The Fanconi anemia protein FANCM can promote branch migration of Holliday junctions and replication forks.

Fanconi anemia (FA) is a genetically heterogeneous cancer-prone disorder associated with chromosomal instability and cellular hypersensitivity to DNA crosslinking agents. The FA pathway is suspected to play a crucial role in the cellular response to DNA replication stress. At a molecular level, however, the function of most of the FA proteins is unknown. FANCM displays DNA-dependent ATPase activity and promotes the dissociation of DNA triplexes, but the physiological significance of this activity remains elusive. Here we show that purified FANCM binds to Holliday junctions and replication forks with high specificity and promotes migration of their junction point in an ATPase-dependent manner. Furthermore, we provide evidence that FANCM can dissociate large recombination intermediates, via branch migration of Holliday junctions through 2.6 kb of DNA. Our data suggest a direct role for FANCM in DNA processing, consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks.

Functional analysis of a conserved DNA methyltransferase in caulobacter crescentus

CcrM is a DNA methyltransferase that methylates the adenine in GANTC motifs in the chromo-some of the bacterial model Caulobacter crescentus. The loss of the CcrM homolog is lethal in C. crescentus and in several other species of Alphaproteobacteria. In this research, we used different experimental and bioinformatic approaches to determine why CcrM is so critical to the physiology of C. crescentus. We first showed that CcrM is a resident orphan DNA methyltransferase in non-Rickettsiales Alphaproteobacteria and that its gene is strictly conserved in this clade (with only one ex¬ception among the genomes sequenced so far). In C. crescentus, cells depleted in CcrM in rich medium quickly lose viability and present an elongated phenotype characteristic of an im¬pairment in cell division. Using minimal medium instead of rich medium as selective and main¬tenance substrate, we could generate a AccrM mutant that presents a viability comparable to the wild type strain and only mild morphological defects. On the basis of a transcriptomic ap¬proach, we determined that several genes essential for cell division were downregulated in the AccrM strain in minimal medium. We offered decisive arguments to support that the efficient transcription of two of these genes, ftsZ and mipZ, coding respectively for the Z-ring forming GTPase FtsZ and an inhibitor of FtsZ polymerization needed for the correct positioning of the Z- ring at mid-cell, requires the methylation of an adenine in a conserved GANTC motif located in their core promoter region. We propose a model, according to which the genome of C. crescentus encodes a transcriptional activator that requires a methylated adenine in a GANTC context to bind to DNA and suggest that this transcriptional regulator might be the global cell-cycle regulator GcrA. In addition, combining a classic genetic approach and in vitro evolution experiments, we showed that the mortality and cell division defects of the AccrM strain in rich medium are mainly due to limiting intracellular levels of the FtsZ protein. We also studied the dynamics of GANTC methylation in C. crescentus using the SMRT technol¬ogy developed by Pacific Biosciences. Our findings support the commonly accepted model, accord¬ing to which the methylation state of GANTC motifs varies during the cell cycle of C. crescentus: before the initiation of DNA replication, the GANTC motifs are fully-methylated (methylated on both strands); when the DNA gets replicated, the GANTC motifs become hemi-methylated (methyl¬ated on one strand only) and this occurs at different times during replication for different loci along the chromosome depending on their position relative to the origin of replication; the GANTC mo¬tifs are