Biomedical subjects

M Dutreix

Publications and source records attributed to M Dutreix.

At least 19 recordsLinked to original sources

PubMed2005

A haploid-specific transcriptional response to irradiation in Saccharomyces cerevisiae.

Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair. We used global transcriptome analysis to investigate the role of ploidy and mating-type in inducing the response to damage in various Saccharomyces cerevisiae strains. We observed a response to DNA damage specific to haploid strains that seemed to be controlled by chromatin regulatory proteins. Consistent with these microarray data, we found that mating-type factors controlled the chromatin-dependent silencing of a reporter gene. Both these analyses demonstrate the existence of an irradiation-specific response in strains (haploid or diploid) with only one mating-type factor. This response depends on the activities of Hdf1 and Sir2. Overall, our results suggest the existence of a new regulation pathway dependent on mating-type factors, chromatin structure remodeling, Sir2 and Hdf1 and independent of Mec1 kinase.

Antigens, Nuclear↗

PubMed2004

Biological detection of low radiation doses by combining results of two microarray analysis methods.

The accurate determination of the biological effects of low doses of pollutants is a major public health challenge. DNA microarrays are a powerful tool for investigating small intracellular changes. However, the inherent low reliability of this technique, the small number of replicates and the lack of suitable statistical methods for the analysis of such a large number of attributes (genes) impair accurate data interpretation. To overcome this problem, we combined results of two independent analysis methods (ANOVA and RELIEF). We applied this analysis protocol to compare gene expression patterns in Saccharomyces cerevisiae growing in the absence and continuous presence of varying low doses of radiation. Global distribution analysis highlights the importance of mitochondrial membrane functions in the response. We demonstrate that microarrays detect cellular changes induced by irradiation at doses that are 1000-fold lower than the minimal dose associated with mutagenic effects.

Analysis of Variance↗

PubMed2002

A new method (GOREC) for directed mutagenesis and gene repair by homologous recombination.

Directed mutagenesis in mammalian cells has been the focus of intense research because of its promising application for gene correction and engineering. Both natural and modified oligonucleotides (ODN), RNA-DNA chimeric oligonucleotide (RDO) and small fragment DNA (SFHR), as well as vector DNA were used for promoting homologous replacement with varying success. It was recently shown that a triple helix-forming oligonucleotide (TFO) tethered to an oligonucleotide (donor DNA) can enhance mutagenesis by homologous recombination in cells. The basic idea is to accelerate homology search by oligonucleotide-directed triple helix formation in the vicinity of the target site for donor DNA. Here we describe a new method named GOREC (guided homologous recombination) which shares similar gene targeting, but has notable difference in the concept with the previous method. It is made of a homing device (TFO) and a donor DNA for effecting distinct functions. They are linked together by non-covalent or covalent interaction. This modular concept allows guidance of either an oligonucleotide (ODN, RDO) or a small DNA fragment to the target site for homologous replacement. Therefore, the triple helix site can be hundreds of base pairs away from the target site. An episomal assay for proof-of-principle study will be presented and discussed.

Animals↗

PubMed2001

Transcriptional induction of repair genes during slowing of replication in irradiated Saccharomyces cerevisiae.

We investigated the inhibition of cell-cycle progression and replication and the induction of the transcriptional response in diploid budding yeast populations exposed to two different doses of gamma-rays resulting in 15 and 85% survival respectively. We studied the kinetics of the cellular response to ionizing treatment during the period required for all of the surviving cells to achieve at least one cell division. The length of these periods increased with the dose. Irradiated populations arrested as large-budded cells containing partially replicated chromosomes. The extent of the S-phase was proportional to the amount of damage and lasted 3 or 7h depending on the irradiation dose. In parallel to the division study, we carried out a kinetic analysis of the expression of 126 selected genes by use of dedicated microarrays. About 26 genes were induced by irradiation and displayed various pattern of expression. Interestingly, 10 repair genes (RAD51, RAD54, CDC8, MSH2, RFA2, RFA3, UBC5, SRS2, SPO12 and TOP1), involved in recombination and DNA synthesis, display similar regulation of expression in the two irradiated populations. Their pattern of expression were confirmed by Northern analysis. At the two doses, the expression of this group of genes closely followed the extended replication period, and their expression resumed when replication restarted. These results suggest that the damage-induced response and DNA synthesis are closely regulated during repair. The analysis of the promoter regions indicates a high occurrence of the three MCB, HAP and UASH regulatory boxes in the promoters of this group of genes. The association of the three boxes could confer an irradiation-replication specific regulation.

