Biomedical subjects
Publications and source records attributed to B Hingerty.
A new model is presented for acetylaminofluorene (AAF)-modified Z-DNA, in which the carcinogen is in a base-displaced position, inserted into the helix interior and parallel with an adjacent base.
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Cyclodextrins (CD's) have proved useful as model systems for the study of hydrogen bonding. They are torus-shaped molecules composed of six(alpha), seven(beta) or eight(gamma) (1----4) linked glucoses. Because of their particular geometry, they are able to act as a "host" to form inclusion complexes with "guest" molecules very much like enzymes. Cyclodextrins have been shown to exert catalytic activity on suitable included-substrate molecules; they catalyze the hydrolysis of phenylacetates, of organic pyrophosphates and of penicillin derivatives. They also accelerate aromatic chlorinations and diazo coupling by means of their primary and/or secondary hydroxyl groups, so that the rates of hydrolysis are enhanced by up to a factor of 400. In order to understand the hydrogen bonding in these enzyme models, neutron diffraction data were collected to unambiguously determine the hydrogen atom positions, which could not be done from the x-ray diffraction data. alpha-CD has been shown to have two different structures with well-defined hydrogen bonds, one "tense" and the other "relaxed". An "induced-fit"-like mechanism for alpha-CD complex formation has been proposed. Circular hydrogen bond networks have also been found for alpha-CD due to the energetically favored cooperative effect. beta-CD with a disordered water structure possesses an unusual flip-flop hydrogen bonding system of the type O-H...H-O representing an equilibrium between two states: O-H...O in equilibrium O...H-O. gamma-CD with a disordered water structure similar to beta-CD also possesses the flip-flop hydrogen bond. This study demonstrates that hydrogen bonds are operative in disordered systems and display dynamics even in the solid state.
Minimized conformational potential energy calculations have been performed for the 7-iodo (AAIF) and 7-fluoro (AAFF) derivatives of N-2-acetylaminofluorene (AAF), linked covalently to guanine C-8 in dCpdG. Both the iodo and the fluoro derivatives are carcinogenic and mutagenic. The lowest energy forms on the dinucleoside monophosphate level have syn guanine and fluorene-cytidine stacking. However, the iodo adduct cannot adopt this conformation in larger polymers, according to earlier experimental studies (Fuchs et al., Biochemistry, 15 (1976) 3347) and model building, because of iodine's large Van der Waal's radius. Therefore, a model consistent with all the experimental evidence, incorporating the second lowest energy conformation in B form duplex (dCdG)3 was constructed. In this model the modified guanine is syn, yet still stacked with the adjacent cytidine in one direction, the fluorene is located primarily at the helix interior between the base pairing sites, rupturing two base pairs, and the iodine atom and its adjoining ring protrude to the helix exterior.
Minimized conformational potential energy calculations have been performed for AAF linked to dCpdG at the guanine amino group. This is a model for the minor AAF adduct observed in DNA, whose conformational influence has been difficult to ascertain. A global minimum energy conformation was computed with torsion angles like those of the dCpdG residue of Z-DNA. This conformation was incorporated into a larger polymer model at a B-Z junction, with the carcinogen residing in the groove in the Z direction. Local minimum energy conformations of the B type were also computed. In addition, two minima were found with fluorenecytidine stacking. These results suggest that existing B-Z junctions may be vulnerable to modification by AAF at the guanine amino group, or that such junctions may be induced by the carcinogen if the base sequence is appropriate. Otherwise the carcinogen can be located in the minor groove of the B helix (5, 10, 11) or covalently intercalated (13-15).
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The (+) anti isomer of benz[a]pyrene diol epoxide (BPDE), 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenz [a] pyrene has been identified as the probable tumorigenic lesion in mammalian systems. It forms a predominant adduct with DNA at N2 of guanine. In order to elucidate its conformation in atomic resolution detail, minimized conformational potential energy calculations were performed for the adduct with dCpdG. A global conformation search involving about 1000 trials was made. The lowest energy conformation had stacking between the hydrocarbon and the adjacent cytidine, in agreement with CD studies on modified GpU and UpG. This conformer differed from the B form most notably in the guanine glycosidic torsion, which is high anti. The next lowest energy form had torsion angles like the B form, with guanine-cytidine stacking. These two conformers differ in energy by only 2.1 kcal./mole, suggesting that their relative stability could easily be reversed in larger polymers, or under specific environmental conditions. Other conformations, with base-hydrocarbon or base-base stacking are also found, at somewhat higher energies. The Z form is at 7.8 kcal./mole. Thus, this adduct stabilizes the B form, in contrast with the N2 linked AAF adduct, which stabilizes the Z conformation.