only remethylated after DNA replication has finished as a consequence of the massive and short-lived expression of CcrM in predivisional cells. About 30 GANTC motifs in the C. crescentus chromosome were found to be undermethylated in most of the bacterial population; these might be protected from CcrM activity by DNA binding proteins and some of them could be involved in methylation-based bistable transcriptional switches. - CcrM est une ADN méthyltransférase qui méthyle les adénines dans le contexte GANTC dans le génome de la bactérie modèle Caulobacter crescentus. La perte de l'homologue de CcrM chez C. crescentus et chez plusieurs autres espèces d'Alphaproteobactéries est létale. Dans le courant de cette recherche, nous tentons de déterminer pourquoi la protéine CcrM est cruciale pour la survie de C. crescentus. Nous démontrons d'abord que CcrM est une adénine méthyltransférase orpheline résidente, dont le gène fait partie du génome minimal partagé par les Alphaprotéobactéries non-Rickettsiales (à une exception près). Lorsqu'une souche de C. crescentus est privée de CcrM, sa viabilité décroît rapi¬dement et ses cellules présentent une morphologie allongée qui suggère que la division cellulaire est inhibée. Nous sommes parvenus à créer une souche AccrM en utilisant un milieu minimum, au lieu du milieu riche classiquement employé, comme milieu de sélection et de maintenance pour la souche. Lorsque nous avons étudié le transcriptome de cette souche de C. crescentus privée de CcrM, nous avons pu constater que plusieurs gènes essentiels pour le bon déroulement de la division cellulaire bactérienne étaient réprimés. En particulier, l'expression adéquate des gènes ftsZ et mipZ - qui codent, respectivement, pour FtsZ, la protéine qui constitue, au milieu de la cellule, un anneau protéique qui initie le processus de division et pour MipZ, un inhibiteur de la polymérisation de FtsZ qui est indispensable pour le bon positionnement de l'anneau FtsZ - est dépendante de la présence d'une adénine méthylée dans un motif GANTC conservé situé dans leur région promotrice. Nous présentons un modèle selon lequel le génome de C. crescentus code pour un facteur de transcription qui exige la présence d'une adénine méthylée dans un contexte GANTC pour s'attacher à l'ADN et nous suggérons qu'il pourrait s'agir du régulateur global du cycle cellulaire GcrA. En outre, nous montrons, en combinant la génétique classique et une approche basée sur l'évolution expérimentale, que la mortalité et l'inhibition de la division cellulaire caractéristiques de la souche àccrMeη milieu riche sont dues à des niveaux excessivement bas de protéine FtsZ. Nous avons aussi étudié la dynamique de la méthylation du chromosome de C. crescentus sur la base de la technologie SMRT développée par Pacific Biosciences. Nous confirmons le modèle communément accepté, qui affirme que l'état de méthylation des motifs GANTC change durant le cycle cellulaire de C. crescentus: les motifs GANTC sont complètement méthylés (méthylés sur les deux brins) avant de début de la réplication de l'ADN; ils deviennent hémi-méthylés (méthylés sur un brin seulement) une fois répliqués, ce qui arrive à différents moments durant la réplication pour différents sites le long du chromosome en fonction de leur position par rapport à l'origine de répli-cation; finalement, les motifs GANTC sont reméthylés après la fin de la réplication du chromosome lorsque la protéine CcrM est massivement, mais très transitoirement, produite. Par ailleurs, nous identifions dans le chromosome de C. crescentus environ 30 motifs GANTC qui restent en perma-nence non-méthylés dans une grande partie de la population bactérienne; ces motifs sont probable-ment protégés de l'action de CcrM par des protéines qui s'attachent à l'ADN et certains d'entre eux pourraient être impliqués dans des mécanismes de régulation générant une transcription bistable.

