Initiator Protein Research Articles

AfsK-Mediated Site-Specific Phosphorylation Regulates DnaA Initiator Protein Activity in Streptomyces coelicolor.

In all organisms, chromosome replication is regulated mainly at the initiation step. Most of the knowledge about the mechanisms that regulate replication initiation in bacteria has come from studies on rod-shaped bacteria, such as Escherichia coli and Bacillus subtilisStreptomyces is a bacterial genus that is characterized by distinctive features and a complex life cycle that shares some properties with the developmental cycle of filamentous fungi. The unusual lifestyle of streptomycetes suggests that these bacteria use various mechanisms to control key cellular processes. Here, we provide the first insights into the phosphorylation of the bacterial replication initiator protein, DnaA, from Streptomyces coelicolor We suggest that phosphorylation of DnaA triggers a conformational change that increases its ATPase activity and decreases its affinity for the replication origin, thereby blocking the formation of a functional orisome. We suggest that the phosphorylation of DnaA is catalyzed by Ser/Thr kinase AfsK, which was shown to regulate the polar growth of S. coelicolor Together, our results reveal that phosphorylation of the DnaA initiator protein functions as a negative regulatory mechanism to control the initiation of chromosome replication in a manner that presumably depends on the cellular localization of the protein.IMPORTANCE This work provides insights into the phosphorylation of the DnaA initiator protein in Streptomyces coelicolor and suggests a novel bacterial regulatory mechanism for initiation of chromosome replication. Although phosphorylation of DnaA has been reported earlier, its biological role was unknown. This work shows that upon phosphorylation, the cooperative binding of the replication origin by DnaA may be disturbed. We found that AfsK kinase is responsible for phosphorylation of DnaA. Upon upregulation of AfsK, chromosome replication occurred further from the hyphal tip. Orthologs of AfsK are exclusively found in mycelial actinomycetes that are related to Streptomyces and exhibit a complex life cycle. We propose that the AfsK-mediated regulatory pathway serves as a nonessential, energy-saving mechanism in S. coelicolor.

Dynamics of DNA nicking and unwinding by the RepC-PcrA complex.

The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s−1, while a typical velocity of 50 bp s−1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.

Specific binding of DnaA to the DnaA box motif in the cyanobacterium Synechococcus elongatus PCC 7942.

In bacterial DNA replication, the initiator protein DnaA binds to the multiple DnaA box sequences located at oriC to facilitate the unwinding of duplex DNA strands. The cyanobacterium Synechococcus elongatus PCC 7942, which contains multiple chromosomal copies per cell, has DnaA box (like sequences around the oriC region, which is located upstream of dnaN. We previously observed the binding of DnaA around the oriC region; however, the DNA-binding specificity of DnaA to DnaA box sequences has not been examined. Here, we analyzed the binding specificity of DnaA protein to the DnaA box in S. elongatus by using bio-layer interferometry (BLI), a method for monitoring intermolecular interactions. We observed that recombinant DnaA protein recognized specifically the DnaA box sequence TTTTCCACA in vitro. In addition, DNA binding activity was significantly increased by R328H mutation of DnaA. This is the first report to characterize DnaA binding to the DnaA box sequence in cyanobacteria.

Characterization of the diverse plasmid pool harbored by the blaNDM-1-containing Acinetobacter bereziniae HPC229 clinical strain.

Acinetobacter bereziniae is an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting. A. bereziniae plasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two different habitats. Here we characterized the whole plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOBP111 relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOBQ1 relaxases and also carrying TA systems, were found. The three latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein genes, and functional analysis of their transfer regions revealed the mobilizable nature of them. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against deposited Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.

Helicobacter pylori helicase loader protein Hp0897 shows unique functions of N- and C-terminal regions.

