Biomedical subjects

Marie Dutreix

Publications and source records attributed to Marie Dutreix.

8 recordsLinked to original sources

PubMed2006

Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation.

The mechanisms of RecA-mediated three-strand homologous recombination are investigated at the single-molecule level, using magnetic tweezers. Probing the mechanical response of DNA molecules and nucleoprotein filaments in tension and in torsion allows a monitoring of the progression of the exchange in real time, both from the point of view of the RecA-bound single-stranded DNA and from that of the naked double-stranded DNA (dsDNA). We show that strand exchange is able to generate torsion even along a molecule with freely rotating ends. RecA readily depolymerizes during the reaction, a process presenting numerous advantages for the cell's 'protein economy' and for the management of topological constraints. Invasion of an untwisted dsDNA by a nucleoprotein filament leads to an exchanged duplex that remains topologically linked to the exchanged single strand, suggesting multiple initiations of strand exchange on the same molecule. Overall, our results seem to support several important assumptions of the monomer redistribution model.

Biophysical Phenomena↗

PubMed2005

Impact of ionizing radiation on the life cycle of Saccharomyces cerevisiae Ty1 retrotransposon.

Ty1 elements, LTR-retrotransposons of Saccharomyces cerevisiae, are known to be activated by genetic and environmental stress. Several DNA-damaging agents have been shown to increase both Ty1 transcription and retrotransposition. To explore further the relationship between Ty1 mobility and DNA damage, we have studied the impact of ionizing radiation at different steps of the Ty1 life cycle. We have shown that Ty1 transposition is strongly activated by gamma-irradiation and we have analysed its effect on Ty1 transcription, TyA1 protein and Ty1 cDNA levels. The activation of transposition rises with increasing doses of gamma-rays and is stronger for Ty1 elements than for the related Ty2 elements. Ty1 RNA levels are markedly elevated upon irradiation; however, no significant increase of TyA1 protein was detected as measured by TYA1-lacZ fusions and by Western blot. A moderate increase in Ty1 cDNA levels was also observed, indicating that ionizing radiation can induce the synthesis of Ty1 cDNA. In diploid cells and ste12 mutants, where both Ty1 transcription and transposition are repressed, gamma-irradiation is able to activate Ty1 transposition and increases Ty1 RNA levels. These results suggest the existence of a specific regulatory pathway involved in Ty1 response to the gamma-irradiation that would be independent of Ste12 and mating-type factors. Our findings also indicate that ionizing radiation acts on several steps of the Ty1 life cycle.

Blotting, Northern↗

PubMed2005

Numerical investigation of sequence dependence in homologous recognition: evidence for homology traps.

During the initial phase of RecA-mediated recombination, known as the search for homology, a single-stranded DNA coated by RecA protein and a homologous double-stranded DNA have to perfectly align and pair. We designed a model for the homology search between short molecules, and performed Monte Carlo Metropolis computer simulations of the process. The central features of our model are 1), the assumption that duplex DNA longitudinal thermal fluctuations are instrumental in the binding; and 2), the explicit consideration of the nucleotide sequence. According to our results, recognition undergoes a first slow nucleation step over a few basepairs, followed by a quick extension of the pairing to adjacent bases. The formation of the three-stranded complex tends to be curbed by heterologies but also by another possible obstacle: the presence of partially homologous stretches, such as mono- or polynucleotide repeats. Actually, repeated sequences are observed to trap the molecules in unproductive configurations. We investigate the dependence of the phenomenon on various energy parameters. This mechanism of homology trapping could have a strong biological relevance in the light of the genomic instability experimentally known to be triggered by repeated sequences.

Algorithms↗

PubMed2004

Model of RecA-mediated homologous recognition.

