]pyrene-dG adducts positioned at a DNA template-primer junction.

The structural features of a chemically modified DNA template strand may promote error-prone DNA synthesis during replication. The resulting higher incidence of mutations, in turn, can eventually lead to tumor initiation. Structural insights into this process can be monitored by studying chemically modified base adducts of defined stereochemistry positioned site-specifically at a single strand--duplex template--primer junction. We have used a NMR-molecular mechanics approach to obtain the solution conformations of the covalent adducts derived from trans additions at the [BP]C10 position of the highly tumorigenic (+)-anti-benzo[a]pyrene diol epoxide [(+)-anti-BPDE] and nontumorigenic (-)-anti-benzo-[a]pyrene diol epoxide [(-)-anti-BPDE] to the N2 position of guanine [(+) and (-)-trans-anti-[BP]dG, respectively] in the d(A1-A2-C3-[BP]G4-C5-T6-A7-C8-C9-A10-T11-C12-C13).d (G14-G15-A16-T17-G18-G19-T20-A 21-G22) 13/9-mer DNA sequence. The modified 13-mer strand constitutes the template strand, while the complementary 9-mer strand constitutes a primer which has been synthesized from the 3'-end of the template toward the 5'-end up to the base preceding, but not including, the modified guanine. The modified guanine (denoted by [BP]dG4) is positioned at the junction site between the single-stranded and duplex segments. Structural features of the (+)-trans-anti-[BP]dG 13/9-mer have been determined by incorporating proton--proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The 3'-side duplex segment retains a minimally perturbed B-DNA conformation with all nine base pairs in Watson--Crick hydrogen-bonded alignments. Conformational heterogeneity is detected at the single-stranded d(A1-A2-C3) segment located 5' to the modified (+)-trans-anti-[BP]dG lesion which contrasts with an unperturbed alignment of these same residues in the unmodified control 13/9-mer. The modified guanine adopts a syn glycosidic torsion angle, is displaced into the major groove, and no longer stacks over the adjacent dC5.dG22 base pair. Such a base displacement is accompanied by stacking of one face of the pyrenyl ring with the dC5.dG22 base pair located on the duplex segment proximate to the modified guanine, while the other face of BP is exposed to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)

Solution conformation of [AF]dG opposite a -1 deletion site in a DNA duplex: intercalation of the covalently attached aminofluorene ring into the helix with base displacement of the C8-modified Syn guanine into the major groove.

This paper reports on the solution structure of the [AF]dG adduct positioned opposite a deletion site in a DNA oligomer duplex that defines the alignment of the covalent aminofluorene--C8-guanine adduct relative to the deletion site. The combined NMR molecular mechanics computational studies were undertaken on the [AF]dG adduct embedded in the d(C5-[AF]G6-C7).d(G16-G17) sequence context in a duplex containing 11 residues on the modified strand and 10 on the partner, with no base opposite the modification. The exchangeable and nonexchangeable protons of the aminofluorene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution conformation of the [AF]G.del 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminofluorene ring of [AF]dG6 is intercalated between intact Watson-Crick dC5.dG17 and dC7.dG16 base pairs with the guanine base of [AF]dG6 in a syn alignment displaced into the major groove. The syn glycosidic torsion angle at [AF]dG6 is supported by both carbon and proton chemical shift data for the sugar resonances of the modified guanine residue. The long axis of the aminofluorene ring is parallel to the long axis of the flanking dG.dC base pairs with the AF ring undergoing rapid 180 degrees flips on the NMR time scale. The intercalation site is wedge shaped with a pronounced propeller-twisting and buckling of the dC5.dG17 base pair. The guanine base of [AF]dG6, which is positioned in the major groove, is inclined relative to the helix axis and stacks over the 5'-flanking dC5 residue in the solution structure. The intercalative-base displacement structure of the [AF]dG.del 11-mer duplex exhibits several unusually shifted proton resonances that can be readily accounted for by the ring current contributions of the guanine purine and carcinogen fluorene aromatic rings of the [AF]dG6 adduct. There are similarities between this structure of the AF-C8-dG covalent adduct positioned opposite a deletion site and the (+)-trans-anti-BP-N2-dG covalent adduct positioned opposite a deletion site in the same sequence context reported previously from this laboratory [Cosman et al. (1994) Biochemistry 33, 11507-11517]. The chromophores are intercalated into the helix opposite the deletion site with displacement of the modified guanine into the major groove in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Solution conformation of the (-)-trans-anti-5-methylchrysene-dG adduct opposite dC in a DNA duplex: DNA bending associated with wedging of the methyl group of 5-methylchrysene to the 3'-side of the modification site.

