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Accueil > Départements > Biologie des Génomes > Bénédicte MICHEL : Stabilité de l’ADN bactérien

Publications de l’équipe


  • B. Michel, A. K. Sinha, et D. R. F. Leach, « Replication Fork Breakage and Restart in Escherichia coli », Microbiology and molecular biology reviews: MMBR, vol. 82, nᵒ 3, sept. 2018.
    Résumé : In all organisms, replication impairments are an important source of genome rearrangements, mainly because of the formation of double-stranded DNA (dsDNA) ends at inactivated replication forks. Three reactions for the formation of dsDNA ends at replication forks were originally described for Escherichia coli and became seminal models for all organisms: the encounter of replication forks with preexisting single-stranded DNA (ssDNA) interruptions, replication fork reversal, and head-to-tail collisions of successive replication rounds. Here, we first review the experimental evidence that now allows us to know when, where, and how these three different reactions occur in E. coli. Next, we recall our recent studies showing that in wild-type E. coli, spontaneous replication fork breakage occurs in 18% of cells at each generation. We propose that it results from the replication of preexisting nicks or gaps, since it does not involve replication fork reversal or head-to-tail fork collisions. In the recB mutant, deficient for double-strand break (DSB) repair, fork breakage triggers DSBs in the chromosome terminus during cell division, a reaction that is heritable for several generations. Finally, we recapitulate several observations suggesting that restart from intact inactivated replication forks and restart from recombination intermediates require different sets of enzymatic activities. The finding that 18% of cells suffer replication fork breakage suggests that DNA remains intact at most inactivated forks. Similarly, only 18% of cells need the helicase loader for replication restart, which leads us to speculate that the replicative helicase remains on DNA at intact inactivated replication forks and is reactivated by the replication restart proteins.
    Mots-clés : BDG, chromosome terminus, double-strand break, PriA, RecA, RecBC, RecBCD, RecG, recombination, replication fork reversal, replication restart, RuvAB, STABAC.

  • A. K. Sinha, C. Possoz, A. Durand, J. - M. Desfontaines, F. - X. Barre, D. R. F. Leach, et B. Michel, « Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome », PLOS Genetics, vol. 14, nᵒ 3, p. e1007256, mars 2018.


  • B. Michel et S. J. Sandler, « Replication Restart in Bacteria », Journal of Bacteriology, p. JB.00102-17, mars 2017.

  • B. Michel et A. K. Sinha, « The inactivation of rfaP, rarA or sspA gene improves the viability of the Escherichia coli DNA polymerase III holD mutant », Molecular Microbiology, mars 2017.
    Résumé : The Escherichia coli holD mutant is poorly viable because the stability of holoenzyme polymerase III (Pol III HE) on DNA is compromised. Consequently, the SOS response is induced and the SOS polymerases DinB and Pol II further hinder replication. Mutations that restore the holD mutant viability belong to two classes, those that stabilize Pol III on DNA and those that prevent the deleterious effects of DinB over-production. We identified a dnaX mutation and the inactivation of rfaP and sspA genes as belonging to the first class of holD mutant suppressors. dnaX encodes a Pol III clamp loader subunit that interacts with HolD. rfaP encodes a lipopolysaccharide kinase that acts in outer membrane biogenesis. Its inactivation improves the holD mutant growth in part by affecting potassium import, previously proposed to stabilize Pol III HE on DNA by increasing electrostatic interactions. sspA encodes a global transcriptional regulator and growth of the holD mutant in its absence suggests that SspA controls genes that affect protein-DNA interactions. The inactivation of rarA belongs to the second class of suppressor mutations. rarA inactivation has a weak effect but is additive with other suppressor mutations. Our results suggest that RarA facilitates DinB binding to abandoned forks.
    Mots-clés : DBG, STABAC.

  • A. K. Sinha, A. Durand, J. - M. Desfontaines, I. Iurchenko, H. Auger, D. R. F. Leach, F. - X. Barre, et B. Michel, « Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme », PLoS genetics, vol. 13, nᵒ 10, p. e1006895, oct. 2017.
    Résumé : Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.
    Mots-clés : DBG, EMC2, NGS, PF, STABAC.


  • A. Durand, A. K. Sinha, C. Dard-Dascot, et B. Michel, « Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant », PLoS genetics, vol. 12, nᵒ 6, p. e1006114, juin 2016.
    Résumé : Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo.
    Mots-clés : DBG, STABAC.


  • E. Bentchikou, C. Chagneau, E. Long, M. Matelot, J. - F. Allemand, et B. Michel, « Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli? », PloS One, vol. 10, nᵒ 8, p. e0134892, 2015.
    Résumé : In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.
    Mots-clés : Bacterial Proteins, DBG, DNA Helicases, DNA Repair, DNA replication, DNA, Bacterial, DNA, Single-Stranded, DNA-Binding Proteins, DnaB Helicases, Escherichia coli, Escherichia coli Proteins, Luminescent Proteins, Microscopy, Fluorescence, Nucleic Acid Conformation, Protein Binding, STABAC.
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Principales Publications avant 2015

- Duigou, S., Silvain, M., Viguera, E., and Michel, B. (2014) ssb gene duplication restores the viability of holC and holD Escherichia coli mutants. Plos Genet in press

- De Septenville, A.L., Duigou, S., Boubakri, H., and Michel, B. (2012). Replication fork reversal after replication-transcription collision. PLoS Genet 8, e1002622.

- Baharoglu, Z., Lestini, R., Duigou, S., Michel, B. (2010) RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases. Mol Microbiol, 77 (2) 324-36.

- Boubakri, H., de Septenville, A.-L., Viguera, E. and Michel, B. (2010) The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo. EMBO J, 29 (1) 145-57. Erratum in : EMBO J. 2010, 29 (1) 278.

- Le Masson, M., Baharoglu, Z. and Michel, B. (2008) ruvA and ruvB mutants specifically impaired for replication fork reversal. Mol Microbiol, 70 (2) 537-48.

- Baharoglu, Z., Bradley, A.-S., Le Masson, M., Tsaneva, I. and Michel, B. (2008) ruvA mutants that resolve Holliday junctions but do not reverse replication forks. PLoS Genet, 4 (3) e1000012.

- Lestini, R. and Michel, B. (2007) UvrD controls the access of recombination proteins to blocked replication forks. EMBO J, 26 (16) 3804-14.

- Michel, B., Boubakri, H., Baharoglu, Z., Lemasson, M. and Lestini, R. (2007) Recombination proteins and rescue of arrested replication forks. DNA Repair (Amst). 6 (7) 967-80, Review.

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