Termination Of Replication Research Articles

Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells.

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.

Dinoroseobacter shibae Outer Membrane Vesicles Are Enriched for the Chromosome Dimer Resolution Site dif.

Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions.IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.

Post-Translational Modifications of PCNA in Control of DNA Synthesis and DNA Damage Tolerance-the Implications in Carcinogenesis.

The faithful DNA replication is a critical event for cell survival and inheritance. However, exogenous or endogenous sources of damage challenge the accurate synthesis of DNA, which causes DNA lesions. The DNA lesions are obstacles for replication fork progression. However, the prolonged replication fork stalling leads to replication fork collapse, which may cause DNA double-strand breaks (DSB). In order to maintain genomic stability, eukaryotic cells evolve translesion synthesis (TLS) and template switching (TS) to resolve the replication stalling. Proliferating cell nuclear antigen (PCNA) trimer acts as a slide clamp and encircles DNA to orchestrate DNA synthesis and DNA damage tolerance (DDT). The post-translational modifications (PTMs) of PCNA regulate these functions to ensure the appropriate initiation and termination of replication and DDT. The aberrant regulation of PCNA PTMs will result in DSB, which causes mutagenesis and poor response to chemotherapy. Here, we review the roles of the PCNA PTMs in DNA duplication and DDT. We propose that clarifying the regulation of PCNA PTMs may provide insights into understanding the development of cancers.

The Neutralizing Antibody Response against Severe Acute Respiratory Syndrome Coronavirus 2 and the Cytokine/Chemokine Release in Patients with Different Levels of Coronavirus Diseases 2019 Severity: Cytokine Storm Still Persists Despite Viral Disappearance in Critical Patients.

Patients with coronavirus disease 2019 (COVID-19) exhibit a wide clinical spectrum ranging from mild respiratory symptoms to critical and fatal diseases, and older individuals are known to be more severely affected. The underlying mechanism of this phenomenon is unknown. A neutralizing antibody against viruses is known to be important to eliminate the virus. In addition, this antibody is induced at high levels in patients with severe COVID-19, followed by a termination of virus replication. Severe COVID-19 patients exhibit high levels of cytokines/chemokines, even after the disappearance of the virus. This indicates that cytokines/chemokines play significant roles in disease severity. These findings also suggest that antiviral therapy (monoclonal antibody and/or convalescent plasma therapy) should be administered early to eliminate the virus, followed by steroid treatment after viral genome disappearance, especially in patients with severe symptoms.

Ritonavir may inhibit exoribonuclease activity of nsp14 from the SARS-CoV-2 virus and potentiate the activity of chain terminating drugs

SARS-CoV-2is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. The nsp14 protein of SARS-CoV-2 houses a 3′ to 5′ exoribonuclease activity responsible for removing mismatches that arise during genome duplication. A homology model of nsp10-nsp14 complex was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp14. This exercise showed that ritonavir might bind to the exoribonuclease active site of the nsp14 protein. A model of the SARS-CoV-2-nsp10-nsp14 complex bound to substrate RNA showed that the ritonavir binding site overlaps with that of the 3′ nucleotide of substrate RNA. A comparison of the calculated energies of binding for RNA and ritonavir suggested that the drug may bind to the active site of nsp14 with significant affinity. It is, therefore, possible that ritonavir may prevent association with substrate RNA and thus inhibit the exoribonuclease activity of nsp14. Overall, our computational studies suggest that ritonavir may serve as an effective inhibitor of the nsp14 protein. nsp14 is known to attenuate the inhibitory effect of drugs that function through premature termination of viral genome replication. Hence, ritonavir may potentiate the therapeutic properties of drugs such as remdesivir, favipiravir and ribavirin.

Long inverted repeats around the chromosome replication terminus in the model strain Bacillus thuringiensis serovar israelensis BGSC 4Q7