Helicase loaders are required for the loading of helicases at the vicinity of replication origins. In Helicobacter pylori, Hp0897 has been shown to be a potential helicase loader for replicative helicase (HpDnaB) although it does not show any sequence homology with conventional DnaC like helicase loader proteins. Therefore, it is important to investigate the in vivo role of Hp0897 and structure-function analysis with respect to domain mapping of Hp0897 and HpDnaB. Although HporiC is divided into oriC1 and oriC2, the latter has been assigned as functional origin based on loading of initiator protein HpDnaA. Using chromatin immunoprecipitation (ChIP) experiment, we show preferential binding of Hp0897 at oriC2 over oriC1 like HpDnaA highlighting its helicase loader function in vivo. Furthermore, we generated series of deletion mutants for HpDnaB and Hp0897 that enabled us to map the domains of interaction between these two proteins. Interestingly, the C-terminal domain of Hp0897 (Hp0897CTD) shows stronger interaction with HpDnaB over the N-terminal region of Hp0897 (Hp0897NTD). Similar to the full-length protein, Hp0897CTD also stimulates the DNA binding activity of HpDnaB. Furthermore, overexpression of Hp0897 full-length protein in H. pylori leads to an elongated cell phenotype. While the overexpression of Hp0897CTD does not show a phenotype of cell elongation, overexpression of Hp0897NTD shows extensive cell elongation. These results highlight the possible role of Hp0897CTD in helicase loading and Hp0897NTD's unique function linked to cell division that make Hp0897 as a potential drug target against H. pylori.

Denovo designing: a novel signal peptide for tat translocation pathway to transport activin A to the periplasmic space of E. coli.

The twin-arginine translocation (Tat) pathway is one of the bacterial secretory strategies which exports folded proteins across the cytoplasmic membrane. In the present study, we designed a novel Tat-signal peptide for secretion of human activin A used as a recombinant protein model here. In doing so, Haloferax volcanii, Halobacterium salinarum, and Escherichia coli Tat specific signal peptides were aligned by ClustalW program to determine conserved and more frequently used residues. After making the initial signal peptide sequence and doing some mutations, efficiency of this designed signal peptide was evaluated using a set of well-known software programs such as TatP, PRED-TAT, and Phobius. Then the best complex between TatC as an initiator protein in Tat secretory machine and the new designed signal peptide connected to activin A with the lowest binding energy was constructed by HADDOCK server, and ΔΔG value of - 5.5kcal/mol was calculated by FoldX module. After that, efficiency of this novel signal peptide for secretion of human activin A to the periplasmic space of E. coli Rosetta-gami (DE3) strain was experimentally evaluated; to scrutinize the activity of the novel signal peptide, Iranian Bacillus Licheniformis α-Amylase enzyme signal peptide as a Sec pathway signal peptide was used as a positive control. The quantitative analysis of western blotting bands by ImageJ software confirmed the high secretion ability of the new designed signal peptide; translocation of 69% of the produced recombinant activin A to the periplasmic space of E. coli. Circular Dichroism (CD) spectroscopy technique also approved the proper secondary structure of activin A secreted to the periplasmic space. The biological activity of activin A was also confirmed by differentiation of K562 erythroleukemia cells to the red blood cell by measuring the amount of hemoglobin or Fe2+ ion using ICP method. In conclusion, this novel designed signal peptide can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP–PNP–DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP–PNP–DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.

A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria.

One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited. Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria. The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen. We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

Streptomycete origin of chromosomal replication with two putative unwinding elements.

