Etude de la cohésion des chromatides soeurs en réponse à un stress génotoxique chez E. coli

Maintaining genome integrity through replication is an essential process for the cell cycle. However, many factors can compromise this replication and thus the genome integrity. Mitomycin C is a genotoxic agent that creates a covalent link between the two DNA strands. When the replication fork encounters the DNA crosslink, it breaks and creates a DNA double strand break (DSB). Escherichia coli (E.coli) is a widely used model for studying complex DNA mechanisms. When facing a DNA DSB, E. coli activates the SOS response pathway. The SOS response comprises over 50 genes that are under the control of a LexA-repressed promoter. Upon a DSB induction, RecA, a central protein of the SOS response will trigger the degradation of LexA and all the SOS genes will be expressed.We have developed a novel molecular biology tool that reveals contacts between sister chromatids that are cohesive. It has been shown in the lab (Lesterlin et al. 2012) that during a regular cell cycle, the two newly replicated sister chromatids stay in close contact for 10 to 20 min before segregating to separate cell halves thanks to the action of Topoisomerase IV. This step is called sister chromatid cohesion. We have used this molecular biology tool to study sister chromatid cohesion upon a genotoxic stress induced by mitomycin C (MMC). We have shown that sister chromatid cohesion is maintained and prolonged when the cell is facing a DSB. Moreover, this sister chromatid cohesion is dependent on RecN, an SOS induced structural maintenance of chromosome-like (SMC-like) protein. In the absence of RecN, the proximity between both sister chromatids is lost and this has a deleterious effect on cell viability. By tagging the chromosome with fluorescent proteins, we have revealed that RecN can also mediated a progressive regression of two previously segregated sister chromatids and this is coordinated with a whole nucleoid compaction. Further studies showed that this genome compaction is orderly and is not the result of a random compaction in response to DNA damage.Interestingly, inhibiting TopoIV in a recN mutant fully restores viability and sister chromatid cohesion suggesting that RecN’s action is mainly structural. Preserving cohesion through precatenanes is sufficient to favor repair and cell viability even in the absence of RecN.An RNA-seq experiment in a WT strain and a recN mutant revealed that the whole SOS response is downregulated in a recN mutant. This suggests that RecN may have an effect on the induction of the SOS response and thus RecA filament formation. This is in good agreement with the change in RecA-mcherry foci formation we observed. In the WT strain, the RecA-mcherry foci are defined as described in previous work. However, in the recN, the RecA-mcherry foci seemed to form bundle like structures. These RecA bundles were previsously described by Lesterlin et al. in the particular case of a DSB occurring on a chromatid that has already been segregated from its homolog. This could mean that in the absence of recN, the sister chromatids segregate and RecA forms bundle like structures in order to perform a search for the intact homologous sister chromatid.Altogether, these results reveal that RecN is an essential protein for sister chromatid cohesion upon a genotoxic stress. RecN favors sister chromatid cohesion by preventing their segregation. Through a whole nucleoid rearrangement, RecN mediates sister chromatid regression, favoring DNA repair and cell viability.La réplication fidèle de l’ADN au cours du cycle cellulaire est essentielle au maintien de l’intégrité du génome à travers les générations. Toutefois, de nombreux éléments peuvent perturber et compromettre la réplication et donc cette intégrité. La mitomycine C (MMC) est une molécule génotoxique utilisée en chimiothérapie. Elle forme des liaisons covalentes entre les deux brins d’ADN, ce qui est un obstacle à la bonne réplication de l’ADN. La rencontre de la fourche de réplication avec une liaison covalente entre les deux brins d’ADN va aboutir à une cassure double brin. Escherichia coli (E.coli) est un modèle d’étude très étendu car facile d’utilisation, permettant d’aborder des notions complexes. E coli possède divers mécanismes pour réparer ces lésions dont le régulon SOS. Le régulon SOS est un ensemble de gènes sous contrôle d’un promoteur réprimé par la protéine LexA. En réponse à des dommages à l’ADN, LexA est dégradé et les gènes du régulon sont activés.En utilisant une technique de biologie moléculaire qui permet de quantifier l’interaction entre deux chromatides sœurs restées cohésives derrière la fourche de réplication (étape appelée cohésion des chromatides sœurs), nous avons montré qu’en réponse à des cassures double brin générées par la MMC, la cohésion entre les chromatides sœurs nouvellement répliquées est maintenue. Ce phénomène est dépendant de RecN, une protéine induite de façon précoce dans le régulon SOS. RecN est une protéine de type SMC (structural maintenance of chromosomes), un groupe de protéines impliqué dans la dynamique et la structure du chromosome. En parallèle, des techniques de microscopie confocale et de marquage du chromosome par des protéines fluorescentes ont permis de montrer que la protéine RecN est impliquée dans une condensation globale du nucléoide suite à un traitement par la MMC. Cette condensation du nucléoide s’accompagne d’un rapprochement des chromatides sœurs ségrégées. Ces deux phénomènes, médiés par RecN pourraient permettre une stabilisation globale des nucléoides et favoriser l’appariement des chromatides sœurs pour permettre la recombinaison homologue.De façon intéressante, l’inhibition de Topoisomérases de type II (Topoisomerase IV et Gyrase) permettent de restaurer le phénotype d’un mutant recN en viabilité et en cohésion des chromatides sœurs. Les Topoisomérases sont des protéines qui prennent en charge les liens topologiques générés par la réplication et la transcription). Les liens topologiques non éliminés par les Topoisomerases permettraient de garder les chromatides sœurs cohésives et favoriser la réparation, même en l’absence de RecN.De plus, une expérience de RNA seq (séquençage de tout le transcriptome de la bactérie) a révélé que dans un mutant recN, le régulon SOS est moins induit que dans les cellules sauvages. Ceci va de pair avec une déstructuration des foci de réparation RecA. Il est possible que le rapprochement des chromatides sœurs médié par RecN permettrait de stabiliser le filament RecA et donc l’induction du SOS.L’ensemble de ces résultats suggère que RecN, une protéine de type SMC, permet de maintenir la cohésion entre les chromatides sœurs nouvellement répliquées, favorisant la réparation de cassures double brins par recombinaison homologue