Minimized conformational potential energy calculations were performed for dCpdG modified with the carcinogen 2-(acetylamino)fluorene (AAF). The major adduct, linked via a covalent bond between guanine C-8 and N-2 of AAF, was investigated. The 12 variable torsion angles and both deoxyribose puckers were independent flexible parameters in the energy minimizations. Three categories of low-energy conformers were calculated in which the guanine was syn and nearly perpendicular to the plane of the fluorene: (1) forms in which fluorene is stacked with cytidine (included among these is the global minimum energy conformation); (2) conformers which preserve guanine-cytidine stacking while placing the fluorene in a base-pair obstructing position; (3) conformers which maintain guanine-cytidine stacking and place the fluorene at the helix exterior, without interfering with base pairing. The Z form is important in this group. In addition, a low-energy conformation with guanine anti, but still nearly perpendicular to fluorene, was computed. Molecular models were constructed for the most important conformations incorporated into larger polymers. These indicated that the fluorene-cytidine stacked forms induce a severe kink in the B helix. Conformers with guanine-cytidine stacking and AAF in a base-pair obstructing position place the AAF at the B-type helix interior with little distortion in the helix direction. Conformers with the guanine-cytidine stack in which AAF does not affect base pairing place the fluorene at the Z or alternate helix exterior. It is suggested that base sequence, extent of modification, and external conditions such as salt concentration determine which of a number of possible conformational effects is actually induced by AAF. The variety of observed experimental results with AAF-modified DNA may reflect there various conformational possibilities.
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Addition of 3' and 5' terminal phosphates to dApdA causes a decrease in conformational flexibility. pdApdAp has much fewer conformers with energies below 2.5 kcal./mole than dApdA. THE A, B and Watson-Crick (34) helices are the most preferred forms. Other important conformations are in the trans domain of psi. Thus, flexibility in psi as well as in omega and omega, and in the sugar pucker is indicated. The transformation from the B helix to the Watson-Crick helix follows a low energy path. This is significant since Watson-Crick conformations may be important for intercalation into nucleic acid polymers (40-42) above the dimer level. The B helix is preferred over the A form in these large DNA subunits.
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Low energy conformations with dihedral angles similar to those occurring in fibers of the 'A' and 'B' forms of DNAs have been calculated for the deoxydinucleoside phosphates dApdA, dCpdC, dTpdT, dGpdG and dGpdC (1-3). These conformers have been used as building blocks for generating larger single stranded polymers, whose helical parameters we have calculated. We find that single stranded 'A' and 'B' form helices tend to be narrower and more tightly wound than the duplexes obtained in fibers (4,5). This is consistent with experimental observations on single stranded fibers of poly (rC) (6). We also find that the different sequences have different helix geometries. In addition, it is observed that large variations in helix geometry for a given sequence are achievable at little energetic cost.
Helical parameters have been calculated for the 'A' form minimum energy conformations of ApA, CpC, GpG, UpU, GpC and UpA. The helix geometries are base sequence dependent. The single strands are narrower and more tightly wound than that duplex RNA-11 form. 9-12 kcal./mole are needed to convert these single strands to the RNA-11 conformation. However, in some sequences other 'A' type conformers capable of complementary base pairing may be formed at lower energetic cost. There is substantially more base stacking in the calculated single strands than in the RNA-11 conformation. Calculated intrastrand base stacking energies reflect these differences, and also are sequence dependent. The 'A' form RNA subunits differ from the analogous DNAs in possessing a larger rise per residue, needed to accomodate the 2'-OH. RNA-DNA hybrids are consequently more likely to be in the 'A-RNA than in the 'A'-DNA conformation, although the base sequence determines the extent of the preference.
The minimum energy conformations of dApdA have been examined for their suitability as buildings blocks of the single stranded coil form of polynucleotides. Calculations of the characteristic ratio C difference = less than ro greater than 2/n liter2 were made for a polymer generated from all the low energy conformers, as well as for selected combinations. A polymer composed of a conformer with omega', omega = t*,g+,(skewed) psi = t, C-(2)-endo type pucker, in combination with the 'B' form, has a C difference equal to that observed in coils of apurinic acid (6) when the fraction of 'B' form conformers is approximately 25% and approximately 91%. The t*,g+ conformer is the second lowest energy form in the C-(2)-endo puckering domain, following the 'B' form.
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