This paper reports on NMR-molecular mechanics structural studies of the (-)-trans-anti-[MC]dG adduct positioned opposite dC in the sequence context of the d(C1-C2-A3-T4-C5-[MC]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17-G18- A19-T20-G21-G22) duplex [designated (-)-trans-anti-[MC]dG.dC 11-mer duplex]. This adduct is derived from the trans addition at C4 of (-)-anti-1(S),2(R)-dihydroxy-3(R),4(S)-epoxy-1,2,3,4-tetrahydro-5-met hylchrysen e [(-)-anti-5-MeCDE] to the N2 position of dG6 in this duplex sequence. The 5-methyl group is located adjacent to the MC(C4) binding site, with these groups juxtaposed in a sterically crowded bay region in the adduct duplex. The 5-methylchrysenyl and the nucleic acid exchangeable and nonexchangeable protons were assigned following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[MC]dG.dC 11-mer duplex has been determined by incorporating DNA-DNA and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the [MC]dG6.dC17 base pair and flanking dC5.dG18 and dC7.dG16 base pairs retain Watson-Crick alignments upon adduct formation. The aromatic chrysenyl ring is positioned in the minor groove of a right-handed B-DNA helix and stacks predominantly over the sugar of the dC17 residue across from it on the unmodified complementary strand. The chrysenyl ring points toward the 3'-end of the modified strand with its 5-methyl group inserting between the modified [MC]dG6.dC17 and dC7.dG16 base pairs. The adduct duplex bends by approximately 47 degrees as a result of the wedged insertion of the 5-methyl group from the minor groove face of the duplex. The solution structure of the (-)-trans-anti-[MC] dG.dC 11-mer duplex is compared with that of the corresponding (-)-trans-anti-[BP]dG.dC 11-mer [De los Santos et al. (1992) Biochemistry 31, 5245-5252] in which the [BP]dG adduct is derived from the binding of (-)-anti-BPDE [7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene] to the N2 position in the same DNA sequence context. Although the solution structures of the (-)-trans-anti-stereoisomers of 5-methylchrysenyl-dG and benzo[a]pyrenyl-dG adducts opposite dC exhibit many features in common with each other, the [MC]dG adduct which contains a bay region methyl group bends the DNA helix to a greater extent than in the corresponding [BP]dG adduct, which lacks a bay region methyl group.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct synthesis and characterization of site-specific adenosyl adducts derived from the binding of a 3,4-dihydroxy-1,2-epoxybenzo[c]phenanthrene stereoisomer to an 11-mer oligodeoxyribonucleotide.