FANCM regulates DNA chain elongation and is stabilized by S-phase checkpoint signalling.

FANCM binds and remodels replication fork structures in vitro. We report that in vivo, FANCM controls DNA chain elongation in an ATPase-dependent manner. In the presence of replication inhibitors that do not damage DNA, FANCM counteracts fork movement, possibly by remodelling fork structures. Conversely, through damaged DNA, FANCM promotes replication and recovers stalled forks. Hence, the impact of FANCM on fork progression depends on the underlying hindrance. We further report that signalling through the checkpoint effector kinase Chk1 prevents FANCM from degradation by the proteasome after exposure to DNA damage. FANCM also acts in a feedback loop to stabilize Chk1. We propose that FANCM is a ringmaster in the response to replication stress by physically altering replication fork structures and by providing a tight link to S-phase checkpoint signalling.

Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA.

DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5'-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments.

Supercoiling, knotting and replication fork reversal in partially replicated plasmids.

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.

The biogeography of mitochondrial and nuclear discordance in animals.

Combining nuclear (nuDNA) and mitochondrial DNA (mtDNA) markers has improved the power of molecular data to test phylogenetic and phylogeographic hypotheses and has highlighted the limitations of studies using only mtDNA markers. In fact, in the past decade, many conflicting geographic patterns between mitochondrial and nuclear genetic markers have been identified (i.e. mito-nuclear discordance). Our goals in this synthesis are to: (i) review known cases of mito-nuclear discordance in animal systems, (ii) to summarize the biogeographic patterns in each instance and (iii) to identify common drivers of discordance in various groups. In total, we identified 126 cases in animal systems with strong evidence of discordance between the biogeographic patterns obtained from mitochondrial DNA and those observed in the nuclear genome. In most cases, these patterns are attributed to adaptive introgression of mtDNA, demographic disparities and sex-biased asymmetries, with some studies also implicating hybrid zone movement, human introductions and Wolbachia infection in insects. We also discuss situations where divergent mtDNA clades seem to have arisen in the absence of geographic isolation. For those cases where foreign mtDNA haplotypes are found deep within the range of a second taxon, data suggest that those mtDNA haplotypes are more likely to be at a high frequency and are commonly driven by sex-biased asymmetries and/or adaptive introgression. In addition, we discuss the problems with inferring the processes causing discordance from biogeographic patterns that are common in many studies. In many cases, authors presented more than one explanation for discordant patterns in a given system, which indicates that likely more data are required. Ideally, to resolve this issue, we see important future work shifting focus from documenting the prevalence of mito-nuclear discordance towards testing hypotheses regarding the drivers of discordance. Indeed, there is great potential for certain cases of mitochondrial introgression to become important natural systems within which to test the effect of different mitochondrial genotypes on whole-animal phenotypes.

Functional analysis of human POT1 and RPA : insights into the role of single stranded DNA binding proteins at telomeres