Base excision repair accessory factors in senescence avoidance and resistance to treatments

Cancer cells, in which the RAS and PI3K pathways are activated, produce high levels of reactive oxygen species (ROS), which cause oxidative DNA damage and ultimately cellular senescence. This process has been documented in tissue culture, mouse models, and human pre-cancerous lesions. In this context, cellular senescence functions as a tumour suppressor mechanism. Some rare cancer cells, however, manage to adapt to avoid senescence and continue to proliferate. One well-documented mode of adaptation involves increased production of antioxidants often associated with inactivation of the KEAP1 tumour suppressor gene and the resulting upregulation of the NRF2 transcription factor. In this review, we detail an alternative mode of adaptation to oxidative DNA damage induced by ROS: the increased activity of the base excision repair (BER) pathway, achieved through the enhanced expression of BER enzymes and DNA repair accessory factors. These proteins, exemplified here by the CUT domain proteins CUX1, CUX2, and SATB1, stimulate the activity of BER enzymes. The ensued accelerated repair of oxidative DNA damage enables cancer cells to avoid senescence despite high ROS levels. As a by-product of this adaptation, these cancer cells exhibit increased resistance to genotoxic treatments including ionizing radiation, temozolomide, and cisplatin. Moreover, considering the intrinsic error rate associated with DNA repair and translesion synthesis, the elevated number of oxidative DNA lesions caused by high ROS leads to the accumulation of mutations in the cancer cell population, thereby contributing to tumour heterogeneity and eventually to the acquisition of resistance, a major obstacle to clinical treatment