Blotting, Northern↗

PubMed2001

Stimulation of RecA-mediated D-loop formation by oligonucleotide-directed triple-helix formation: guided homologous recombination (GOREC).

Oligonucleotide-directed triple helix formation provides an elegant rational basis for gene-specific DNA targeting and has been widely used to interfere with gene expression ("antigene" strategies) and as a molecular tool for biological studies. Various strategies have been developed to introduce sequence modifications in genomes. However, the low efficiency of the overall process in eucaryotic cells impairs efficient recovery of recombinant genomes. Since one limiting step in homologous recombination is the targeting to the homologous sequence, we have tested the contribution of an oligonucleotide-directed triple helix formation on the RecA-dependent association of an oligonucleotide and its homologous target on duplex DNA (D-loop formation). For this study, the recombinant ssDNA fragment was noncovalently associated to a triple helix-forming oligonucleotide. The physicochemical and biochemical characteristics of the triple helix and D-loop structures formed by the complex molecules in the presence or in the absence of RecA protein were determined. We have demonstrated that the triple helix-forming oligonucleotide increases the efficiency of D-loop formation and the RecA protein speeds up also the triple helix formation. The so-called "GOREC" (for guided homologous recombination) approach can be developed as a novel tool to improve the efficiency of directed mutagenesis and gene alteration in living organisms.

Base Sequence↗

PubMed2001

(CA/TG) microsatellite sequences escape the inhibition of recombination by mismatch repair in Saccharomyces cerevisiae.

Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. In the yeast Saccharomyces cerevisiae, repair of mismatches results in gene conversion or restoration, whereas failure to repair mismatches results in postmeiotic segregation (PMS). By examining the conversion and PMS in yeast strains deficient in various MMR genes and heterozygous for large inserts (107 bp) with either a mixed sequence or a 39 (CA/TG) repetitive microsatellite sequence, we demonstrate that: (1) the inhibition of conversion by large inserts depends upon a complex containing both Msh2 and Pms1 proteins; (2) conversion is not inhibited if the single-stranded DNA loop in the heteroduplex is the microsatellite sequence; and (3) large heteroduplex loops with random sequence or repetitive sequence might be repaired by two complexes, containing either Msh2 or Pms1. Our results suggest that inhibition of recombination by heterologous inserts and large loop repair are not processed by the same MMR complexes. We propose that the inhibition of conversion by large inserts is due to recognition by the Msh2/Pms1 complex of mismatches created by intrastrand interactions in the heteroduplex loop.

Base Pair Mismatch↗

PubMed2000

(CA/GT)(n) microsatellites affect homologous recombination during yeast meiosis.

One of the most common microsatellites in eukaryotes consists of tandem arrays of the dinucleotide GT. Although the study of the instability of such repetitive DNA has been extremely fruitful over the last decade, no biological function has been demonstrated for these sequences. We investigated the genetic behavior of a region of the yeast Saccharomyces cerevisiae genome containing a 39-CA/GT dinucleotide repeat sequence. When the microsatellite sequence was present at the ARG4 locus on homologous chromosomes, diploid cells undergoing meiosis generated an excess of tetrads containing a conversion of the region restricted to the region of the microsatellite close to the recombination-initiation double-strand break. Moreover, whereas the repetitive sequence had no effect on the frequency of single crossover, its presence strongly stimulated the formation of multiple crossovers. The combined data strongly suggest that numerous recombination events are restricted to the initiation side of the microsatellite as though progression of the strand exchange initiated at the ARG4 promoter locus was impaired by the repetitive sequence. This observation corroborates in vitro experiments that demonstrated that RecA-promoted strand exchange is inhibited by CA/GT dinucleotide tracts. Surprisingly, meiotic instability of the microsatellite was very high (>0.1 alterations per tetrad) in all the spores with parental and recombinant chromosomes.