Bacillus thuringiensis serovar israelensis is the most widely used natural biopesticide against mosquito larvae worldwide. Its lineage has been actively studied and a plasmid-free strain, B . thuringiensis serovar israelensis BGSC 4Q7 (4Q7), has been produced. Previous sequencing of the genome of this strain has revealed the persistent presence of a 235 kb extrachromosomal element, pBtic235, which has been shown to be an inducible prophage, although three putative chromosomal prophages have been lost. Moreover, a 492 kb region, potentially including the standard replication terminus, has also been deleted in the 4Q7 strain, indicating an absence of essential genes in this area. We reanalysed the genome coverage distribution of reads for the previously sequenced variant strain, and sequenced two independently maintained samples of the 4Q7 strain. A 553 kb area, close to the 492 kb deletion, was found to be duplicated. This duplication presumably restored the equal sizes of the replichores, and a balanced functioning of replication termination. An analysis of genome assembly graphs revealed a transient association of the host chromosome with the pBtic235 element. This association may play a functional role in the replication of the bacterial chromosome, and the termination of this process in particular. The genome-restructuring events detected may modify the genetic status of cytotoxic or haemolytic toxins, potentially influencing strain virulence. Twelve of the single-nucleotide variants identified in 4Q7 were probably due to the procedure used for strain construction or were present in the precursor of this strain. No sequence variants were found in pBtic235, but the distribution of the corresponding 4Q7 reads indicates a significant difference from counterparts in natural B. thuringiensis serovar israelensis strains, suggesting a duplication or over-replication in 4Q7. Thus, the 4Q7 strain is not a pure plasmid-less offshoot, but a highly genetically modified derivative of its natural ancestor. In addition to potentially influencing virulence, genome-restructuring events can modify the replication termination machinery. These findings have potential implications for the conclusions of virulence studies on 4Q7 as a model, but they also raise interesting fundamental questions about the functioning of the Bacillus genome.

Loss of the Nuclear Protein RTF2 Enhances Influenza Virus Replication.

While hundreds of genes are induced by type I interferons, their roles in restricting the influenza virus life cycle remain mostly unknown. Using a loss-of-function CRISPR screen in cells prestimulated with interferon beta (IFN-β), we identified a small number of factors required for restricting influenza A virus replication. In addition to known components of the interferon signaling pathway, we found that replication termination factor 2 (RTF2) restricts influenza virus at the nuclear stage (and perhaps other stages) of the viral life cycle, based on several lines of evidence. First, a deficiency in RTF2 leads to higher levels of viral primary transcription, even in the presence of cycloheximide to block genome replication and secondary transcription. Second, cells that lack RTF2 have enhanced activity of a viral reporter that depends solely on four viral proteins that carry out replication and transcription in the nucleus. Third, when the RTF2 protein is mislocalized outside the nucleus, it is not able to restrict replication. Finally, the absence of RTF2 leads not only to enhanced viral transcription but also to reduced expression of antiviral factors in response to interferon. RTF2 thus inhibits primary influenza virus transcription, likely acts in the nucleus, and contributes to the upregulation of antiviral effectors in response to type I interferons.IMPORTANCE Viral infection triggers the secretion of type I interferons, which in turn induce the expression of hundreds of antiviral genes. However, the roles of these induced genes in controlling viral infections remain largely unknown, limiting our ability to develop host-based antiviral therapeutics against pathogenic viruses, such as influenza virus. Here, we performed a loss-of-function genetic CRISPR screen in cells prestimulated with type I interferon to identify antiviral genes that restrict influenza A virus replication. Besides finding key components of the interferon signaling pathway, we discovered a new restriction factor, RTF2, which acts in the nucleus, restricts influenza virus transcription, and contributes to the interferon-induced upregulation of known restriction factors. Our work contributes to the field of antiviral immunology by discovering and characterizing a novel restriction factor of influenza virus and may ultimately be useful for understanding how to control a virus that causes significant morbidity and mortality worldwide.

Physiology of Highly Radioresistant Escherichia coli After Experimental Evolution for 100 Cycles of Selection.

Ionizing radiation (IR) is lethal to most organisms at high doses, damaging every cellular macromolecule via induction of reactive oxygen species (ROS). Utilizing experimental evolution and continuing previous work, we have generated the most IR-resistant Escherichia coli populations developed to date. After 100 cycles of selection, the dose required to kill 99% the four replicate populations (IR9-100, IR10-100, IR11-100, and IR12-100) has increased from 750 Gy to approximately 3,000 Gy. Fitness trade-offs, specialization, and clonal interference are evident. Long-lived competing sub-populations are present in three of the four lineages. In IR9, one lineage accumulates the heme precursor, porphyrin, leading to generation of yellow-brown colonies. Major genomic alterations are present. IR9 and IR10 exhibit major deletions and/or duplications proximal to the chromosome replication terminus. Contributions to IR resistance have expanded beyond the alterations in DNA repair systems documented previously. Variants of proteins involved in ATP synthesis (AtpA), iron-sulfur cluster biogenesis (SufD) and cadaverine synthesis (CadA) each contribute to IR resistance in IR9-100. Major genomic and physiological changes are emerging. An isolate from IR10 exhibits protein protection from ROS similar to the extremely radiation resistant bacterium Deinococcus radiodurans, without evident changes in cellular metal homeostasis. Selection is continuing with no limit to IR resistance in evidence as our E. coli populations approach levels of IR resistance typical of D. radiodurans.

