Jean Claude Maurizot

Interaction of aromatic residues of proteins with nucleic acids. Circular dichroism studies of the binding of oligopeptides to poly(adenylic acid).

The binding of peptides containing lysyl and aromatic residues to poly(A) in its single-stranded form at pH 7 leads to a change of its circular dichroism (CD) spectrum, which is mainly due to the stacking of the aromatic amino acid with the bases of poly(A). Comparison is made between the binding of peptides having different primary structures which gives indications on the way the peptides bind to poly(A). A method is described which allows the calculation of the binding parameters from CD data. The magnitude of the association constant depends on the size of the aromatic ring and decreases in the order tryptophan greater than tyrosine greater than phenylalanine. The CD amplitude decreases linearly with the concentration of bound molecules. These results are discussed with respect to the role played by aromatic amino acids in complex formation between nucleic acids and proteins.

Interaction between the peptide lysyl-tryptophanyl-lysine and copolynucleotides of adenine and uracil: Selectivity of interaction

Both complexes involve electrostatic interactions between the phosphate groups of the nucleic acid and the amino groups of the lysyl residues of the peptide. In complex I the fluorescence quantum yield of tryptophan is identical to that of the free peptide. Complex II involves a stacking of the tryptophanyl ring with the nucleic acid bases and the fluorescence of the peptide is completely quenched. The value of K2, which represents the ratio of the concentrations of stacked to unstacked complexes, is much smaller for double stranded than for single stranded nucleic acids [4,6,7]. We report here results on the interaction of LysTrpLys with copolynucleotides of adenine and uracil. Such a study was undertaken to determine whether the binding of the peptide to single strands involved a base sequence specificity. Previous experiments had shown that the constant K, was larger for poly(U) (K2 4) than for poly(A) (K2 2.3) [4]. It was of interest to follow the evolution of K, when the content of U increased in a copoly(AU) since one could think that the interaction of LysTrpLys involved more than one base and would thus be sequence dependent.

Effect of strand orientation on the interaction of berenil with DNA triple helices

Abstract. Using circular dichroism spectroscopy the ability of berenil, a minor groove binding drug, to induce triple helix formation was investigated with two oligonucleotides designed to form two intramolecular triplexes containing T*A:T and G*G:C triplets, which differ only by the orientation of their third strand: 5′-d(G4A4G4-[T4]-C4T4C4-[T4]-G4T4G4), and 5′-d(G4T4G4-[T4]-G4A4G4-[T4]-C4T4C4), where [T4] represents a stretch of four thymine residues. We demonstrate that when added to the duplex form of these oligonucleotides, berenil induces triplex structure formation only if the orientation of third strand is antiparallel to the purine strand.

Thermodynamic parameters of the binding of the tight-binding I12X86 lac repressor to operator and non-operator DNA

The thermodynamic parameters ΔH and ΔS corresponding to the binding of the tight‐binding double mutant lac repressor I12X86 with operator and non‐operator DNA fragments were determined using the nitrocellulose filter binding assay. In both cases the binding processes are entropically driven and accompanied by an unfavorable enthalpy variation. The differences between these parameters and those previously reported for the wild type lac repressor show that the strategy adopted by the mutant to interact with DNA is highly different from that of the wild type repressor and suggest more hydrophobic contacts between the mutant and DNA.

Specific Binding of Cyclic-AMP Receptor Protein to DNA. Effect of the Sequence and of the Introduction of a Nick in the Binding Site

The binding of Escherichia coli Cyclic AMP Receptor Protein (CRP) to several DNA fragments of about 45 base pairs, bearing the natural lactose or galactose sites, as well as several synthetic related sites, was investigated using fluorescence spectroscopy and gel retardation experiments. The salt dependence of the equilibrium binding constant indicates that CRP makes an identical number of ion pairs with the lac, lacL8 and gal sites although the binding constants are drastically different. However increasing the symmetry of the gal site leads to an increase of the number of ion pairs between the protein and the DNA. A single strand nick was introduced at the centre of a symmetrized gal site and this reduces the binding energy of CRP by about 0.6 Kcal. These results are discussed with respect to the bending constraints imposed on the DNA by the binding of CRP. The results are in agreement with the recently published crystal structure of the CRP complexed with DNA [Schutz, S.C., Shields, G.C. and Steitz, T.A., Science 253, 1001-1007 (1991)] showing that the 90 degrees bending of the DNA in the complex results from two kinks.

Spectroscopic Studies on Ethidium Bromide Binding to Intramolecular Parallel and Antiparallel Triple Helices Containing T*A:T and G*G:C Triplets

Abstract The interaction of ethidium bromide (EB), a DNA intercalator, with two intramolecular triplexes 5′d(G4A4G4-[T4]-C4T4C4-[TJ-G4T4G4), 5′d(G4T4G4-[T4]-G4A4G4-[T4]-C4T4C4) ([T4]represents a stretch of 4 thymine residues) and their precursor duplexes has been investigated by circular dichroism, fluorescence and UV absorption spectroscopy. Binding of EB induces a circular dichroism band in the region around 310 nm which is positive for the duplex forms but negative for the triplex forms. We observed that the binding of EB to the duplex form does not induce the formation of the triplex structures. Thermal denaturation experiments demonstrate that EB stabilizes more the parallel triple helix than the antiparallel one. Analysis of the binding process from fluorescence measurements shows that binding constants to the triple helical forms and to the hairpin reference duplex [T4]-G4A4G4-[T4]-C4T4C4) are close. However the binding site size is larger for the triplexes (4–6 base triplets) than for the duplex (2 base pairs).

Molecular Modelling Study of the Netropsin Complexation With a Nucleic Acid Triple Helix

A detailed molecular mechanical study has been made on the complexes of netropsin with the double stranded oligonucleotide (dA)12.(dT)12 and with the triple helix (dA)12.(dT)12.(dT)12. The complexes were built using computer graphics and energy refined using JUMNA program. In agreement with circular dichroism experiments we have shown that 3 netropsins can bind the minor grooves of the triple helix and of the double helix. The groove geometry in the duplex and in the triplex is very similar. However a detailed analysis of the energetic terms shows, in agreement with thermal denaturation studies, that the affinity of netropsin toward the double helices is larger than towards triple helices.