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Biomedical subjects

R P Fuchs

Publications and source records attributed to R P Fuchs.

At least 127 records · Page 7Linked to original sources
PubMed1981

Mechanism of inhibition of enzymatic deoxyribonucleic acid methylation by 2-(acetylamino)fluorene bound to deoxyribonucleic acid.

Binding of 2-(acetylamino)fluorene (AAF) to C-8 of guanine induces a local destabilization of the DNA helix. A relationship was observed where the degree of DNA modification by AAF was inversely proportional to its methyl acceptor capacity from S-adenosyl-L-methionine in the presence of rat brain DNA cytosine 5-methyltransferase. Moreover, substituted DNA (DNA-AAF) behaves as a methylation inhibitor of native DNA. This inhibition is of the mixed type. The substituted DNAs have higher affinities for the enzyme than native DNA. The inhibition is irreversible. Addition of DNA-AAF to the enzyme preincubated with native DNA inhibits methylation, but only after a lag period. This agrees with the model in which the methylase "walks" along the strand to methylate cytosine residues before being detached from the DNA. AAF bound to guanine residues may block the movement of the enzyme along the helix. Single-stranded DNA has an affinity for the methylase 1.6 times lower than that of native double-stranded DNA. On the other hand, single-stranded DNA-AAF is more methylated than double-stranded DNA-AAF. A tentative model taking into account these observations is presented under Discussion. The in vitro hypomethylation of DNA-AAF could explain the in vivo observations made by several authors.

2-Acetylaminofluorene↗
PubMed1981

Comparative carcinogenicities and reactivities of N-myristoyloxy-N-acetyl-2-aminofluorene and its 7-iodo derivative.

Earlier studies showed that N-acetyl-2-aminofluorene (AAF) is much more carcinogenic than N-acetyl-2-amino-7-iodofluorene (AAIF). Subsequently it was found that substitution of C-8 of guanine bases in DNA with AAF residues resulted in displacement of the guanine bases outside the DNA helix. This did not occur after similar substitution with AAIF residues. As one approach to assessing the possible importance of this gross conformational difference to the carcinogenicity of AAF, the carcinogenic activities of two electrophilic esters, N-myristoyloxy-AAF and its 7-iodo derivative, were compared by s.c. injection into male Fischer rats. On injection of a total of 64 mumol, each ester induced a high incidence of sarcomas, and the latent periods were similar. N-Myristoyloxy-AAIF was solvolyzed in aqueous media at about one-half the rate of N-myristoyloxy-AAF, and it was less than 10% as reactive with native DNA as N-myristoyloxy-AAF. N-Myristoyloxy-AAF and N-myristoyloxy-AAIF were each less reactive than the corresponding acetoxy derivatives. These data suggest that the low carcinogenicity of AAIF as compared to that of AAF may not be associated with the conformations of their adducts in the DNA. This difference in carcinogenicity may be related to differences in the rates of metabolic activation and inactivation of these two amides.

2-Acetylaminofluorene↗
PubMed1981

Structural modification and protein recognition of DNA modified by N-2-fluorenylacetamide, its 7-iodo derivative, and by N-2-fluorenamine.

Several experimental approaches were used to compare the structural modifications introduced by the binding to DNA of N-hydroxy-N-2-fluorenamine and of N-acetoxy-N-2-fluorenylacetamide and its 7-iodo derivative. In the three binding reactions, the major part of the substitution occurred at C-8 of guanine. Formaldehyde unwinding, hydrolysis by endonuclease S(1), linear electric dichroism, circular dichroism, use of antibodies, unwinding of covalently closed circular DNA, and binding of tripeptide were thus successively examined. Most of the techniques showed marked differences between the different modified DNA's. However, some experiments failed to show discrimination between the induced structural alterations. These specific points are discussed. On the basis of all the available experimental evidence and after examination of the Cory-Pauling-Koltun molecular model, we proposed three models of DNA binding at the C-8 of the guanine residue with 1) the 7-iodo derivative (outside binding) 2), N-acetoxy-N-2-fluorenylacetamide (insertion-denaturation) and 3) N-hydroxy-N-2-fluorenamine (insertion without denaturation).

2-Acetylaminofluorene↗
PubMed1980

Binding of a tryptophan-containing peptide (lysyltryptophyllysine) to deoxyribonucleic acid modified by 2-(N-acetoxyacetylamino)fluorene.