Argininosuccinate Lyase↗

PubMed1999

Conserved sequence preference in DNA binding among recombination proteins: an effect of ssDNA secondary structure.

Repetitive sequences have been proposed to be recombinogenic elements in eukaryotic chromosomes. We tested whether dinucleotide repeats sequences are preferential sites for recombination because of their high affinity for recombination enzymes. We compared the kinetics of the binding of the scRad51, hsRad51 and ecRecA proteins to oligonucleotides with repeats of dinucleotides GT, CA, CT, GA, GC or AT. Since secondary structures in single-stranded DNA (ssDNA) act as a barrier to complete binding we measured whether these oligonucleotides are able to form stable secondary structures. We show that the preferential binding of recombination proteins is conserved among the three proteins and is influenced mainly by secondary structures in ssDNA.

Base Sequence↗

PubMed1997

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

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.

Bacterial Proteins↗

PubMed1997

Differences and similarities between various tandem repeat sequences: minisatellites and microsatellites.

Tandemly repetitive DNA sequences are abundantly interspersed in the genome of practically all eukaryotic species studied. The relative occurrence of one type of repetitive sequence and its location in the genome appear to be species specific. A common property of repetitive sequences within the living world is their ability to give rise to variants with increased or reduced number of repeats. This instability depends upon numerous parameters whose exact role is unclear: the number of repeats, their sequence content, their chromosomal location, the mismatch repair capability of the cell, the developmental stage of the cell (mitotic or meiotic) and/or the sex of the transmitting parent. It is now apparent that mutations in repetitive sequences are a common cause of human disease, including cancer and disorders which may exhibit a dominant mode of inheritance. Two mechanisms have been proposed to explain the instability of repetitive sequences: DNA polymerase slippage, which may account for the instability of short repeats and unequal recombination which reshuffles repeat variants and maintains repeat heterogeneity in minisatellites. The purpose of this review is to show that no general rule can explain the instability of repetitive sequence. Each sequence of repeats is under the influence of local and general biological activities that determine its level of instability.

Animals↗

PubMed1995

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

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.

Bacterial Proteins↗

PubMed1993

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

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).

Bacteriophage M13↗

PubMed1993

Partial characterization of an rpoD-like gene of Lactococcus lactis subsp. lactis ML3 with a polymerase chain reaction-based approach.

With degenerated oligonucleotide primers for conserved regions of bacterial sigma factor proteins, a 117-bp internal DNA fragment of an rpoD-like gene of Lactococcus lactis subsp. lactis ML3 was amplified by the polymerase chain reaction (PCR). The DNA sequence of this PCR product was determined by cycle sequencing, and the deduced amino acid sequence of this internal fragment showed an extensive homology with the known sigma factor sequences from six other microorganisms and present a 13-amino acid region corresponding to the typical "RpoD box" of primary sigma factors. This PCR product was used as a probe to specifically detect sigma homologs in Pediococcus acidilactici, Leuconostoc lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Enterococcus faecalis, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris. These data are consistent with the existence of a high similarity between the primary sigma factors from diverse Gram-positive microorganisms.

Amino Acid Sequence↗

PubMed1992

A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments.

The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein. If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction. In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction. Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein. The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step. In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype. Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities. RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody. However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI. RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis. These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments.

Adenosine Triphosphatases↗

PubMed1991

The effects on strand exchange of 5' versus 3' ends of single-stranded DNA in RecA nucleoprotein filaments.