DNA replication is controlled mostly at the initiation step. In bacteria, replication of the chromosome starts at a single origin of replication called oriC. The initiator protein, DnaA, binds to specific sequences (DnaA boxes) within oriC and assembles into a filament that promotes DNA double helix opening within the DNA unwinding element (DUE). This process has been thoroughly examined in model bacteria, including Escherichia coli and Bacillus subtilis, but we have a relatively limited understanding of chromosomal replication initiation in other species. Here, we reveal new details of DNA replication initiation in Streptomyces, a group of Gram-positive soil bacteria that possesses a long linear (8-10 Mbps) and GC-rich chromosome with a centrally positioned oriC. We used comprehensive in silico, in vitro and in vivo analyses to better characterize the structure of Streptomyces oriC. We identified 14 DnaA-binding motifs and determined the consensus sequence of the DnaA box. Unexpectedly, our in silico analysis using the WebSIDD algorithm revealed the presence of two putative Streptomyces DUEs (DUE1 and DUE2) located very near one another toward the 5' end of the oriC region. In vitro P1 nuclease assay revealed that DNA unwinding occurs at both of the proposed sites, but using an in vivo replication initiation point mapping, we were able to confirm only one of them (DUE2). The previously observed transcriptional activity of the Streptomyces oriC region may help explain the current results. We speculate that transcription itself could modulate oriC activity in Streptomyces by determining whether DNA unwinding occurs at DUE1 or DUE2.

The effects of manipulating levels of replication initiation factors on origin firing efficiency in yeast.

Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome.

Prematurity blunts the feeding-induced stimulation of translation initiation signaling and protein synthesis in muscle of neonatal piglets.

Postnatal growth of lean mass is commonly blunted in preterm infants and may contribute to short- and long-term morbidities. To determine whether preterm birth alters the protein anabolic response to feeding, piglets were delivered at term or preterm, and fractional protein synthesis rates (Ks) were measured at 3 days of age while fasted or after an enteral meal. Activation of signaling pathways that regulate protein synthesis and degradation were determined. Relative body weight gain was lower in preterm than in term. Gestational age at birth (GAB) did not alter fasting plasma glucose or insulin, but when fed, plasma insulin and glucose rose more slowly, and reached peak value later, in preterm than in term. Feeding increased Ks in longissimus dorsi (LD) and gastrocnemius muscles, heart, pancreas, and kidney in both GAB groups, but the response was blunted in preterm. In diaphragm, lung, jejunum, and brain, feeding increased Ks regardless of GAB. Liver Ks was greater in preterm than term and increased with feeding regardless of GAB. In all tissues, changes in 4EBP1, S6K1, and PKB phosphorylation paralleled changes in Ks. In LD, eIF4E·eIF4G complex formation, phosphorylation of TSC2, mTOR, and rpS6, and association of mammalian target of rapamycin (mTOR1) complex with RagA, RagC, and Rheb were increased by feeding and blunted by prematurity. There were no differences among groups in LD protein degradation markers. Our results demonstrate that preterm birth reduces weight gain and the protein synthetic response to feeding in muscle, pancreas, and kidney, and this is associated with blunted insulin- and/or amino acid-induced translation initiation signaling.

Occurrence and distribution of begomoviruses infecting tomatoes, peppers and cucurbits in Thailand

Incidence of begomoviruses infecting tomatoes, peppers and cucurbits was investigated in major production fields in the north, northeastern, central and southern regions of Thailand between January 2015 and June 2017. Of 585 samples subjected to begomovirus detection by polymerase chain reaction (PCR), 57% were positive for begomovirus infection. Nucleotide sequence analysis of coat protein (CP) and replication initiation protein (Rep) genes identified 15 previously known begomovirus species, 7 of which were reported infecting these crops in Thailand for the first time. A candidate novel bipartite begomovirus designated Melon yellow mosaic virus was also identified in melons from a northeastern province of Thailand. The predominant begomovirus species were different depending on the host plant (Tomato yellow leaf curl Thailand virus [TYLCTHV] for tomato, Pepper yellow leaf curl Thailand virus [PepYLCTHV] for pepper, Tomato leaf curl New Delhi virus [ToLCNDV] for loofah, bitter gourd, cucumber and melon, and Squash leaf curl China virus [SLCCNV] for pumpkin and wax gourd) and location. The update on diversity and distribution of begomoviruses obtained from this study provides useful information for further epidemiological study and support development of effective disease management through selective use of resistant cultivars suitable for particular plants and locations. Additionally, these data can be applied for development of diagnostic assays to specifically detect begomoviruses for continuous monitoring of viral diversity in these economic crops.

Nicotiana benthamiana phosphatidylinositol 4-kinasetype II regulates chilli leaf curl virus pathogenesis.