Abstract Telomeres, the natural ends of chromosomes, need to be protected from chromosome end fusions, aberrant homologous recombination and degradation. In humans, chromosome ends are specified through arrays of tandemly repeated 5'-TTAGGG-3' hexamers, ending in a 3' overhang. A complex formed by the six proteins TRF1, TRF2, hRap1, TIN2, TPP1 and POT1 specifically assocìates with and protects telomeres. Telomeres are maintained by semiconservative DNA replication and by a specialized reverse transcriptase, telomerase, that carries an RNA subunit which templates new telomeric repeat synthesis. The telomeric single stranded (ss) DNA binding protein POT1 protects the telomeric 3' overhang and modulates telomerase-mediated telomere elongation. It is possible that POT1 also influences DNA synthesis during semiconservative DNA replication, which is initiated by the DNA polymerase alpha-primase complex. The heterotrimeric ss DNA-binding protein RPA plays essential roles during DNA replication. RPA binds to ss DNA with high affinity in order to stabilize ss DNA and facilitate nascent strand synthesis at the replication fork. Here we investigate how the two proteins RPA and POT1 contribute to telomere maintenance by regulating semi-conservative DNA replication and telomerase. Using chromatin immunoprecipitation experiments, we show that RPA associates with telomeres during S-phase. Analysis of telomere structure in cells shRNA-depleted for RPA and POT1 reveals that loss of RPA and POT1 causes exposure of single-stranded DNA at telomeres, suggestive of incomplete DNA replication. Biochemical experiments using purified recombinant POT1 and RPA show that saturating telomeric oligonucleotides with POT1 or RPA reduces the primase activity of the DNA polymerase alpha-primase complex and the overall activity of telomerase. POT1 and RPA also increase the primer extension by DNA polymerase alpha-primase complex and the processivity of telomerase under certain conditions, although POT1 increases the activities to a greater extent than RPA. We propose that POT1 is required for proper replication of the lagging strand of telomeres and that some phenotypes observed in POT1-depleted cells may stern from incomplete DNA replication rather than de-protection of the single-stranded overhang. Résumé Les télomères, les extrémités normales des chromosomes linéaires, doivent être protégés des fusions chromosomiques, d'événements de recombinaison homologue aberrants et de phénomènes de dégradation. Chez l'Homme, les extrémités des chromosomes sont constitués d'ADN double brin répétitif de séquence 5'-TTAGGG-3', d'une extension simple brin 3' sortante et d'un complexe protéique formé des six facteurs TRF1, TRF2, hRap1, TIN2, TPP1 et POT1 qui, s'associant à cette séquence, protègent l'ADN télomèrique. Les télomères sont maintenus par la télomérase, une transcriptase inverse capable d'allonger l'extension 3' sortante télomérique. POT1 lie l'ADN simple brin télomérique et module l'élongation des télomères par la télomérase. POT1 pourrait en théorie également influencer la réplication semi-conservative de l'ADN. L'ADN-polymérase Pal alpha-primase amorce et initie la synthèse d'ADN. Pendant la réplication, l'ADN simple brin est stabilisé par RPA, un complexe hétérotrimèrique qui lie l'ADN simple brin. RPA facilite la synthèse du brin naissant à la fourche de réplication. Ici nous avons étudié comment ces deux protéines qui lient l'ADN simple brin, RPA et POT1, régulent la réplication des télomères par la télomérase et la machinerie classique de réplication de l'ADN. Par immunoprécipitation de chromatine (ChIP), nous montrons que RPA est localisé aux télomères lors de la phase S du cycle cellulaire. De plus, l'analyse de la structure des télomeres indique que !a perte de RPA ou de POT1 conduit à l'apparition d'ADN simple brin télomérique, suggérant une réplication incomplète de l'ADN télomérique in vivo. Par une approche complémentaire biochimique utilisant les protéines POT1 et RPA recombinantes purifiées, nous montrons également que la liaison de POT1 ou de RPA à des oligonucléotides télomériques bloque l'activité primase du complexe polymérase alpha/primase et réduit l'activité télomérase sur ces substrats. En revanche, leur liaison augmente l'activité ADN-polymérase du complexe polymérase alpha/primase, ainsi que fa processivité de la télomérase dans certaines conditions, POT1 étant le plus efficace des deux facteurs. Nous proposons que POT1 est nécessaire à la réplication du brin retardé au niveau des télomères, ce qui suggère que certains phénotypes des cellules déplétés en POT1 puissent résulter d'une réplication incomplète de l'ADN télémétrique plutôt que d'une déprotection de l'extrémité sortante des télomères.

The SOS screen in Arabidopsis: a search for functions involved in DNA metabolism.

The SOS screen, as originally described by Perkins et al. (1999) [7], was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS(+) candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS(+) candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS(+) candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.

Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice.

Mitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata. Cells with mitochondrial DNA deficiency (so-called "petite mutants") upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabrata mutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrata isolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1 (CgCDR1), CgCDR2, and CgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitro growth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitro growth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivo under azole therapy, even if strongly affecting in vitro growth characteristics, can confer a selective advantage under host conditions, allowing the C. glabrata mutant to be more virulent than wild-type isolates.

Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter.

Chromosome replication in Caulobacter crescentus is tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. The timing of replication initiation is controlled by both CtrA and DnaA. CtrA binds to and silences the origin. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. Here, we identify an additional layer of replication initiation control that is mediated by the HdaA protein. In Escherichia coli, the Hda protein inactivates DnaA after replication initiation. We show that the Caulobacter HdaA homologue is necessary to restrict the initiation of DNA replication to only once per cell cycle and that it dynamically colocalizes with the replisome throughout the cell cycle. Moreover, the transcription of hdaA is directly activated by DnaA, providing a robust feedback regulatory mechanism that adjusts the levels of HdaA to inactivate DnaA.

Interplay of DNA supercoiling and catenation during the segregation of sister duplexes.

The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo.

Spatial organization of nucleotide excision repair proteins after UV-induced DNA damage in the human cell nucleus.