The DNA replication fork suppresses CMG unloading from chromatin before termination.

When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2Lrr1 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in Xenopus egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading.

The Role of Ubiquitination and SUMOylation in DNA Replication.

DNA replication is a tightly regulated conserved process that ensures the faithful transmission of genetic material to define heritable phenotypic traits. Perturbations in this process result in genomic instability, mutagenesis, and diseases, including malignancy. Proteins involved in the initiation, progression, and termination of DNA replication are subject to a plethora of reversible post-translational modifications (PTMs) to provide a proper temporal and spatial control of replication. Among these, modifications involving the covalent attachment of the small protein ubiquitin or the small ubiquitin-like modifier (SUMO) to replication and replication-associated proteins are particularly important for the proper regulation of DNA replication as well as for optimal cellular responses to replication stress. In this article, we describe how the ubiquitination and SUMOylation processes impact DNA replication in eukaryotes and highlight the consequences of deregulated signals emanating from these two versatile regulatory pathways on cellular activities.

Direct observation of independently moving replisomes in Escherichia coli

The replication and transfer of genomic material from a cell to its progeny are vital processes in all living systems. Here we visualize the process of chromosome replication in widened E. coli cells. Monitoring the replication of single chromosomes yields clear examples of replication bubbles that reveal that the two replisomes move independently from the origin to the terminus of replication along each of the two arms of the circular chromosome, providing direct support for the so-called train-track model, and against a factory model for replisomes. The origin of replication duplicates near midcell, initially splitting to random directions and subsequently towards the poles. The probability of successful segregation of chromosomes significantly decreases with increasing cell width, indicating that chromosome confinement by the cell boundary is an important driver of DNA segregation. Our findings resolve long standing questions in bacterial chromosome organization.

Light-powered Escherichia coli cell division for chemical production

Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 μm−1 and acetoin titer to 67.2 g·L−1. Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 μm3 and poly(lactate-co-3-hydroxybutyrate) titer to 14.31 g·L−1. Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories.

Novel Divisome-Associated Protein Spatially Coupling the Z-Ring with the Chromosomal Replication Terminus in Caulobacter crescentus.

Cell division requires proper spatial coordination with the chromosome, which undergoes dynamic changes during chromosome replication and segregation. FtsZ is a bacterial cytoskeletal protein that assembles into the Z-ring, providing a platform to build the cell division apparatus. In the model bacterium Caulobacter crescentus, the cellular localization of the Z-ring is controlled during the cell cycle in a chromosome replication-coupled manner. Although dynamic localization of the Z-ring at midcell is driven primarily by the replication origin-associated FtsZ inhibitor MipZ, the mechanism ensuring accurate positioning of the Z-ring remains unclear. In this study, we showed that the Z-ring colocalizes with the replication terminus region, located opposite the origin, throughout most of the C. crescentus cell cycle. Spatial organization of the two is mediated by ZapT, a previously uncharacterized protein that interacts with the terminus region and associates with ZapA and ZauP, both of which are part of the incipient division apparatus. While the Z-ring and the terminus region coincided with the presence of ZapT, colocalization of the two was perturbed in cells lacking zapT, which is accompanied by delayed midcellular positioning of the Z-ring. Moreover, cells overexpressing ZapT showed compromised positioning of the Z-ring and MipZ. These findings underscore the important role of ZapT in controlling cell division processes. We propose that ZapT acts as a molecular bridge that physically links the terminus region to the Z-ring, thereby ensuring accurate site selection for the Z-ring. Because ZapT is conserved in proteobacteria, these findings may define a general mechanism coordinating cell division with chromosome organization.IMPORTANCE Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. In enteric bacteria, ZapA associates with the cytoskeletal Z-ring and establishes a physical linkage to the chromosomal replication terminus through its interaction with ZapB-MatP-DNA complexes. However, because ZapB and MatP are found only in enteric bacteria, it remains unclear how the Z-ring and the terminus are coordinated in the vast majority of bacteria. Here, we provide evidence that a novel conserved protein, termed ZapT, mediates colocalization of the Z-ring with the terminus in Caulobacter crescentus, a model organism that is phylogenetically distant from enteric bacteria. Given that ZapT facilitates cell division processes in C. crescentus, this study highlights the universal importance of the physical linkage between the Z-ring and the terminus in maintaining cell integrity.

