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This paper reports on the solution structure of the (+)-cis-anti-[BP]dG adduct positioned opposite dC in a DNA oligomer duplex which provides the first experimentally based solution structure of an intercalative complex of a polycyclic aromatic hydrocarbon covalently bound to the N2 of deoxyguanosine. The combined NMR-energy minimization computation studies were undertaken on the (+)-cis-anti-[BP]dG adduct embedded in the same d(C5-[BP]G6-C7).d(G16-C17-G18) trinucleotide segment of the complementary 11-mer duplex studied previously with the stereoisomeric trans adducts. The exchangeable and nonexchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY data sets as restraints in energy minimization computations. The benzo[a]pyrene ring of [BP]dG6 is intercalated between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs in a right-handed DNA helix. The benzylic ring is in the minor groove while the pyrenyl ring sacks with flanking dC5 and dC7 bases on the same strand. The deoxyguanosine ring of [BP]dG6 is not Watson-Crick base paired but displaced into the minor groove with its plane parallel to the helix axis and stacks over the sugar ring of dC5. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove by the intercalated benzo[a]pyrene ring. This intercalative structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex exhibits several unusually shifted proton resonances which can be readily accounted for by the ring current contributions of the deoxyguanosine and pyrenyl rings of the [BP]dG6 adduct. Several phosphorus resonances are shifted to low and high field of the unperturbed phosphorus spectral region and have been assigned to internucleotide phosphates centered about the [BP]dG6 modification site. These studies define the changes in the helix at the central trinucleotide segment needed to generate the intercalation site for the covalently bound (+)-cis-anti-[BP]dG adduct.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzo[a]pyrene (BP) is an environmental genotoxin, which, following metabolic activation to 7,8-diol 9,10-epoxide (BPDE) derivatives, forms covalent adducts with cellular DNA. A major fraction of adducts are derived from the binding of N2 of guanine to the C10 position of BPDE. The mutagenic and carcinogenic potentials of these adducts are strongly dependent on the chirality at the four asymmetric benzylic carbon atoms. We report below on the combined NMR-energy minimization refinement characterization of the solution conformation of (-)-trans-anti-[BP]G positioned opposite C and flanked by G.C base pairs in the 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. Two-dimensional NMR techniques were applied to assign the exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid in the modified duplex. These results establish Watson-Crick base pair alignment at the [BP]G6.C17 modification site, as well as the flanking C5.G18 and C7.G16 pairs within a regular right-handed helix. The solution structure of the (-)-trans-anti-[BP]G.C 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOE buildup curves as constraints in energy minimization computations. The BP ring spans both strands of the duplex in the minor groove and is directed toward the 3'-end of the modified strand in the refined structure. One face of the BP ring of [BP]G6 stacks over the C17 residue across from it on the partner strand while the other face is exposed to solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

The linear dichroism spectra of complexes of tetrakis(N-methyl-4-pyridinio)prophine (H2TMpyP) and its zinc(II) derivative (ZnTMpyP) with DNA oriented in a flow gradient have been investigated. The dichroism of H2TMpyP determined within the Soret band and the Qy band system is consistent with an intercalative conformation in which the plane of the porphyrin ring system is nearly parallel to the planes of the DNA bases. In the case of ZnTMpyP on the other hand, the porphyrin ring system is inclined at angles of 62-67 degrees with respect to the axis of the DNA helix. The pyridyl groups in both cases are characterized by a low degree of orientation with respect to the axis of the helix. In contrast to H2TMpyP which does not significantly affect the degree of alignment of the DNA in the flow gradient, the binding of ZnTMpyP causes a significant decrease (about 50% for a base pair/ZnTMpyP ratio of 20) in the intrinsic dichroism at 260 nm due to the oriented DNA bases; the binding of ZnTMpyP to DNA either gives rise to regions of higher flexibility or causes bends or kinks at the binding sites. Increasing the ionic strength has little influence on the linear dichroism of the ZnTMpyP-DNA complexes, but the number of molecules bound at intercalation sites diminishes in the case of the H2TMpyP-DNA complexes; the accompanying changes in the linear dichroism characteristics suggest that external H2TMpyP complexes are formed at the expense of intercalation complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

In the presence of native DNA the hydrolysis of benzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE) to tetrols (BPT) is markedly accelerated (by a factor of up to approximately 80 at 25 degrees C, pH 7.0, in 5 mM sodium cacodylate buffer solution). When stopped-flow kinetic techniques are utilized, it is shown that the pseudo-first-order hydrolysis rate constant kH is smaller by a factor of approximately 3 in the presence of equivalent concentrations of denatured DNA, by a factor of 8-25 in the presence of nucleotides, and by a factor of 35-45 in the presence of nucleosides (depending on the nucleotide or nucleoside). In the presence of native DNa, kH increases with increasing DNA concentration and reaches a limiting value of kH = 0.684 +/- 0.04 s-1 at DNA concentrations in excess of approximately 5 x 10(-4) M (expressed in concentration of nucleotides). A kinetic model based on (1) rapid formation of a noncovalent BPDE-DNA complex followed by (2) slower hydrolysis of BPDE to BPT at these binding sites is consistent with the experimental data. It is shown furthermore that the DNA concentration dependence of kH and of noncovalent intercalative binding of BPDE to DNA is similar and that addition of magnesium ions (which is known to reduce intercalative binding of planar aromatic molecules to DNA) also reduces kH. These results suggest, but do not necessarily prove, that the DNA binding sites at which the hydrolysis of BPDE (to BPT) is catalyzed are intercalative in nature.