Site-specifically modified oligonucleotides were obtained in milligram quantities by reacting racemic 3t,4r-dihydroxy-1,2t-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (B[c]PhDE-2, or anti-B[c]PhDE) with the single deoxyadenosine (dA) residue in the oligodeoxynucleotide d(CTCTCACTTCC). Enzyme digestion of the covalently modified oligonucleotides with the exonuclease spleen phosphodiesterase yielded covalently linked B[ca]PhDE-N6-deoxyadenosyl monophosphate (dAMP) adducts. Comparisons of the reverse phase HPLC retention times and CD spectra of these B[c]PhDE-3'-dAMP mononucleotide adducts, with those of standards derived from the reaction of the enantiomers (+)- and (-)-anti-B[c]PhDE with 3'-dAMP, show that two major oligonucleotide adducts (I and II) were obtained upon reacting racemic anti-B[c]PhDE with d(CTCTCACTTCC). In oligonucleotide adduct I, the lesion is a (+)-trans-anti-B[c]PhDE-N6-dA residue, and in oligonucleotide adduct II it is a (-)-trans-anti-B[c]PhDE-N6-dA residue. These assignments were further confirmed using a standard 32P postlabeling assay of B[c]PhDE-3'-dAMP mononucleotide adducts obtained from the digestion of oligonucleotides I and II by spleen phosphodiesterase. The melting points (Tm) of duplexes of modified oligonucleotides I and II and their natural complementary strands are not affected significantly by the presence of the covalently bound benzo[c]phenanthrenyl residues. Opposite stereoselective resistance to enzyme digestion by the exonucleases snake venom phosphodiesterase and spleen phosphodiesterase is exhibited by the stereoisomeric (+)-trans- and (-)-trans-anti-B[c]PhDE-modified oligonucleotide adducts I and II; these results are consistent with the intercalative insertion of the benzo[c]phenanthrenyl residues on the 5'-side of the modified dA residue in adduct I, and its insertion on the 3'-side of the dA residue in adduct II, as observed in the duplexes by high resolution NMR techniques [Cosman et al. (1993) Biochemistry 32, 12488-12497, and Cosman et all, Biochemistry, in press.

Solution conformation of the (-)-trans-anti-benzo[c]phenanthrene-dA ([BPh]dA) adduct opposite dT in a DNA duplex: intercalation of the covalently attached benzo[c]phenanthrenyl ring to the 3'-side of the adduct site and comparison with the (+)-trans-anti-[BPh]dA opposite dT stereoisomer.

This paper reports on NMR-molecular mechanics structural studies of the (-)- trans-anti-benzo[c]phenanthrene-dA adduct positioned opposite dT in the sequence context of the d(C1-T2-C3-T4-C5-[BPh]A6-C7-T8-T9-C10-C11).d(G12- G13-A14-A15-G16-T17-G18-A19-G20-A21- G22) duplex (designated as the (-)-trans-anti-[BPh]dA.dT 11-mer duplex). This adduct is derived from the covalent binding of (-)-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene [(-)-anti-BPhDE] to N6 of dA6 in this duplex sequence. The benzo[c]phenanthrenyl and nucleic acid exchangeable and nonexchangeable protons were assigned in the predominant conformation following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[BPh]dA.dT 11-mer duplex has been determined by incorporating intramolecular and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results show that the [BPh]dA6.dT17 base pair propeller twists and buckles slightly to permit the covalently attached benzo[c]phenanthrenyl ring to intercalate between the [BPh]dA6.dT17 and dC7.dG16 base pairs to the 3'-side of the [BPh]dA6 lesion site without disrupting the Watson-Crick hydrogen bond alignments in the modified duplex. The strain in the highly sterically hindered fjord region of the benzo[c]phenanthrenyl moiety is relieved by the propeller-like nonplanar geometry of the aromatic phenanthrenyl ring system, which stacks predominantly with the dG16 and dT17 bases on the unmodified strand. The benzylic ring adopts a distorted half-chair form, in which the H1 and H2 protons are pseudo-diequatorial and the H3 and H4 protons are pseudodiaxial. The current observation that the (-)-trans-anti-[BPh]dA positioned opposite dT intercalates to the 3'-side of the intact modified base pair contrasts with our previous demonstration that the stereoisomeric (+)-trans-anti-[BPh]dA adduct positioned opposite dT intercalates to the 5'-side of the intact modified base pair [Cosman, M., et al. (1993b) Biochemistry 32, 12488-12497]. These stereochemically induced structural differences between isomeric [BPh]dA lesions derived from the binding of chiral (+)- and (-)-anti-BPhDE enantiomers may in turn profoundly influence the interactions of the carcinogen-modified DNA with repair and replication enzymes in the cell.