Functional Link Between Mitochondria and Rnr3, the Minor Catalytic Subunit of Yeast Ribonucleotide Reductase

International audienceRibonucleotide reductase (RNR) is an essential holoenzyme required for de novo synthesis of dNTPs. The Saccharomyces cerevisiae genome encodes for two catalytic subunits, Rnr1 and Rnr3. While Rnr1 is required for DNA replication and DNA damage repair, the function(s) of Rnr3 is unknown. Here, we show that carbon source, an essential nutrient, impacts Rnr1 and Rnr3 abundance: Non-fermentable carbon sources or limiting concentrations of glucose down regulate Rnr1 and induce Rnr3 expression. Oppositely, abundant glucose induces Rnr1 expression and down regulates Rnr3. The carbon source dependent regulation of Rnr3 is mediated by Mec1, the budding yeast ATM/ATR checkpoint response kinase. Unexpectedly, this regulation is independent of all currently known components of the Mec1 DNA damage response network, including Rad53, Dun1, and Tel1, implicating a novel Mec1 signalling axis. rnr3Δ leads to growth defects under respiratory conditions and rescues temperature sensitivity conferred by the absence of Tom6, a component of the mitochondrial TOM (translocase of outer membrane) complex responsible for mitochondrial protein import. Together, these results unveil involvement of Rnr3 in mitochondrial functions and Mec1 in mediating the carbon source dependent regulation of Rnr3

Replication Fork Reactivation in a dnaC2 Mutant at Non-Permissive Temperature in Escherichia coli

Replicative helicases unwind double-stranded DNA in front of the polymerase and ensure the processivity of DNA synthesis. In Escherichia coli, the helicase loader DnaC as well as factors involved in the formation of the open complex during the initiation of replication and primosomal proteins during the reactivation of arrested replication forks are required to recruit and deposit the replicative helicase onto single-stranded DNA prior to the formation of the replisome. dnaC2 is a thermosensitive allele of the gene specifying the helicase loader; at non-permissive temperature replication cannot initiate, but most ongoing rounds of replication continues through to completion (18% of dnaC2 cells fail to complete replication at non-permissive temperature). An assumption, which may be drawn from this observation, is that only a few replication forks are arrested under normal growth conditions. This assumption, however, is at odds with the severe and deleterious phenotypes associated with a null mutant of priA, the gene encoding a helicase implicated in the reactivation of arrested replication forks. We developed an assay that involves an abrupt inactivation of rounds of synchronized replication in a large population of cells, in order to evaluate the ability of dnaC2 cells to reactivate arrested replication forks at non-permissive temperature. We compared the rate at which arrested replication forks accumulated in dnaC2 priA+ and dnaC2 priA2 cells and observed that this rate was lower in dnaC2 priA+ cells. We conclude that while replication cannot initiate in a dnaC2 mutant at non-permissive temperature, a class of arrested replication forks (PriA-dependent and DnaC-independent) are reactivated within these cells

Caractérisation de cycC, un nouveau gène impliqué dans le programme de réplication d'Escherichia coli

Dans Escherichia coli la Dam Methyl Transferase (DamMT) est responsable du transfert d un groupement méthyle sur les adénosines situés au cœur du tétranucléotide GATC; il s agit donc d une activité post réplicative. Ainsi, après le passage de la fourche de réplication, le brin d ADN nouvellement synthétisé est non méthylé l ADN est dit hémimethylé. L ADN reste hémimethylé pendent une brève période - de l ordre de la minute - avant d être reméthylé par la DamMT. L hypothèse de l implication de la méthylation de l ADN dans le contrôle général du programme de maintenance de l ADN repose essentiellement sur cette observation, puisque l ADN hemimethyle exception faite de l origine de réplication et de la région promotrice du gène dnaA est diagnostique du passage récent de la fourche de réplication. Cette hypothèse, et le criblage phylogénomique qui en a découlé a conduit a l identification de plusieurs gènes dont les produits sont supposes être impliqués dans la maintenance de l ADN. yjaG est l un de ces gènes. Il a été renomme cycC en raison des dérèglements de la progression du cycle cellulaire associés a un mutant nul de ce gène. L étude effectuée au cours de ma thèse s attachera à expliquer l état actuel de nos connaissances sur la protéine CycC et de son implication dans le processus de réplication de l ADN. Nos résultats montrent que la protéine CycC est impliquée dans la processivité de la réplication lorsqu il y a un dommage au niveau de l ADN. CycC spécifie une activité qui conduit à freiner les fourches de réplication, afin de prévenir des avortements des réplisomes. La surexpression de CycC bloque l initiation de la réplication entre l ouverture de la molécule d ADN et le chargement de l hélicase réplicative. Nous proposons que CycC interagisse avec le complexe réplicative et ralentit les fourches de réplication. Ce ralentissement prévient de nouvelles collisions lorsque les cellules sont dans des conditions de stress-qui cause des arrêts de la réplication.In Escherichia coli the Dam Methyl Transferase (DamMT) is responsible for the transfer of a methyl group on the adenosine located in tetranucleotide GATC, so this is a post-replicative activity. Thus, after the passage of the replication fork, the newly synthesized DNA strand is unmethylated - DNA is called hemimethylated. DNA remains hemimethylated in a brief period - about a minute - before being reméthylé by DamMT. The hypothesis of the involvement of DNA methylation in the general control of the maintenance program of the DNA is essentially on this observation, since the hemimethylated DNA - except the origin of replication and the region dnaA gene promoter - is diagnostic of the recent passage of the replication fork. This assumption and phylogenomics screening has led to the identification of several genes whose protein are supposed to be involved in the maintenance of DNA. yjaG is one of these genes. It was renamed cycC, the cell cycle progression is deregulated with a null mutant of this gene. The study in my thesis will focus on explaining the current state of our knowledge of the cycC protein and its involvement in the process of DNA replication. Our results show that the CycC protein is involved in the processivity of replication when there is damage into the DNA. CycC specifies an activity that leads to slow replication forks to prevent abortions of replisomes. CycC overexpression blocks the initiation of replication between the open complex of the DNA at oriC and the loading of the replicative helicase. We propose that CycC interacts with the replicative complex and slows replication forks. This slowdown replication prevents new collisions when cells are under stress, causing replication stops.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Experimental design.