Nucleotide excision repair (NER) is an evolutionary conserved DNA repair system that is essential for the removal of UV-induced DNA damage. In this study we investigated how NER is compartmentalized in the interphase nucleus of human cells at the ultrastructural level by using electron microscopy in combination with immunogold labeling. We analyzed the role of two nuclear compartments: condensed chromatin domains and the perichromatin region. The latter contains transcriptionally active and partly decondensed chromatin at the surface of condensed chromatin domains. We studied the distribution of the damage-recognition protein XPC and of XPA, which is a central component of the chromatin-associated NER complex. Both XPC and XPA rapidly accumulate in the perichromatin region after UV irradiation, whereas only XPC is also moderately enriched in condensed chromatin domains. These observations suggest that DNA damage is detected by XPC throughout condensed chromatin domains, whereas DNA-repair complexes seem preferentially assembled in the perichromatin region. We propose that UV-damaged DNA inside condensed chromatin domains is relocated to the perichromatin region, similar to what has been shown for DNA replication. In support of this, we provide evidence that UV-damaged chromatin domains undergo expansion, which might facilitate the translocation process. Our results offer novel insight into the dynamic spatial organization of DNA repair in the human cell nucleus.

Formation of knots in partially replicated DNA molecules.

Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo. The knots formed are localised within the newly replicated DNA regions. Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings. We propose that helical winding of replication bubbles in vivo leads to topoisomerase-mediated formation of knots on partially replicated DNA molecules.

Cross-species analysis of the replication complex of Old World arenaviruses reveals two nucleoprotein sites involved in L protein function.

Lassa virus (LASV) causing hemorrhagic Lassa fever in West Africa, Mopeia virus (MOPV) from East Africa, and lymphocytic choriomeningitis virus (LCMV) are the main representatives of the Old World arenaviruses. Little is known about how the components of the arenavirus replication machinery, i.e., the genome, nucleoprotein (NP), and L protein, interact. In addition, it is unknown whether these components can function across species boundaries. We established minireplicon systems for MOPV and LCMV in analogy to the existing LASV system and exchanged the components among the three systems. The functional and physical integrity of the resulting complexes was tested by reporter gene assay, Northern blotting, and coimmunoprecipitation studies. The minigenomes, NPs, and L proteins of LASV and MOPV could be exchanged without loss of function. LASV and MOPV L protein was also active in conjunction with LCMV NP, while the LCMV L protein required homologous NP for activity. Analysis of LASV/LCMV NP chimeras identified a single LCMV-specific NP residue (Ile-53) and the C terminus of NP (residues 340 to 558) as being essential for LCMV L protein function. The defect of LASV and MOPV NP in supporting transcriptional activity of LCMV L protein was not caused by a defect in physical NP-L protein interaction. In conclusion, components of the replication complex of Old World arenaviruses have the potential to functionally and physically interact across species boundaries. Residue 53 and the C-terminal domain of NP are important for function of L protein during genome replication and transcription.

Regulation of chromosomal replication in Caulobacter crescentus.

The alpha-proteobacterium Caulobacter crescentus is characterized by its asymmetric cell division, which gives rise to a replicating stalked cell and a non-replicating swarmer cell. Thus, the initiation of chromosomal replication is tightly regulated, temporally and spatially, to ensure that it is coordinated with cell differentiation and cell cycle progression. Waves of DnaA and CtrA activities control when and where the initiation of DNA replication will take place in C. crescentus cells. The conserved DnaA protein initiates chromosomal replication by directly binding to sites within the chromosomal origin (Cori), ensuring that DNA replication starts once and only once per cell cycle. The CtrA response regulator represses the initiation of DNA replication in swarmer cells and in the swarmer compartment of pre-divisional cells, probably by competing with DnaA for binding to Cori. CtrA and DnaA are controlled by multiple redundant regulatory pathways that include DNA methylation-dependent transcriptional regulation, temporally regulated proteolysis and the targeting of regulators to specific locations within the cell. Besides being critical regulators of chromosomal replication, CtrA and DnaA are also master transcriptional regulators that control the expression of many genes, thus connecting DNA replication with other events of the C. crescentus cell cycle.