Christian Cazenave

Anti-messenger oligodeoxynucleotides: specific inhibition of rabbit β-globin synthesis in wheat germ extracts and Xenopus oocytes

Oligodeoxyribonucleotides complementary to the initiation region of rabbit beta-globin messenger RNA were used to selectively inhibit translation in a wheat germ extract and in injected Xenopus oocytes. The oligonucleotides interacted specifically with their RNA target as shown by thermal denaturation studies of hybrids on nitrocellulose filters. The longest oligonucleotide used (17-mer) efficiently blocked translation both in vitro and in vivo. In contrast the shortest one (8-mer) exhibited only a limited effect. The translation block was specific. The synthesis of endogenous proteins in oocytes and that of alpha-globin in the in vitro system were not affected by anti-beta-globin oligonucleotides. A non-complementary oligonucleotide had no inhibitory effect.

Absorption and fluorescence studies of the binding of the recA gene product from E. coli to single-stranded and double-stranded DNA: ionic strength dependence

The binding of the recA gene product from E. coli to double-stranded and single-stranded nucleic acids has been investigated by following the change in melting temperature of duplex DNA and the fluorescence of single-stranded DNA or poly(dA) modified by reaction with chloroacetaldehyde. At low ionic strength, in the absence of Mg2+ ions, RecA protein binds preferentially to duplex DNA or poly(dA-dT). This leads to an increase of the DNA melting temperature. Stabilization of duplex DNA decreases when ionic strength or pH increases. In the presence of Mg2+ ions, preferential binding to single-stranded polynucleotides is observed. Precipitation occurs when duplex DNA begins to melt in the presence of RecA protein. From competition experiments, different single-stranded and double-stranded polydeoxynucleotides can be ranked according to their ability to bind RecA protein. Structural changes induced in nucleic acids upon RecA binding are discussed together with conformational changes induced in RecA protein upon magnesium binding.

RNA AND N3′ → P5′ KISSING APTAMERS TARGETED TO THE TRANS-ACTIVATION RESPONSIVE (TAR) RNA OF THE HUMAN IMMUNODEFICIENCY VIRUS-1

We used in vitro selection to identify RNA aptamers able to selectively bind to the TAR RNA motif of HIV-1, an unperfect RNA hairpin involved in the transcription of the retroviral genome. We selected aptameric RNA hairpins giving rise to kissing complexes with TAR. The N3′ → P5′ phosphoramidate variant of the aptamer bind to TAR with a Kd in the low nanomolar range. However, only the RNA-RNA loop-loop complex is recognized by the Rop protein of E. coli which is specific for kissing complexes.

Mechanisms for the recognition of chemically-modified DNA by peptides and proteins.

Resume La proteine codee par le gene 32 du bacteriophage T4 (specifique des acides nucleiques en simple brin, SSB) et des oligopeptides contenant des residus basiques et aromatiques (par exemple Lys-Trp-Lys) ont ete utilises pour analyser la structure locale des regions du DNA modifiees soit par des agents cancerogenes (aminofluorene, agents alkylants), soit par des substances antitumorales (derives du platine). La fluorescence du tripeptide Lys-Trp-Lys est completement inhibee quand le residu tryptophane est empile avec les bases nucleiques ; cette interaction se fait preferentiellement dans les regions localement desappariees du DNA. La proteine du gene 32 du phage T4 (gp32) se fixe de facon non-cooperative sur les regions localement ouvertes de la double helice alors quelle se fixe de facon cooperative sur les acides nucleiques en simples brins. La fixation covalente de derives de laminofluorene sur les guanines du DNA induit une ouverture locale de la double helice qui est reconnue a la fois par Lys-Trp-Lys et par la proteine gp 32. Des trois derives du platine etudies (cis-Pt, trans-Pt, Pt-dien), seul le cis-Pt induit des structures locales capables de fixer fortement le peptide et la proteine gp 32. La methylation du DNA par le dimethylsulfate na pratiquement aucun effet sur la fixation de Lys-Trp-Lys et de gp 32. Les sites apuriniques crees par elimination des purines methylees sont des sites tres forts de fixation du peptide alors que la proteine gp 32, au contraire, ne les reconnait pas. Lincubation du complexe DNA apurinique Lys-Trp-Lys a 37°C entraine une coupure de la liaison phosphodiester au niveau des sites apuriniques. Ce tripeptide, et dautres oligopeptides contenant des residus lysine et tryptophane ou tyrosine, se comportent donc comme des endonucleases specifiques des sites apuriniques : le residu aromatique permet la reconnaissance selective de ces sites par insertion a la place de la purine manquante et les groupes NH2 des lysines catalysent la coupure de la liaison phosphodiester par un mecanisme de β-elimination. Apres coupure de la chaine au voisinage des sites apuriniques, le tripeptide Lys-Trp-Lys perd une partie de son affinite. Au contraire la proteine gp 32 se fixe fortement sur ces sites apuriniques coupes. Un mecanisme est propose pour expliquer la reconnaissance des sites apuriniques dans le DNA et laction sequentielle des endonucleases apuriniques et des proteines specifiques de simple brin (SSB) au cours de la reparation du DNA contenant des sites apuriniques. La proteine codee par le gene recA dE. coli se fixe sur les acides nucleiques en simple brin ; cette fixation est responsable de lapparition de deux activites enzymatiques : ATPase et protease. Des experiences preliminaires indiquent que la fixation de la proteine recA sur le DNA en simple brin et sur le polydT est accompagnee dune inhibition de fluorescence de la proteine. La taille apparente du site de fixation est cependant tres differente dans les deux cas. La proteine recA pourrait donc posseder une selectivite de fixation vis-a-vis de certaines sequences. Son mode de fixation sur des zones localement ouvertes dun DNA modifie par des agents mutagenes est discute.

Binding of recA protein from E. Coli to double-stranded DNA: influence of the degree of superhelicity

The binding of the recA protein from E. coli to supercoiled double-stranded DNA is strongly dependent upon the superhelical density of the DNA molecule. A threshold of superhelical density is required for strong binding in the presence of ATP. This finding is consistent with a model in which recA protein first binds to unpaired regions and then polymerises on the contiguous double-stranded lattice.