<p>A – Synchronization procedure. A dilution of an overnight culture of <i>dnaC2</i> or <i>dnaA46</i> cells was grown during 3 generations at 30°C until exponential phase (exp) and then incubated at 40°C for 90 minutes (synchro) to synchronize the cells with respect to the initiation of replication. Replication was initiated by an abrupt downshift of the temperature of the culture to 30°C. 6 minutes after the temperature downshift, cultures were reincubated at 40°C to prevent the initiation of new rounds of replication. Samples were taken 40 minutes after initiation of replication.Time in minutes is indicated in brackets B – DNA histograms of a culture of exponentially growing <i>dnaC2</i> cells at 30°C before (0′) and after being shifted to 40°C for 45 (45′) and 90 minutes (90′). Similar DNA histograms were obtained with cultures of <i>dnaA46</i> cells (data not shown). After 90 minutes of incubation at non-permissive temperature, <i>dnaC2</i> and <i>dnaA46</i> cells are synchronized with respect to the initiation of replication. The dashed line indicates the position of the peak on a DNA histogram of stationnary phase cells (i.e., cells containing one genome). C – DNA histograms of <i>dnaC2</i> cells harvested at different time points after the temperature downshift (0′, 10′, 20′, 30′ and 40′). The dashed line indicates the position of the peak on a DNA histogram of stationnary phase cells (i.e., cells containing one genome). D – DNA histograms of synchronized <i>dnaC2</i> (left) and <i>dnaA46</i> cells (right) before initiation of replication (t0) and 40 minutes after replication initiation (t40). Replication was initiated by shifting abruptly the temperature from 40° to 30°C. After 6 minutes of incubation at a permissive temperature for the initiation of replication (30°C), the cells were brought back to 40°C. 40 minutes after initiation of replication, cells with active RFcontain around two genomes.</p

Assessment of the proportion of cells with active RF in a population of synchronized cells.

<p>Contour plot of cytograms (FL3 [DNA] vs. FSC [mass], top) and DNA histograms (bottom) of <i>dnaC2</i> synchronized cells before (t0) and 40 minutes after replication initiation (t40). Cells with one genome at t0 and at t40 (a₁⁰ and a₁⁴⁰) are circled in red on cytograms. The corresponding peaks are delineated by dashed lines on DNA histogram. The total amount of cells within the samples (a_tot⁰ and a_tot⁴⁰) is circled in blue on cytograms. The formula above the cytograms were used to calculate the proportion of cells with one genome at t0 and at t40 (α₀ and α₄₀) and then the proportion of cells with active RF (ρ₄₀) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033613#s2" target="_blank">Materials and Methods</a>).</p

Strains and plamids used in this study.

<p>Strains and plamids used in this study.</p

Effect of Novobiocin on RF inactivation.

