Réparation, Radiations et Thérapies innovantes anticancer

Publications de l’équipe

Année de publication : 1997

M Dutreix (1997 Nov 21)

(GT)n repetitive tracts affect several stages of RecA-promoted recombination.

Journal of molecular biology : 105-13 En savoir plus
Résumé

The effect of GT/CA dinucleotide repeat tracts on RecA-dependent homologous recombination was examined in vitro. By measuring the binding of RecA protein to oligonucleotides containing GT or CA repeats using the surface plasmon resonance (BIAcore), we show that the efficiency of RecA protein binding is sequence dependent: the protein binds to non-repetitive, poly(CA) or poly(GT) sequences with an increasing affinity. This preferential binding to repetitive sequences is specific for RecA protein and is not observed with the single-strand DNA binding (SSB) protein. Despite the fact that RecA filaments formed on GT tracts efficiently bind duplex DNAs, they are unable to promote stable joint formation. Moreover, strand exchange between a duplex DNA and a fully homologous single-stranded DNA (ssDNA) containing dinucleotide repeats, is inhibited as a function of the length of the repetitive tract. The number of molecules which underwent a complete strand exchange decreased from 100% to 80% and 30% for DNA containing seven, 16 and 39 GT repeats, respectively. The inhibition is less pronounced when the ssDNA contains CA instead of GT dinucleotide repeats. We propose that the high affinity of RecA protein for (CA)n or (GT)n tracts prevents strand exchange from progressing across such sequences. Thus, our results suggest that repetitive tracts of dinucleotides CA/GT could influence recombinational activity potentially leading to an increase in genomic rearrangements.

Replier

Année de publication : 1995

H Szpilewska, P Bertrand, A Bailone, M Dutreix (1995 Jan 1)

In vitro inhibition of RecA-mediated homologous pairing by UmuD’C proteins.

Biochimie : 848-53 En savoir plus
Résumé

The process of SOS mutagenesis in Escherichia coli requires: i) the replisome enzymes; ii) RecA protein; and iii) the formation of the UmuD’C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. It has recently been shown that the UmuD’C complex, if overproduced, inhibits recombinational repair of a UV-damaged plasmid DNA as well as homologous recombination in an Hfr x F- cross. Since UmuD’C proteins might inhibit an early recombination step by interacting with a RecA nucleo-protein filament, we checked whether UmuD’C proteins will inhibit RecA promoted homologous pairing in vitro. We tested the inhibitory action of UmuD’C proteins in a crude bacterial extract containing possible cofactors such as chaperone proteins that ensure the proper folding of UmuC and the assembly of the UmuD’C complex in vivo. We used a novel recombination assay in which RecA protein promotes the formation of a stable plectonemic joint between a circular single-stranded DNA immobilized onto a membrane and an incoming homologous linear duplex DNA. Under these conditions we show that UmuD’C proteins inhibit the formation of joint molecules.

Replier

Année de publication : 1993

P Bertrand, E Corteggiani, M Dutreix, J Coppey, B S Lopez (1993 Aug 11)

Homologous pairing between single-stranded DNA immobilized on a nitrocellulose membrane and duplex DNA is specific for RecA activity in bacterial crude extract.

Nucleic acids research : 3653-7 En savoir plus
Résumé

Reaction between a circular single stranded and a linear double stranded DNA molecule (ssDNA and dsDNA) provides an efficient system to study recombination mediated by RecA protein. However, classical assays using reaction in solution require highly purified enzymes. This limits biochemical studies of mutant RecA proteins from Escherichia coli or of RecA proteins from other organisms. We describe here an assay that is specific for RecA activity even in bacterial crude extracts. In this assay, the ssDNA is bound to a nitrocellulose membrane, proteins are loaded on this membrane and it is then incubated with a labeled homologous dsDNA. Joint molecules are visualized by autoradiography. We have shown that, despite the reduced mobility of the DNA due to its binding to the membrane, RecA protein is able to promote formation of stable plectonemic joints, in a homology dependent manner. Fourteen other proteins involved in DNA metabolism were checked and did not produce a signal in our assay. Moreover, in Dot blot analysis as well as after native electrophoresis and electrotransfer on a ssDNA coated membrane, production of a signal was strictly dependent on the presence of active RecA protein in the bacterial crude extracts used. We named this assay Pairing On Membrane blot (POM blot).

Replier