The effects of the aliphatic acid series of salts, formate, acetate, propionate, butyrate, valarate, and caproate, on the conformation of sperm whale myoglobin, human hemoglobin A, and horse heart cytochrome c were investigated by spectral measurements in the Soret region, optical rotation, and intrinsic viscosity measurements. The effectiveness of the aliphatic acid salts as unfolding reagents for proteins is found to increase with increasing hydrocarbon content of the alkyl chains of the salts, which is analogous in behavior to effects of the urea, amide, and alcohol series of protein denaturants. The denaturation midpoints, Sm, as a function of the unfolding reagent were analyzed using the equations of Peller (Peller, L. (1959), J. Phys, Chem- 63, 1199) and Flory (Flory, P.J. (1957), J. Cell. Comp. Physiol. 49, 175) with binding constants based in part on the Scherage-Nemethy theory of hydrophobic bonding or evaluated from free-energy transfer data of nonpolar amino acid side chains from aqueous to nonaqueous solvents. The summation of the polar KP and hydrophobic KHphi contributions of solvent to protein amino acid side chain interactions were found to give best account of the protein denaturation data. Intrinsic viscosity and optical rotation data obtained on hemoglobin and myoglobin at high salt concentrations, above the unfolding transition regions, indicate that the product of denaturation by the aliphatic acid salts is less unfolded than in 6 M guanidine hydrochloride solutions. Residual elements of the helical regions of the proteins seem to either escape unfolding or are reformed at high concentrations of the denaturing salts.

The subunit dissociation of human hemoglobin A by the aliphatic acid salts at neutral pH has been investigated by light-scattering molecular-weight measurements at 630 nm. Dissociation of hemoglobin tetramers to alphabeta dimers is observed in essentially all experiments at low to intermediate levels of salt concentrations, below the denaturation transitions, described in the accompanying paper (Ibanez, V.S., and Herskovits, T.T. (1976), Biochemistry 15, preceding paper in this issue). The effectiveness of the salts as subunit dissociating agent, reflected by the slopes, s, of the plots of deltaGDdegrees, the standard free energy of dissociation, vs. [D], the salt concentration, is found to increase with increasing alkyl chain length or hydrocarbon content of the salt. Estimates of the apparent number of amino acid sites at the areas of contact per alphabeta dimer formed, N', based on the slopes of the higher members of the series have been obtained using the equation, deltaGDdegrees = deltaGD,Wdegrees - 2N'RTKB[D]. Independent estimates of the binding constant, KB, required for these calculations were based on free-energy transfer data of hydrophobic amino acid alkyl groups and protein denaturation data. Our estimates of N' denaturation data. Our estimates of N' obtained with the more reliable data of the higher members of salt series are in the ranges of 19 and 27 amino acid groups, shown by the x-ray crystallographic structure of horse and human hemoglobin of Perutz (Perutz, M.F., et al. (1968), Nature (London) 219, 131) and Fermi ((1975) J. Mol. Biol. 97, 237) for the smaller alpha1 beta2 contact areas in the tetrameric structure. The lower estimates than 27 based on our dissociation of human hemoglobin suggest that several of the amino acid residues in the contact areas of the subunits are partially exposed to solvent. The increasing effectiveness of the higher mn imporant source of stabilization of the tetrameric structure of hemoglobin.

The base-sequence selectivity of the noncovalent binding of (+/-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyr ene (BPDE) to a series of synthetic polynucleotides in aqueous solutions (5 mM sodium cacodylate buffer, 20 mM NaCl, pH 7.0, 22 degrees C) was investigated. The magnitude of a red-shifted absorbance at 353 nm, attributed to intercalative complex formation, was utilized to determine values of the association constant Kic. Intercalation in the alternating pyridine-purine polymers poly(dA-dT).(dA-dT) (Kic = 20,000 M-1), poly(dG-dC).(dG-dC) (4200 M-1), and poly(dA-dC).(dG-dT) (9600 M-1) is distinctly favored over intercalation in their nonalternating counterparts poly(dA).(dT) (780 M-1), poly(dG).(dC) (1800 M-1), and poly(dA-dG).(dT-dC) (5400 M-1). Methylation at the 5-position of cytosine gives rise to a significant enhancement of intercalative binding, and Kic is 22,000 M-1 in poly(dG-m5dG).(dG-m5dC). In a number of these polynucleotides, values of Kic for pyrene qualitatively follow those exhibited by BPDE, suggesting that the pyrenyl residue in BPDE is a primary factor in determining the extent of intercalation. Both BPDE and pyrene exhibit a distinct preference for intercalating within dA-dT and dG-m5dC sequences. The catalysis of the chemical reactions of BPDE (hydrolysis to tetrols and covalent adduct formation) is enhanced significantly in the presence of each of the polynucleotides studied, particularly in the dG-containing polymers. A model in which catalysis is mediated by physical complex formation accounts well for the experimentally observed enhancement in reaction rates of BPDE in the alternating polynucleotides; however, in the nonalternating polymers a different or more complex catalysis mechanism may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)