<p>A – <i>priA⁺</i> (x) and <i>priA2</i> cells (Δ) were grown to log phase in minimal Glucose medium, diluted and plated on minimal Glucose plates to which Novobiocin was added. Colonies were counted after 3 days of incubation at 30°C, normalized to the cfu calculated in absence of Novobiocin and plotted over the concentration of Novobiocin. Error bars indicate the standard deviation around the mean in three independent experiments. B – Contour plots of <i>dnaC2</i> cells before replication initiation (t0) and 40 minutes after initiation of replication (t40). Cells that initiated replication were incubated 10 minutes after initiation of replication with different concentrations of Novobiocin for 30 minutes. The final concentration of Novobiocin (in µg/ml) is indicated above the arrows. Cells containing one genome at t0 or at t40 are circled in red. C – The proportion of <i>dnaC2</i> cells with active RF under each condition tested (ρ₄₀, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033613#s2" target="_blank">Materials and Methods</a>) was plotted over the range of concentrations of Novobiocin tested.</p

RF reactivation in <i>dnaC2</i> cells at non-permissive temperature.

<p>A – Schematic description of the experimental procedure. Replication is initiated synchronously at t0. 10 minutes after replication initiation, Novobiocin is added to the cultures at an inhibiting concentration (dark grey) before being brought back to a permissive concentration (light grey) until the end of the experiment. A control sample, not treated with Novobiocin, (−N) was also analyzed. Samples incubated with Novobiocin (N_i) are identified by the time ‘i’ (in minutes) of incubation at a concentration of Novobiocin of 10 µg/ml. B – Contour plots of <i>dnaA⁺ dnaC2</i> cells before (t0) and after initiation of replication (−N, N₀, N₁, N₂, N₅ and N₁₀) in <i>priA⁺</i> (top) and <i>priA2</i> cells (bottom). The fraction of cells with active RF (ρ₄₀) under each condition tested was normalized to that of cells that were not incubated with Novobiocin (ρ₄₀⁰), and the logarithm value of these ratios was plotted over the time of incubation with Novobiocin at 10 µg/ml (plot on the right). Circles (<i>priA⁺</i>) and triangles (<i>priA2</i>) identify the average value for a given time point, and error bars correspond to the standard deviation around the mean in three independent experiments. C –Same as B except that the cells analyzed are <i>dnaA46 dnaC⁺</i> and that error bars for <i>priA⁺</i> cells correspond to the standard deviation around the mean in two independent experiments.</p

Recovery of Vibrio cholerae polarized cellular organization after exit from a non-proliferating spheroplast state.

Vibrio cholerae, the causative agent of cholera epidemics, is a rod-shaped bacterium with a highly polarized cellular organization. It can survive harmful growth conditions by entering a non-proliferating spheroplast state, which involves loss of the cell envelope and polarity. How polarized rod organization cells are formed when the spheroplasts exit the non-proliferating state remains largely uncharacterized. To address this question, we investigated how L-arabinose-induced V. cholerae spheroplasts return to growth. We found that de novo morphogenesis started with the elimination of an excess of periplasm, which was immediately followed by cell elongation and the formation of cell branches with a diameter similar to that of normal V. cholerae cells. Periplasm elimination was driven by bifunctional peptidoglycan synthases involved in cell-wall maintenance, the aPBPs. Elongation and branching relied on the MreB-associated monofunctional peptidoglycan synthase PBP2. The cell division monofunctional peptidoglycan synthase FtsI was not involved in any of these processes. However, the FtsK cell division protein specifically targeted the sites of vesicle extrusion. Genetic material was amplified by synchronous waves of DNA replication as periplasmic elimination began. The HubP polarity factor targeted the tip of the branches as they began to form. However, HubP-mediated polarization was not involved in the efficiency of the recovery process. Finally, our results suggest that the positioning of HubP and the activities of the replication terminus organizer of the two V. cholerae chromosomes, MatP, are independent of cell division. Taken together, these results confirm the interest of L-arabinose-induced V. cholerae spheroplasts to study how cell shape is generated and shed light on the de novo establishment of the intracellular organization and cell polarization in V. cholerae

Vibrio cholerae Chromosome Partitioning without Polar Anchoring by HubP

International audiencePartition systems are widespread among bacterial chromosomes. They are composed of two effectors, ParA and ParB, and cis acting sites, parS, located close to the replication origin of the chromosome (oriC). ParABS participate in chromosome segregation, at least in part because they serve to properly position sister copies of oriC. A fourth element, located at cell poles, is also involved in some cases, such as HubP for the ParABS1 system of Vibrio cholerae chromosome 1 (ch1). The polar anchoring of oriC of ch1 (oriC1) is lost when HubP or ParABS1 are inactivated. Here, we report that in the absence of HubP, ParABS1 actively maintains oriC1 at mid-cell, leading to the subcellular separation of the two ch1 replication arms. We further show that parS1 sites ectopically inserted in chromosome 2 (ch2) stabilize the inheritance of this replicon in the absence of its endogenous partition system, even without HubP. We also observe the positioning interference between oriC1 and oriC of ch2 regions when their positionings are both driven by ParABS1. Altogether, these data indicate that ParABS1 remains functional in the absence of HubP, which raises questions about the role of the polar anchoring of oriC1 in the cell cycle