High-resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells

BackgroundDNA replication in mammalian cells occurs in a defined temporal order during S phase, known as the replication timing (RT) programme. Replication timing is developmentally regulated and correlated with chromatin conformation and local transcriptional potential. Here, we present RT profiles of unprecedented temporal resolution in two human embryonic stem cell lines, human colon carcinoma line HCT116, and mouse embryonic stem cells and their neural progenitor derivatives.ResultsFine temporal windows revealed a remarkable degree of cell-to-cell conservation in RT, particularly at the very beginning and ends of S phase, and identified 5 temporal patterns of replication in all cell types, consistent with varying degrees of initiation efficiency. Zones of replication initiation (IZs) were detected throughout S phase and interacted in 3D space preferentially with other IZs of similar firing time. Temporal transition regions were resolved into segments of uni-directional replication punctuated at specific sites by small, inefficient IZs. Sites of convergent replication were divided into sites of termination or large constant timing regions consisting of many synchronous IZs in tandem. Developmental transitions in RT occured mainly by activating or inactivating individual IZs or occasionally by altering IZ firing time, demonstrating that IZs, rather than individual origins, are the units of developmental regulation. Finally, haplotype phasing revealed numerous regions of allele-specific and allele-independent asynchronous replication. Allele-independent asynchronous replication was correlated with the presence of previously mapped common fragile sites.ConclusionsAltogether, these data provide a detailed temporal choreography of DNA replication in mammalian cells.

Two chromatographic schemes for protein purification involving the biotin/avidin interaction under native conditions

The strength of the biotin/avidin interaction makes it an ideal tool for the purification of biotin-labeled proteins via avidin-coupled resin with high specificity and selectivity. Nevertheless, this tight binding comes at an extra cost of performing the elution step under denaturing conditions. Weakening the biotin/avidin interaction improves the elution conditions, but only to mild or harsh denaturing buffers with the drawback of reducing the specificity and selectivity of this interaction. Here, we present two chromatographic protein purification schemes that are well-suited for application under native conditions thus preserving the strength of the biotin/avidin interaction. In the first scheme, we introduce a biotin-labeled SUMO-tag to each of human flap endonuclease 1 and Escherichia coli replication termination protein Tus, and elute both proteins by performing on-resin cleavage using SUMO protease. In the second scheme, we immobilize biotin-labeled human proliferating cell nuclear antigen (PCNA) on the avidin-coupled resin and use the resulting resin as a tag-free affinity method to purify the PCNA-binding protein human DNA Ligase 1. Furthermore, we streamlined the protein biotinylation protocol by constructing a single plasmid expression system that ensures high level of expression and solubility for each of the target protein bearing the biotin-tag and the enzyme responsible for the in vivo biotinylation reaction. Both chromatographic schemes resulted in a high yield of pure proteins in their native form.

Heteromorphism of Persistence of Sapronosis Causative Agents in Cells in Various Environmental Conditions

The paper discusses the issues of morphofunctional variability of sapronoses pathogens in stressful environment. In the current century, sapronoses infections attract increasing attention. Under unfavorable environmental conditions the pathogens use the strategy for the formation of resting (stable) states, that is: viable but non-culturable cell formes and persistent bacteria, which are characterized by reduced metabolism and changes in the morphology and physiology of the microorganisms, termination of replication. Possibility of sapronoses pathogens survival in interepidemic period and antibiotic resistance formation, which play an important role in chronic infections, are associated with the formation of persistent forms of bacteria. The literature extensively discusses mechanisms and conditions of the pathogenic bacteria stable states formation and their pathogenetic contribution to infectious pathology, whereas ultrastructural organization and morphological variability of persistent cell forms, as well as their differentiation, causing the pathogens population heterogeneity, is still insufficiently illuminated. Based on the analysis of current data and their own experience, the authors evaluate the morphological and functional changes of bacteria stable cellular forms and their role in sapronoses pathogens adaptation strategies.