SummaryGeminiviruses are single‐stranded DNA viruses that can cause significant losses in economically important crops. In recent years, the role of different kinases in geminivirus pathogenesis has been emphasized. Although geminiviruses use several host kinases, the role of phosphatidylinositol 4‐kinase (PI4K) remains obscure. We isolated and characterized phosphatidylinositol 4‐kinase type II from Nicotiana benthamiana (NbPI4KII) which interacts with the replication initiator protein (Rep) of a geminivirus, chilli leaf curl virus (ChiLCV). NbPI4KII‐mGFP was localized into cytoplasm, nucleus or both. NbPI4KII‐mGFP was also found to be associated with the cytoplasmic endomembrane systems in the presence of ChiLCV. Furthermore, we demonstrated that Rep protein directly interacts with NbPI4KII protein and influenced nuclear occurrence of NbPI4KII. The results obtained in the present study revealed that NbPI4KII is a functional protein kinase lacking lipid kinase activity. Downregulation of NbPI4KII expression negatively affects ChiLCV pathogenesis in N. benthamiana. In summary, NbPI4KII is a susceptible factor, which is required by ChiLCV for pathogenesis.

Changing Perspectives on the Role of DnaA-ATP in Orisome Function and Timing Regulation.

Bacteria, like all cells, must precisely duplicate their genomes before they divide. Regulation of this critical process focuses on forming a pre-replicative nucleoprotein complex, termed the orisome. Orisomes perform two essential mechanical tasks that configure the unique chromosomal replication origin, oriC to start a new round of chromosome replication: (1) unwinding origin DNA and (2) assisting with loading of the replicative DNA helicase on exposed single strands. In Escherichia coli, a necessary orisome component is the ATP-bound form of the bacterial initiator protein, DnaA. DnaA-ATP differs from DnaA-ADP in its ability to oligomerize into helical filaments, and in its ability to access a subset of low affinity recognition sites in the E. coli replication origin. The helical filaments have been proposed to play a role in both of the key mechanical tasks, but recent studies raise new questions about whether they are mandatory for orisome activity. It was recently shown that a version of E. coli oriC (oriCallADP), whose multiple low affinity DnaA recognition sites bind DnaA-ATP and DnaA-ADP similarly, was fully occupied and unwound by DnaA-ADP in vitro, and in vivo suppressed the lethality of DnaA mutants defective in ATP binding and ATP-specific oligomerization. However, despite their functional equivalency, orisomes assembled on oriCallADP were unable to trigger chromosome replication at the correct cell cycle time and displayed a hyper-initiation phenotype. Here we present a new perspective on DnaA-ATP, and suggest that in E. coli, DnaA-ATP is not required for mechanical functions, but rather is needed for site recognition and occupation, so that initiation timing is coupled to DnaA-ATP levels. We also discuss how other bacterial types may utilize DnaA-ATP and DnaA-ADP, and whether the high diversity of replication origins in the bacterial world reflects different regulatory strategies for how DnaA-ATP is used to control orisome assembly.

Development of a PCR method for detection of Pseudoalteromonas marina associated with green spot disease in Pyropia yezoensis

Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease (GSD) in Pyropia yezoensis. To prevent GSD from development and spread, an effective method to detect the pathogen at early GSD infection stages need to be established. In this research, PCR methods were established targeting the dnaA gene (encoding chromosome replication initiator protein) and the dnaN gene (encoding β sliding clamp of DNA polymerase III protein) to detect P. marina with three primer pairs pws-dnaA2 (Forward, 5′-ACCGCATTAACGAACTACTCGTG-3′; Reverse, 5′-TGCCATTACCTACAGCATGG-3′), pcs-dnaN2 (Forward, 5′-CTTACAACGTTATCAGCGGC-3′; Reverse, 5′-GTTGAGTATTAAGTGATTGAGTAAGC-3′) or pws-dnaN3 (Forward, 5′-ACTTACAACGTTATCAGCGGC-3′; Reverse, 5′-ACTGCTGTTTGAGTCTGCTAAC-3′). Three PCR methods corresponding to the three primer pairs sufficiently distinguished P. marina from 22 bacterial species, thus resulting in detection limits of 4 to 4×102 CFU cells or 2.37×101 to 2.37×103 fg of P. marina DNA per PCR reaction. In an artificial infection experiment of P. yezoensis infected with P. marina, all established PCRs successfully detected P. marina at early GSD infection stages. The results show that the established PCRs are specific and sensitive, and are potential for applications in early diagnosis of GSD in Pyropia.