CUT Domain Proteins in DNA Repair and Cancer

Recent studies revealed that CUT domains function as accessory factors that accelerate DNA repair by stimulating the enzymatic activities of the base excision repair enzymes OGG1, APE1, and DNA pol β. Strikingly, the role of CUT domain proteins in DNA repair is exploited by cancer cells to facilitate their survival. Cancer cells in which the RAS pathway is activated produce an excess of reactive oxygen species (ROS) which, if not counterbalanced by increased production of antioxidants, causes sustained oxidative DNA damage and, ultimately, cell senescence. These cancer cells can adapt by increasing their capacity to repair oxidative DNA damage in part through elevated expression of CUT domain proteins such as CUX1, CUX2, or SATB1. In particular, CUX1 overexpression was shown to cooperate with RAS in the formation of mammary and lung tumors in mice. Conversely, knockdown of CUX1, CUX2, or SATB1 was found to be synthetic lethal in cancer cells exhibiting high ROS levels as a consequence of activating mutations in KRAS, HRAS, BRAF, or EGFR. Importantly, as a byproduct of their adaptation, cancer cells that overexpress CUT domain proteins exhibit increased resistance to genotoxic treatments such as ionizing radiation, temozolomide, and cisplatin

Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome

International audienceCatenation links between sister chromatids are formed progressively during DNA replica-tion and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chro-matin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle

The SMC-like RecN protein is at the crossroads of several genotoxic stress responses in Escherichia coli

International audienceIntroduction DNA damage repair (DDR) is an essential process for living organisms and contributes to genome maintenance and evolution. DDR involves different pathways including Homologous recombination (HR), Nucleotide Excision Repair (NER) and Base excision repair (BER) for example. The activity of each pathway is revealed with particular drug inducing lesions, but the repair of most DNA lesions depends on concomitant or subsequent action of the multiple pathways. Methods In the present study, we used two genotoxic antibiotics, mitomycin C (MMC) and Bleomycin (BLM), to decipher the interplays between these different pathways in E. coli . We combined genomic methods (TIS and Hi-SC2) and imaging assays with genetic dissections. Results We demonstrate that only a small set of DDR proteins are common to the repair of the lesions induced by these two drugs. Among them, RecN, an SMC-like protein, plays an important role by controlling sister chromatids dynamics and genome morphology at different steps of the repair processes. We further demonstrate that RecN influence on sister chromatids dynamics is not equivalent during the processing of the lesions induced by the two drugs. We observed that RecN activity and stability requires a pre-processing of the MMC-induced lesions by the NER but not for BLM-induced lesions. Discussion Those results show that RecN plays a major role in rescuing toxic intermediates generated by the BER pathway in addition to its well-known importance to the repair of double strand breaks by HR

Determinants of Topo IV activity at <i>dif</i>.

<p>A) Southern blot analysis of the Topo IV cleavage at the <i>dif</i> site. Genomic DNA extracted from WT, Δ<i>dif</i>::<i>Tc</i>, Δ<i>dif</i>, Δ<i>xerD</i> box, Δ<i>xerC</i> box, Δ<i>xerD</i>, and Δ<i>xerC</i> strains was digested by <i>Pst</i>I; the size of the fragment generated by Topo IV cleavage at <i>dif</i> is marked by an arrow. The average percentage of cleavage observed in two independent experiments is presented. B) Southern blot analysis of the Topo IV cleavage at the <i>dif</i> site. Genomic DNA extracted from WT, Δ<i>xerC</i>, Δ<i>xerC pUCxerC</i>, Δ<i>xerC pUCxerCK172A</i>, Δ<i>xerC pUCxerCK172Q</i> strains was digested with <i>Pst</i>I; the size of the cleaved fragment in <i>dif</i> is marked by an arrow. C) Topo IV cleavage at the 1.9Mb site in the WT, Δ<i>xerC and</i> Δ<i>dif</i>. D) Plating of <i>parEts</i>, <i>parEts xerC</i>, <i>parEts xerD and parEts recN</i> mutants at 30 and 37°C. E) Colony Forming Unit (CFU) analysis of the WT and <i>nalR</i> strains deleted for the <i>dif</i> site, the <i>xerC</i>, <i>xerD</i> genes or the C-terminal domain of FtsK in the presence of ciprofloxacin. F) EMSA on a 250 bp CY3 probe containing <i>dif</i> (green) and a 250 bp CY5 control probe (red). The amount of Topo IV, XerC or XerD proteins present in each line is indicated above the gel. G) Quantification of Topo IV EMSA presented in C, data are an average of three experiments. H) Southern blot analysis of Topo IV cleavage at <i>dif</i> and position 1.92Mb in a strain overexpressing the C-terminal domain of ParC.</p

Role of the <i>dif</i> site for the management of circular chromosomes.

