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F Hamy

Publications and source records attributed to F Hamy.

26 records · Page 2Linked to original sources
PubMed1994

Localized chemical reactivity in DNA associated with the sequence-specific bisintercalation of echinomycin.

Four complementary footprinting and probing techniques utilizing DNAse I, methidiumpropyl EDTA (MPE).FeII, diethyl pyrocarbonate (DEPC) and KMnO4 as DNA-cleaving or DNA-modifying agents have been applied to investigate the sequence-specific binding to DNA of the antitumour antibiotic echinomycin. A 265 bp EcoRI-PvuII DNA restriction fragment excised from plasmid pBS was used as a substrate. Six regions of protection against DNAase I cleavage were located on the 265-mer: three sites encompass the sequences 5'-TCGA or 5'-GCGT and the three others contain 5'-GpG (CpC) dinucleotide sequences where the inhibition of DNAase I cutting by echinomycin is less pronounced. In contrast, MPE.FeII cleavage allows identification of only three echinomycin-binding sites on the 265-mer: two sites contain the sequence 5'-TCGA and one encompasses the sequence 5'-ACCA. Cleavage of DNA by MPE.FeII in the presence of echinomycin remains practically unaffected at the sequence 5'-GCGT, despite its identification by DNAase I as a strong site for binding the antibiotic, as well as at the two other sequences containing GpG steps. With both DNAase I and MPE.FeII, enhanced DNA cleavage is evident at AT-rich sequences in the presence of echinomycin. Enhanced reactivity towards KMnO4 and DEPC provides clear evidence for sequence-dependent conformational changes in DNA induced by the antibiotic. The experiments reveal that KMnO4 reacts most strongly with thymines located around, but not necessarily adjacent to, an echinomycin-binding site, whereas the carbethoxylation reactions caused by DEPC occur primarily at the adenine residues lying immediately 5' or 3' to the dinucleotide that denotes an echinomycin-binding site. The results reported here demonstrate that DEPC and KMnO4 serve as sensitive probes for different states of the DNA helix. It seems that the reaction with KMnO4 involves transient unstacking events, whereas the carbethoxylation reaction of DEPC requires larger-scale helix opening.

Base Sequence↗
PubMed1993

High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region.

The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations. The relative dissociation constants for the mutant TAR RNAs were measured using a dual-label competition filter binding assay in which 35S-labelled wild-type TAR RNA (K1) was competed against 3H-labelled mutant TAR RNA (K2). The error in the self-competition experiment was usually less than 10% (e.g. K2/K1 = 1.07 +/- 0.05, n = 19) and the experimental data accurately matched theoretical curves calculated with fitted dissociation constants. Mutations in U23, a critical residue in the U-rich "bulge" sequence, or in either of the two base-pairs immediately above the "bulge", G26.C39 and A27.U38 reduced that affinity by 8- to 20-fold. Significant contributions to tat binding affinity were also made by the base-pairs located immediately below the bulge. For example, mutation of A22.U40 to U.A reduced tat affinity 5-fold, and mutation of G21.C41 to C.G reduced tat affinity 4-fold. The binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays. Each of the peptides showed significantly reduced affinities for wild-type TAR RNA compared to the tat protein. The ADP-2 (residues 43 to 72), ADP-3 (residues 48 to 72) and ADP-5 (residues 49 to 86) peptides were unable to discriminate between wild-type TAR RNA and TAR RNA mutants with the same fidelity as the tat protein. For example, these peptides showed no more than 3-fold reductions in affinity relative to wild-type TAR RNA for the U23-->C mutation in the bulge, or G26.G39-->C.G mutation in the stem of TAR RNA. By contrast, the ADP-I (residues 37 to 72), ADP-4 (residues 32 to 62) and ADP-6 (residues 32 to 72) peptides, which each carry amino acid residues from the "core" region of the tat protein have binding specificities that more closely resemble the protein. The ADP-4 and ADP-6 peptides showed between 4- and 7-fold reductions in affinity for the U23-->C or G26.C39-->C.G mutations. The ADP-1 peptide most closely resembles the protein in its binding specificity and showed 9-fold and 14-fold reductions in affinity for the two mutants, respectively. Chemical-modification interference assays using diethylpyrocarbonate (DEPC) and ethylnitrosourea (ENU) were also used to compare the binding properties of the tat protein and the tat-derived peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA.

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.

Base Sequence↗
PubMed1993

HIV gene regulatory proteins tat and rev and their interactions with synthetic RNA.

Synthetic oligoribonucleotides have been prepared and annealed to form model RNA duplexes that mimic the high affinity RNA recognition sites for the HIV-1 tat and rev proteins. The contributions of individual functional groups on the model RNAs to the specificity of binding by their respective proteins were studied by use of oligoribonucleotides containing site-specifically modified nucleotides. Both tat and rev appear to recognise specifically a limited number of functional groups in the major groove of an RNA double helix distorted by virtue of unpaired or non-Watson-Crick paired nucleotides.

Base Sequence↗
PubMed1992

Comparison between synthetic nuclear localization signal peptides from the steroid/thyroid hormone receptors superfamily.

The main objective of the study is to demonstrate that short basic peptides from the steroid/thyroid hormone receptors superfamily act as Nuclear Localization Signals out of receptors context. Such synthesized peptides, chemically coupled to Bovine Serum Albumin, were shown to enable the corresponding BSA-conjugate to be transported to the nucleus. A second objective is to demonstrate the utility of viral cointernalization as a good method for rapid quantitation, comparison and competition in nuclear entry.

Adenoviridae↗
PubMedDate not supplied

Rational optimization of a HIV-1 Tat inhibitor: rapid progress on combinatorial lead structures.

Lead molecules identified by combinatorial chemistry approaches are preferred starting points for straightforward improvements of compound profiles. Structure-guided rationales can be supported and complemented by systematic variations based on the modular nature of the molecules. A peptoidic compound (CGP 64222), previously identified from a sequential unrandomization process, was shown to specifically inhibit the interaction between the HIV-1 trans-activator Tat and its RNA response element TAR. To improve the compound's pharmaceutical attractiveness an approach to reduce both, size and number of charges was pursued. Because this resulted in activity decrease, parallel synthesis with variations on one rationally defined position aimed at the identification of structural determinants was undertaken to regain in vitro activity in biochemical and cellular Tat-TAR interaction assays. As a result CGP74026 was identified, a drastically simplified but highly active Tat antagonist, which is able to block HIV-1 replication even in primary human cells.

Acridines↗
PubMedDate not supplied

[PKC 412].

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Antineoplastic Agents↗