Michel Daune

Antibodies to DNA modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene

Several studies have shown that the carcinogen N-acetoxy-N-2-acetylaminofluorene (AAAF) reacts in vivo and in vitro with native DNA and that the DNA contains a major (80%) adduct N(deoxyguanosin8-yl)-acetylaminofluorene (dGuo8-AAF) and a minor (20%) adduct 3{deoxyguanosin-N2-yl)-acetylaminofluorene (dGuo-N2-AAF) (reviewed [1,2]). It has been shown that the major alteration induced by the fluorene ring is the creation of locally disorganized regions inside the double helical structure [3-81 . Methods sensitive enough to assay the regions of DNAmodified by a carcinogen at the levels of modification occuring in the ‘in vivo’ carcinogenesis experiments would be of great value. The immunological method has already been shown to be able to detect small modifications in DNA (see e.g. [9,10]). On the other hand, the study of the specificity of the antibodies can bring some knowledge on the conformation of the antigen. For these reasons, we have undertaken a study of the immunogenicity of native DNA after reaction with AAAF. In this paper, we show that native DNA slightly modified by AAAF can induce in rabbits the synthesis of specific antibodies which selectively recognize AAF-substituted DNA. A methods of purification of these antibodies is described. Also, the association constants for the binding of the antibodies and several ligands are reported.

Interaction between hydroxystilbamidine and DNA: I. Binding isotherms and thermodynamics of the association

Isotherms describing the binding of hydroxystilbamidine to DNA and polydeoxyribonucleotides were obtained by means of sedimentation or dialysis experiments and fluorescence measurements, over a large range of ionic strengths, temperatures and base compositions. Two different sets of binding sites are necessary to explain the shapes of the isotherms. The first one is characterized by a higher binding constant, a topological specificity for the A-T pair, exclusion of four base pairs per bound dye molecule, the involvement of two ion-pairs, an almost purely entropic free energy of binding and a large enhancement of the blue fluorescence (450 nm) when the site corresponds to three adjacent A-T pairs. The latter does not present any specificity nor enhancement of fluorescence and only one ion-pair is formed. From the geometry of the dye and its selective binding to a double stranded structure, the hydroxystilbamidine molecule in the first set of sites is likely to be situated in the small groove astride the two complementary strands and slightly distorting the helical structure. The angle of the dye axis with the helix axis has a value close to 47 degrees. No definite explanation could be given for the specific binding of hydroxystilbamidine but the phenolic hydroxyl group is likely to play a major role. The hydroxystilbamidine molecule can be considered as a useful tool for checking the accessibility of the small groove.

Changes of stability and conformation of DNA following the covalent binding of a carcinogen.

Recently Miller [ 1 ] has thrown some light on the effects of many carcinogenic chemicals. He has shown that the activity of these compounds or their metabolic derivatives is related to their strong electrophilic reactivity. The active form of those carcinogens is covalently bound to nucleic acids and proteins in induced tumors [ 11. In the case of acetylaminofluorene (AAF) Miller et al. [2] have shown that the last metabolic derivative was an ester of N-hydroxy-AAF. We have therefore studied the effects of N-acetoxy-AAF (NAcO-AAF) on native DNA. The analysis of melting profiles of modified DNA gives evidence for a destabilizing effect which is interpreted as the opening of G-C base pairs. Furthermore it is likely that the carcinogen reacts preferentially with G-C rich regions.

Études des complexes de colorants basiques avec le RNA

Abstract Complexes of basic dyes with RNA The binding of certain basic dyes (proflavine, pyronine, toluidine blue, acridine orange) by polyribonucleotides (ribosomal RNA, (A) n ·(U) n , (A) n ·(U) n ·(U) n , (I) n ·(C) n , which causes a red shift in the absorption spectra of the dyes, has been studied. The number of dye molecules bound per nucleotide and the association constants were determined as well as the influence of proflavine and acridine orange on the melting temperature of the polyribonucleotides. Evidence is presented showing that the first dye molecules are intercalated between the base pairs of the organized regions of polyribonucleotides. These dye molecules cause a partial disorganization of the macromolecule which permits subsequent binding of other dye molecules with smaller binding constant but the same spectral effect. Toluidine blue is specifically bound by (I) n ·(C) n , pyronine by (A) n ·(U) n . The secondary structure of the polyribonucleotides is generally stabilized by the dyes as shown by the difference between the absorption temperature of the dye and the melting temperature of the polyribonucleotide. When the secondary structure of the polyribonucleotides disappears at high temperatures, the dye is bound to the phosphates as evidenced by the blue shift of the absorption spectra of the dyes.