<p>A) Southern blot analysis of Topo IV cleavage at the <i>dif</i> and 2.56 Mb sites in the <i>mukB</i> mutant grown in minimal medium at 22°C. B) Southern blot analysis of Topo IV cleavage at the <i>dif</i> and 2.56 Mb sites in the <i>seqA</i> mutant grown in minimal medium at 37°C. C) Southern blot analysis of Topo IV cleavage at the <i>dif</i> and 1.9 Mb sites in the <i>matP</i> mutant grown in LB at 37°C. D) Colony Forming Unit (CFU) analysis of the WT and <i>nalR</i> strains deleted for the <i>dif</i> site, the <i>xerC</i> and <i>matP</i> genes in the presence of ciprofloxacin. E) Colony Forming Unit (CFU) analysis of the WT, <i>parEts</i> and <i>gyrBts</i> strains deleted for the <i>matP</i> at a semi permissive temperature (38°C). F) Southern blot analysis of the Topo IV cleavage at the <i>dif</i> and 1.9Mb sites in cells with a circular or linearized chromosome. G) Phenotypes observed during exponential growth in LB in the <i>matP</i> mutant strains with circular or linear chromosome (DNA is labeled with DAPI, green). Scale bar is 5μm.</p

Topo IV binding pattern of replicating chromosome.

<p>A) Circos plot of the ChIP-seq experiments for ParC-flag and ParE-flag. The IP / input ratio over the entire <i>E</i>. <i>coli</i> genome is presented for three independent experiments, one IP on the <i>parC-flag</i> strain and two IPs on the <i>parE-flag</i> strain. From the center to the outside, circles represent: genomic coordinates, macrodomain map, position of tRNA genes and ribosomal operons, ParE-Flag 1 ChIP-seq (untreated data, orange), ParE-Flag 1 ChIP-seq (filtered data, red), ParE-Flag 2 ChIP-seq (untreated data, orange), ParE-Flag 2 ChIP-seq (filtered data, red), ParC-Flag ChIP-seq (untreated data, orange), ParC-Flag ChIP-seq (filtered data, red), position of the 19 validated Topo IV binding sites. The right panels represent magnifications for four specific Topo IV binding sites, position 1.25 Mb, position 1.58 Mb (<i>dif</i>), position 1.85 Mb and position 2.56Mb. The three first rows correspond to filtered IP/Input ratio for ParC-Flag, ParE-Flag1 and ParE-Flag2 IPs, the fourth and fifth rows correspond respectively to the forward and reverse raw read numbers of the <i>parC-flag</i> experiment. The position and orientation of genes are illustrated at the bottom of each panel. B) Sliding averages of the IP (blue, left Y axis), Input (red, left Y axis) and IP/input (green, right Y axis) data for the <i>parC-flag</i> experiment over 60 kb regions along the genome. To facilitate the reading, <i>oriC</i> is positioned at 0 and 4.639 Mb. C) Analysis of Topo IV binding during the bacterial cell cycle. Marker frequency analysis was used to demonstrate the synchrony of the population at each time point. Stars represent the position of the selected Topo IV sites. D) IP/input ratio for 7 regions presenting specific Topo IV enrichment during S and G2 phases. For each genomic position the maximum scale is set to the maximum IP/Input ratio observed.</p

Topo IV cleavage at the Topo IV binding sites.