Since the ends of DNA chains are thought to be important in homologous recombination, the way in which RecA protein and similar recombination enzymes process ends is important. We analyzed the effects of ends both on the formation of joints, and the progression of strand exchange. When the only homologous end was provided by a single strand, there was no significant difference between the formation of joints at a 5' end or a 3' end; but in agreement with the report of Konforti & Davis, Escherichia coli single-stranded DNA binding protein (SSB) selectively inhibited the activity of 5' ends. Complete strand exchange, assessed by study of linear single-stranded and double-stranded substrates, took place only in the 5' to 3' direction relative to DNA in the nucleoprotein filament. These observations pose a paradox: in the presence of SSB, of which there are about 800 tetramers per cell, the formation of homologous joints by RecA protein is favored at a 3' end, from which, however, authentic strand exchange appears not to occur. Since observations reported here and elsewhere show that joints have different properties when formed at a 5' versus a 3' end, we suggest that they may be processed differently in vivo.

DNA, Circular↗

PubMed1991

Stable three-stranded DNA made by RecA protein.

When RecA protein, in the form of a nucleoprotein filament containing circular single-stranded DNA (plus strand only), reacts with homologous linear duplex DNA, a directional transfer ensues of a strand from the duplex DNA to the nucleoprotein filament, resulting in the displacement of the linear plus strand in the 5' to 3' direction. The initial homologous synapsis, however, can occur at either end of the duplex DNA, or anywhere in between, and when homology is restricted to different regions of the duplex DNA, the joint molecules that form in each region show striking differences in stability upon deproteinization: distal joints greater than proximal joints much greater than medial joints. In the deproteinized distal joints, which are thermostable, 2000 nucleotide residues of the circular plus strand are resistant to P1 nuclease; both strands of the original duplex DNA remain resistant to P1 nuclease, and the potentially displaceable linear plus strand, which has a 3' homologous end, remains resistant to Escherichia coli exonuclease I. These observations suggest that RecA protein promotes homologous pairing and strand exchange via long three-stranded DNA intermediates and, moreover, that, once formed, such triplex structures in natural DNA are stable even when RecA protein has been removed.

DNA↗

PubMed1991

Production of triple-stranded recombination intermediates by RecA protein, in vitro.

During the directional strand exchange that is promoted by RecA protein between linear duplex DNA and circular single-stranded DNA, a triple-stranded DNA intermediate was formed and persisted even after the completion of strand transfer followed by deproteinization. In the deproteinized three-stranded DNA complexes, the sequestered linear third strand resisted digestion by E coli exonuclease I. In relation to polarity of strand exchange which defines the proximal and distal ends of the duplex DNA, when homology was restricted to the distal region of duplex substrate, the joints formed efficiently and were stable even upon complete deproteinization. Enzymatic probing of deproteinized distal joints with nuclease P1 revealed that the joints consist of long three-stranded structures that at neutral pH lack significant single-stranded character in any of the three strands. Instead of circular single-stranded DNA, when a linear single strand is recombined with partially homologous duplex DNA, in the presence of SSB, the formation of homologous joints by RecA protein, is significantly more efficient at distal end than at the proximal. Taken together, these observations suggest that with any single-stranded DNA (circular or linear), RecA protein efficiently promotes the formation of distal joints, from which, however, authentic strand exchange may not occur. Moreover, these joints might represent an intermediate which is trapped into a stable triple stranded state.

DNA Replication↗

PubMed1991

A RecA protein mutant deficient in its interaction with the UmuDC complex.

recA1730 is a dominant point mutation preventing SOS mutagenesis. We demonstrate here that: i) RecA1730 fails to produce mutagenesis even though UmuD' is formed, ii) recA1730, when complemented by recA+, can cleave LexA protein and it displays a UmuDC- phenotype in spite of adequate concentrations of matured UmuD' and UmuC proteins, iii) the Mut- phenotype caused by RecA1730 is partially alleviated by MucAB proteins, functional analogs of UmuDC. To explain the mutant phenotype, we postulate that recA1730 impairs a RecA function required for the positioning of the UmuD'C complex within the replisome at the site of lesions.

Bacterial Proteins↗