The binding of the tripeptide Lys-Trp-Lys to DNA modified by reaction with the chemical carcinogen 2-(N-acetoxyacetylamino)fluorene (AAAF) has been investigated by fluorescence spectroscopy. A quenching of tryptophan fluorescence was observed which increased when the degree of base substitution by AAAF increased. Similar results were obtained with the 7-iodo derivative of AAAF (AAAIF). Two hypotheses are discussed which could account for the experimental results: (1) stacking interactions of the tryptophyl residue of the peptide with nucleic acid bases in locally unpaired regions in the vicinity of modified bases; (2) energy transfer from the tryptophyl residue of the peptide to acetylaminofluorene bound to guanine bases without direct interaction of this residue with nucleic acid bases (outside binding). The results obtained with denatured DNA in the absence and the presence of chemical modifications by AAAF or AAAIF allow us to conclude that energy transfer contributes to fluorescence quenching in the case of AAIF but not in that of AAF. Stacking interactions are therefore responsible for fluorescence quenching of Lys-Trp-Lys when bound to AAF-modified DNA. In the case of DNA-AAIF, fluorescence quenching is due both to energy transfer and to stacking of the tryptophan ring with bases inside the helix. These results are discussed in relation to what is already known in terms of local structure and with respect to the role that could be played by aromatic residues of proteins in the recognition of chemically damaged DNA.

2-Acetylaminofluorene↗
PubMed1979

Reactivity of the antibodies to DNA modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene.

Rabbits were immunized with native DNA modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. The interactions between the purified antibodies to nDNA-AAF (or the Fab fragments) and several ligands have been studied. By radioimmunoassay, nDNA-AAF, dDNA-AAF, and GMP-AAF were found to bind to the antibodies with about the same affinity. GMP-AF interacts slightly less, and GMP and N-OH-AAF do not interact. The values of the association constants deduced from fluorescence measurements for the binding of the Fab fragments to nDNA-AAF, dDNA-AAF, and GMP-AAF, in 50 mM NaCl, pH 7.5, are of the same order of magnitude. The values of the association constants with nDNA-AAF and dDNA-AAF depend upon salt concentration. From this variation; it is deduced that 1-1.5 phosphate groups interact by charge--charge interactions with the Fab fragments. The absorption and circular dischroism spectra of GMP-AAF, nDNA-AAF, and dDNA-AAF bound to the Fab fragments show that the Fab fragments induce similar perturbation to the three ligands. These results lead to the conclusion that the immunodeterminant group is the dGMP-AAF residue.

Acetoxyacetylaminofluorene↗
PubMed1979

Unwinding of supercoiled Col E1-DNA after covalent binding of the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene and its 7-iodo derivative.

The unwinding of superhelical Col E1-DNA was studied by means of gel electrophoresis and electron microscopy after covalent binding of N-acetoxy-N-2-[14C]acetylaminofluorene (N-Aco-[14C]AAF) and its 7-iodo derivative (N-Aco-[14C]AAIF). Studies with both compounds indicated that complete unwinding of the supercoiled DNA required the binding of hydrocarbon residue to about 3% of the bases. Thus the unwinding angle per residue of N-2-acetylaminofluorene (AAF) and its 7-iodo derivative was of 22 degrees +/- 3 and 18 degrees +/- 3 respectively. Our results are in good agreement with those obtained by Drinkwater et al. [9]. Precedent studies from this laboratory have shown that N-Aco-AAF and its 7-iodo derivative induce different local conformation change in native DNA (insertion-denaturation model and outside binding model respectively). The unexpected ability of the 7-iodo derivative to unwind supercoiled DNA is discussed.

2-Acetylaminofluorene↗
PubMed1979

Electron microscopic visualization of N-acetoxy-N-2-acetylaminofluorene binding sites in ColE1 DNA by means of specific antibodies.

ColE1 DNA has been allowed to react in vitro with N-acetoxy-N-2-[14C]acetylaminofluorene in the range of 0-15 N-2-[14C]acetylaminofluorene residues bound per molecule of DNA, at the C8 of guanine residues. Purified rabbit antibodies to both N-2-(guanosine-8-yl)-acetylaminofluorene and native DNA that had reacted with N-acetoxy-N-2-acetylaminofluorene were shown by electron microscopy to recognize specifically the acetylaminofluorene-modified ColE1 DNA. The antibodies bound to DNA were visualized either per se or after reaction with goat anti-rabbit immunoglobulins coupled with ferritin. There was a linear relationship between the average number of antibodies bound per DNA molecule and the number of N-2-(deoxyguanosine-8yl)-acetylaminofluorene residues per DNA molecule. The slope of this straight line was equal to 0.4. Due to the bivalence of the immunoglobulins one would expect a value of 0.5; we actually observed an important fraction of the bound antibodies crosslinking two parts of the same (or of another) DNA molecule.

2-Acetylaminofluorene↗
PubMed1978

Comparative studies on the 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene: binding sites on DNA and conformational change of modified deoxytrinucleotides.