<p>A) Norfloxacin mediated DNA cleavage revealed by Southern blot with a radiolabeled probe near the <i>dif</i> site, the 1.25 Mb, 1.85 Mb, 2.56 Mb and the 3.24 Mb site. The size of the expected fragment generated by Topo IV cleavage is marked by an arrow. Topo IV cleavage can be differentiated from gyrase cleavages because of their presence in a <i>nalR</i> strain. B) Genome browser image of a 15kb region representative of Topo IV cleavage frequency (purple). These cleavage sites are not correlated with Topo IV enrichment in the ChIP-seq experiments described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.g001" target="_blank">Fig 1</a> (red). C) Circos plot of the NorflIP experiments. From the center to the outside, circles represent: genomic coordinates, macrodomain map, position of tRNA genes and ribosomal operons, ParC-Flag 1 NorflIP (untreated data, orange), ParC-Flag 1 NorflIP (filtered data, purple), ParE-Flag NorflIP(filtered data, purple), ParC-Flag 2 NorflIP (filtered data, purple), validated TopoIV sites present in the ParC-Flag 1, ParE-Flag and ParC-Flag 2 experiments. For visualization purpose, the maximum scale of NorflIP data has been fixed to an IP/input ratio of 10. D) Peak calling procedure, dedicated to DNA cleavage mediated by TopoIV in the presence of norfloxacin (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.s006" target="_blank">S6 Fig</a>), revealed 571 sites in total, in three experiments. Venn diagrams of common Topo IV cleavage sites in two experiments. About 200 common sites are observed in each pair of experiments. E) Genome browser zooms on the <i>dif</i>, 1.85, 1.92 and 2.56 Mb regions for Topo IV cleavage (purple) and Topo IV binding revealed by ChIP-seq (red). F) DNA cleavage mediated by TopoIV in the presence of norfloxacin revealed by Southern blot with a radiolabeled probe at 0.02, 1.92 Mb and the 3.2 Mb sites. G) Cleavage experiments performed on synchronized cultures, revealed a replication dependency (AS asynchronous, NR not replicating, S20 20 min after the initiation of replication (IR), S40 40 min after IR, S60 60 min after IR. H) Distribution of the ParC-Flag 1 NorflIP validated sites on the genome by 50 kb bins.</p

Targeting Topo IV cleavage sites along the <i>E</i>. <i>coli</i> genome.

<p>A) Circos plot of the ParC-Flag Chipseq and ParC-Flag 1 NorflIP experiments. From the center to the outside, circles represent: genomic coordinates, macrodomain map, Fis binding sites in mid exponential phase, % of bases bound by Fis per 20 kb windows of genomic DNA, H-NS binding sites in mid exponential phase, % of bases bound by H-NS per 20 kb windows of genomic DNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.ref033" target="_blank">33</a>], ParC-Flag ChIP-seq (depleted regions blue, IP/input <1), ParC-Flag ChIP-seq (enriched regions red, IP/input >1), ParC-Flag 1 NorflIP (depleted regions blue, IP/input <1), ParC-Flag 1 NorflIP (enriched regions red, IP/input >1), gene expression data (RNA-seq results performed in the ChIP-seq and NorflIP conditions). For visualization purpose, the maximum scale of RNAseq data has been fixed to 500 reads which approximately corresponds to the 400 transcription units that were the most expressed (the distribution of read counts scaled from 0 to 30 000). B) Correlation between the localization of Topo IV cleavages and chromatin markers. The NUST [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.ref032" target="_blank">32</a>] hypergeometric test was used to compare Topo IV and chromatin markers localization. The set of 172 validated Topo IV cleavage sites was used. The number of common localizations over the total number of chromatin marker localization is indicated. The P value of a Fisher’s exact test is indicated. C) Genome browser magnifications of the panel A’s pink and yellows regions. Mid log phase Fis and H-NS binding sites are respectively indicated with burgundy and black boxes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.ref033" target="_blank">33</a>]. D) Magnification of the 2.56 Mb Topo IV binding and cleavage site that overlaps a Fis binding site. The position of the deleted Topo IV site is marked by vertical lines (<i>frt</i>). Southern blot analysis of Topo IV cleavage at the 2.56 Mb locus, in the <i>nalR strain</i>, the <i>nalR</i> strain with a deletion of the Topo IV cleavage and binding site and the deletion of <i>fis</i>. E) Same as D for the 1.92 Mb Topo IV cleavage site. Southern blot analysis of Topo IV cleavage at the 1.92 Mb locus, in the WT, the <i>nalR strain</i>, the <i>nalR</i> strain with a deletion of the Topo IV cleavage and Fis binding site and the <i>nalR</i> strain with <i>fis</i> deletion. The cleavage was also analyzed following a 20 min treatment with rifampicin (rif).</p