We consider theoretically the homology search between a long double-stranded DNA and a RecA-single-stranded DNA nucleofilament, emphasizing the polymeric nature of the search and the ability of double-stranded DNA to overcome the difference in pitch between itself and the nucleofilament by thermally activated stretching from the canonical B state to the metastable, stretched S state. Our analytical first-passage-time analysis agrees well with experimental data, predicts new dependencies on the intracellular fluid viscosity and ionic strength, and strongly suggests that initial homologous recognition involves a three base-pair seed.

Adenosine Triphosphatases↗

PubMed2004

Twisting and untwisting a single DNA molecule covered by RecA protein.

We study dsDNA-RecA interactions by exerting forces in the pN range on single DNA molecules while the interstrand topological state is controlled owing to a magnetic tweezers setup. We show that unwinding a duplex DNA molecule induces RecA polymerization even at moderate force. Once initial polymerization has nucleated, the extent of RecA coverage still depends on the degree of supercoiling: exerting a positive or negative torsional constraint on the fiber forces partial depolymerization, with a strikingly greater stability when ATPgammaS is used as a cofactor instead of ATP. This nucleofilament's sensitivity to topology might be a way for the bacterial cell to limit consumption of precious RecA monomers when DNA damage is addressed through homologous recombination repair.

Adenosine Triphosphate↗

PubMed2003

Stimulation of D-loop formation by polypurine/polypyrimidine sequences.

Most of the approaches used to correct gene mutations in mammalian cells involve the targeting of short nucleotide molecules to homologous chromosomal sequences and the replacement of resident sequences via homologous recombination and mismatch repair. The limited efficiency and inconsistent reproducibility of these techniques are major constraints to their use in gene therapy. One of the main problems is that it is impossible to obtain reproducible results when the targeted gene loci differ. We investigated the effects of flanking sequences on homologous recombination by means of an in vitro assay of the efficiency of oligonucleotide targeting to its homologous sequence on a large duplex molecule in a reaction catalysed by the Escherichia coli RecA protein. We demonstrated that polypurine.polypyrimidine tracts (PPTs) in duplex DNA strongly stimulate the formation of D-loops with short oligodeoxynucleotides. This result was reproduced with various PPT sequences and oligonucleotides. The stimulatory effect was observed at loci as far as 4000 bp from the PPT. The formation of complexes between the oligonucleotide and the duplex molecule depended on the extent of sequence similarity between the two DNAs and the presence of the RecA protein. The stimulatory effect was inhibited by excess RecA and restored by adding heterologous DNA. We suggest that PPT sequences induce conformational changes in duplex DNA, leading to the aggregation of molecules, facilitating homology searches. We compared, in vivo, the efficiency of the oligonucleotide-mediated correction of a URA3 chromosomal mutation for sequences with and without a PPT sequence in the vicinity. Consistent with our in vitro results, the efficiency of correction was eight times higher in the presence of the PPT sequence.

Base Sequence↗

PubMed2003

[Homologous recombination and gene targeting].

Gene therapy and the production of mutated cell lines or model animals both require the development of efficient, controlled gene-targeting strategies. Classical approaches are based on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. The low frequency of homologous recombination in mammalian cells leads to inefficient targeting. Here, we review the limiting steps of classical approaches and the new strategies developed to improve the efficiency of homologous recombination in gene-targeting experiments.

Animals↗

PubMed2003

Tyrosinase gene correction using fluorescent oligonucleotides.

Gene therapy and production of mutated cell lines or animal models should be improved significantly once efficient controlled gene targeting strategies are developed. We used short single-stranded oligodeoxynucleotides (ODN), in some cases coupled to the fluorescent dye fluorescein isothiocyanate (FITC), to correct an endogenic natural point mutation in melanocytes in culture. The addition of the FITC molecule to the 5' extremity of the ODN did not interfere with the efficiency of the reversion of the mutation and did not have any deleterious side-effects. The use of fluorescent ODN could lead to great improvement in the technique. In particular, it may facilitate sorting of the transfected cells in the treated population, and thereby significantly increase the percentage of corrected cells.

Animals↗