Heteromorphism of Persistence of Sapronosis Causative Agents in Cells in Various Environmental Conditions

The paper discusses the issues of morphofunctional variability of sapronoses pathogens in stressful environment. In the current century, sapronoses infections attract increasing attention. Under unfavorable environmental conditions the pathogens use the strategy for the formation of resting (stable) states, that is: viable but non-culturable cell formes and persistent bacteria, which are characterized by reduced metabolism and changes in the morphology and physiology of the microorganisms, termination of replication. Possibility of sapronoses pathogens survival in interepidemic period and antibiotic resistance formation, which play an important role in chronic infections, are associated with the formation of persistent forms of bacteria. The literature extensively discusses mechanisms and conditions of the pathogenic bacteria stable states formation and their pathogenetic contribution to infectious pathology, whereas ultrastructural organization and morphological variability of persistent cell forms, as well as their differentiation, causing the pathogens population heterogeneity, is still insufficiently illuminated. Based on the analysis of current data and their own experience, the authors evaluate the morphological and functional changes of bacteria stable cellular forms and their role in sapronoses pathogens adaptation strategies.

Organization of the Escherichia coli Chromosome by a MukBEF Axial Core

SummaryStructural maintenance of chromosomes (SMC) complexes organize chromosomes ubiquitously, thereby contributing to their faithful segregation. We demonstrate that under conditions of increased chromosome occupancy of the Escherichia coli SMC complex, MukBEF, the chromosome is organized as a series of loops around a thin (<130 nm) MukBEF axial core, whose length is ∼1,100 times shorter than the chromosomal DNA. The linear order of chromosomal loci is maintained in the axial cores, whose formation requires MukBEF ATP hydrolysis. Axial core structure in non-replicating chromosomes is predominantly linear (1 μm) but becomes circular (1.5 μm) in the absence of MatP because of its failure to displace MukBEF from the 800 kbp replication termination region (ter). Displacement of MukBEF from ter by MatP in wild-type cells directs MukBEF colocalization with the replication origin. We conclude that MukBEF individualizes and compacts the chromosome lengthwise, demonstrating a chromosome organization mechanism similar to condensin in mitotic chromosome formation.

Chromosomal origin of replication coordinates logically distinct types of bacterial genetic regulation

For a long time it has been hypothesized that bacterial gene regulation involves an intricate interplay of the transcriptional regulatory network (TRN) and the spatial organization of genes in the chromosome. Here we explore this hypothesis both on a structural and on a functional level. On the structural level, we study the TRN as a spatially embedded network. On the functional level, we analyze gene expression patterns from a network perspective (“digital control”), as well as from the perspective of the spatial organization of the chromosome (“analog control”). Our structural analysis reveals the outstanding relevance of the symmetry axis defined by the origin (Ori) and terminus (Ter) of replication for the network embedding and, thus, suggests the co-evolution of two regulatory infrastructures, namely the transcriptional regulatory network and the spatial arrangement of genes on the chromosome, to optimize the cross-talk between two fundamental biological processes: genomic expression and replication. This observation is confirmed by the functional analysis based on the differential gene expression patterns of more than 4000 pairs of microarray and RNA-Seq datasets for E. coli from the Colombos Database using complex network and machine learning methods. This large-scale analysis supports the notion that two logically distinct types of genetic control are cooperating to regulate gene expression in a complementary manner. Moreover, we find that the position of the gene relative to the Ori is a feature of very high predictive value for gene expression, indicating that the Ori–Ter symmetry axis coordinates the action of distinct genetic control mechanisms.

Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus

ABSTRACTMembers of the conserved Argonaute (Ago) protein family provide defence against invading nucleic acids in eukaryotes in the process of RNA interference. Many prokaryotes also contain Ago proteins that are predicted to be active nucleases; however, their functional activities in host cells remain poorly understood. Here, we characterize the in vitro and in vivo properties of the SeAgo protein from the mesophilic cyanobacterium Synechococcus elongatus. We show that SeAgo is a DNA-guided nuclease preferentially acting on single-stranded DNA targets, with non-specific guide-independent activity observed for double-stranded substrates. The SeAgo gene is steadily expressed in S. elongatus; however, its deletion or overexpression does not change the kinetics of cell growth. When purified from its host cells or from heterologous E. coli, SeAgo is loaded with small guide DNAs whose formation depends on the endonuclease activity of the argonaute protein. SeAgo co-purifies with SSB proteins suggesting that they may also be involved in DNA processing. The SeAgo-associated small DNAs are derived from diverse genomic locations, with certain enrichment for the proposed sites of chromosomal replication initiation and termination, but show no preference for an endogenous plasmid. Therefore, promiscuous genome sampling by SeAgo does not have great effects on cell physiology and plasmid maintenance.