GMP and native DNA were reacted with 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene. It was shown that the 7-halogeno derivatives react on C-8 of guanine. Furthermore the respective amount of arylamidation (covalent linkage on the C-8 of guanine) and arylation (covalent linkage on 2-NH2 groups of C3 of guanine) addition products was determined in both native and denatured DNA-[14C]AAIF. Two G containing deoxytrinucleotides modified by either AAFF or AAIF were studied comparatively by means of circular dichroism, and as a function of several parameters known to affect the conformation of the deoxytrinucleotides. The induced optical activity in fluorofluorene ring seemed to be very sensible to the conformational changes of the deoxytrinucleotides. On the other hand, the AAIF residue exhibit a lower induced optical activity which remained unchanged when the deoxytrinucleotides conformation was affected. The results presented in this paper led us to conclude that the AAFF and AAIF modified deoxytrinucleotides adopt a conformation which nicely fits with the insertion-denaturation and outside-binding model, respectively.

Acetoxyacetylaminofluorene↗
PubMed1978

Arylamidation and arylation by the carcinogen N-2-fluorenylacetamide: a sensitive and rapid radiochemical assay.

N-acetoxy-N-arylacetamides, which are generally considered as an ultimate carcinogenic form of the corresponding N-arylacetamides, react with the cellular macromolecules (nucleic acids, proteins, etc.) to give two types of adducts: (I) arylamidation and (II) arylation addition products. In this paper, we present a radiochemical determination of the amount of N-2-fluorenylacetamide bound to DNA via arylamidation or arylation, respectively. This assay is based upon the difference of stability under weak alkali hydrolysis conditions (0.1 N NaOH, 75 degrees C, 2 h) of the specifically 14C-labeled N-acetyl group of the N-2-fluorenylacetamide residue linked to the macromolecule either via arylamidation or arylation. Native DNA which has been reacted with N-acetoxy-N-2-[14C]acetylaminofluorene exhibits 16% of the fluorene adducts linked to the bases via arylation. On the other hand, denatured DNA reacts with the fluorene derivative to give almost only arylamidation addition products.

2-Acetylaminofluorene↗
PubMed1977

Secondary structural modifications as a consequence of in vitro acetylation and phenanthrylation of DNA by the ultimate carcinogen N-acetoxy-N-2-acetylaminophenanthrene.

The acetic acid ester of the proximate carcinogen N-hydroxy-N-2-acetylaminophenanthrene was reacted in vitro with native and heat-denatured calf thymus DNA under various conditions. We showed that besides the phenanthrylation of the DNA bases there is an acetylation reaction of the DNA during its reaction with the ultimate carcinogen. Heat-denatured DNA is 5 to 10 times more acetylated than native DNA. This result suggests that most of the acetylation sites are nonreactive in the double-helical structure of DNA. On the other hand, the phenanthrylation of the bases is shown not to depend on the DNA secondary structure, suggesting that the phenanthrylation sites of the bases are accessible in the grooves of the DNA double helix. The influence of the DNA dynamic structure on the reactions of acetylation and phenanthrylation has been investigated by increasing the ionic strength of the incubation buffer. The melting temperature of different DNA samples, which have been reacted with different concentrations of N-acetoxy-N-2-acetylaminophenanthrene, decreases as the extent of the DNA modifications increases. This thermal destabilization of the double helix is tentatively attributed to the phenanthrylation rather than to the acetylation reaction.

Acetylation↗
PubMed1976

Comparative orientation of the fluorene residue in native DNA modified by N-acetoxy-N-2-acetylaminofluorene and two 7-halogeno derivatives.

Native calf thymus DNA was reacted with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) and its 7-fluoro and 7-iodo derivatives. Different ways of purification of the fluorene modified DNA samples were checked in order ot obtain a nucleic acid free from all noncovalently bound fluorene residues. The decrease in melting temperature in DNA samples modified by N-AcO-AAF(DNA-AAF) was carefully reinvestigated. From these experiments, we conclude that the melting temperature decrease is equal to 1.15 degree C per percent of modified bases, in DNA-AAF samples. Electric dichroism measurements on sonicated DNA samples modified by the different fluorene derivatives show the fluorene ring perpendicular to the helix axis in the case of the N-AcO-AAF and its fluoro derivative, and lying alone the phosphate-sugar backbone in the case of the iodo derivative. The results presented in this paper, along with those obtained earlier, led us to propose an "insertion-denaturation model" for the mode of binding of N-Aco-AAF and its fluoro derivative, and an "outside binding model" for the iodo derivative. Discrepancies with the data obtained by Chang et al.((1974) Biochemistry 13,2142-2148) concerning the melting temperature decrease and the electric dichroism results are observed and discussed.

Acetoxyacetylaminofluorene↗