Co-operativity value of DNA-RecA protein interaction. Influence of the protein quaternary structure on the binding analysis

We show that an erroneous estimation of the quaternary structure of free protein distorts the quantitative analysis of its interaction with DNA, affecting especially the co-operativity value found. This could explain the discrepancy reported for the co-operativity value of the RecA-DNA interaction. The large cluster observed by electron microscopy indicates a very high co-operativity, whereas analysis of the binding isotherm indicates a moderate one, on the assumption of monomer. But if RecA is a large oligomer, the latter analysis would give a much higher co-operativity value and the former a smaller one, and they would be in accordance. Our sedimentation and light-scattering experiments suggest an oligomerization of about 30-mer or more, and support this explanation.

Histone phosphorylation in native chromatin induces local structural changes as probed by electric birefringence

In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.

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.

Effect of tetramethylammonium ions on conformational changes of DNA in the premelting temperature range

The reversible conformational change of DNAs and polydeoxyribonucleotides occurring before melting was followed by circular dichroism. deltatheta/deltaT, the rate of change of ellipticity theta with temperature, was used mainly as a measure of this premelting phenomenon. If sodium ions were replaced by tetramethylammonium ions deltatheta/deltaT decreased for poly (dA) poly (dT) and poly (dA.dT) poly (dT.dA), but increased for poly (dG.dC) poly (dC.dG). DNAs of different base composition showed no more premelting (deltatheta/deltaT approximately 0) even at low molarities of TMACl provided the Na/TMA ratio was very small. For all cases studied the theta values at 0 degrees C and at a given ionic strength were smaller in NaCl than in TMACl. When studying the series of ammonium ions from NH4+ to (C2H5)4N+, the deltatheta/deltaT values first decreased, going through zero with TMA+ ions, and then increased again. A tentative and qualitative explanation of our results can be given: (a) Hydration of the polymers increases in presence of TMA ions and their average stability decreases; locally, however, (AT) pairs are preferentially stabilized by TMA ions owing to a specific interaction at the level of O2 of thymine. (b) In order to explain the different behaviour of (AT) polymers and DNA, it is assumed that only the B structure is able to accommodate TMA ions in the small groove of the double stranded helix.

Effect of induction of SOS response on expression of pBR322 genes and on plasmid copy number

Several lines of evidence are presented that indicate that the level of tetracycline resistance of Esherichia coli strains harboring plasmid pBR322 varies according to whether the SOS system of the host bacteria has been induced. These include use of strains in which the SOS system is expressed constitutively (lexA def.), is thermoinducible (recA441) or noninducible (lexA ind-), or is highly repressed (multiple copies of lexA+). Similar induction was observed with the product of another plasmid gene, beta-lactamase. The amounts of extractable plasmid DNA were also increased by SOS induction, and we propose that the SOS-induced increases in levels of tetracycline resistance and beta-lactamase activity are due to an increased plasmid copy number.

Cooperative and salt-resistant binding of lexA protein to non-operator DNA.

The interaction of the lexA repressor of E. coli with poly[d(A‐T)] has been studied by circular dichroism. The binding induces an about 2‐fold increase of the circular dichroism intensity at 263 nm, pointing out a conformational change of the nucleic acid. The observed spectral changes are very similar to those observed for the binding of the lac repressor to poly[d(A‐T)] and natural DNA. At elevated ionic strength the binding isotherms do show a pronounced sigmoidal shape indicating a cooperative mode of binding.