Novel Aspects of Extracellular Vesicles as Mediators of Cancer-Associated Thrombosis.

The establishment of prothrombotic states during cancer progression is well reported but the precise mechanisms underlying this process remain elusive. A number of studies have implicated the presence of the clotting initiator protein, tissue factor (TF), in circulating tumor-derived extracellular vesicles (EVs) with thrombotic manifestations in certain cancer types. Tumor cells, as well as tumor-derived EVs, may activate and promote platelet aggregation by TF-dependent and independent pathways. Cancer cells and their secreted EVs may also facilitate the formation of neutrophil extracellular traps (NETs), which may contribute to thrombus development. Alternatively, the presence of polyphosphate (polyP) in tumor-derived EVs may promote thrombosis through a TF-independent route. We conclude that the contribution of EVs to cancer coagulopathy is quite complex, in which one or more mechanisms may take place in a certain cancer type. In this context, strategies that could attenuate the crosstalk between the proposed pro-hemostatic routes could potentially reduce cancer-associated thrombosis.

Pigeon circoviruses from feral pigeons in Australia demonstrate extensive recombination and genetic admixture with other circoviruses

ABSTRACTLike other avian circovirus species, Pigeon circovirus (PiCV) is known to be genetically diverse with a relatively small circular single-stranded DNA genome of 2 kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Recent paleoviral evidence hints towards a probable Gondwanan origin of avian circoviruses, paralleling the evolution and dispersal of their hosts. Limited availability of PiCV genome sequence data in Australia has hindered phylogeographic studies in this species, so we screened clinically normal rock doves (Columba livia) in regional New South Wales, and demonstrated a high prevalence (76%) of PiCV infection by PCR. We also recovered 12 complete novel PiCV genomes and phylogenetic analyses revealed that PiCV circulating in Australian feral pigeons formed two strongly supported monophyletic clades. One clade resided with PiCV genomes from Poland, Australia, United Kingdom, Belgium, China, and Japan, and another basal clade was more closely related to PiCV genomes from Poland. A novel more distantly-related PiCV rep gene formed a solitary clade with weak posterior support. So we further analysed all selected partial rep gene sequences to demonstrate a likely naturally occurring spillover infection from a passerine circovirus candidate. The findings suggest that there is a high degree of genetic variation within PiCV in Columbiformes with potential greater admixture between avian circoviruses within Australia than previously known.RESEARCH HIGHLIGHTSConfirmed high prevalence rate of PiCV circulating in Australian wild pigeons.Highlighted extensive recombination events within Australian PiCV.Demonstrated a likely naturally occurring spillover infection from a passerine circovirus candidate.

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC.

The stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation in Escherichia coli remains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factor dnaA, but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication, oriC Active transcription normally introduces negative supercoils into oriC to help promote replication initiation, so the accumulation of ppGpp reduces initiation potential at oriC by reducing transcription. We find that maintaining transcription near oriC, either by expressing a ppGpp-blind RNA polymerase mutant or by inducing transcription from a ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting topoisomerase I or by deleting the nucleoid-associated protein gene seqA also relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which ppGpp indirectly creates an unfavorable energy landscape for initiation by limiting the introduction of negative supercoils into oriCIMPORTANCE To survive bouts of starvation, cells must inhibit DNA replication. In bacteria, starvation triggers production of a signaling molecule called ppGpp (guanosine tetraphosphate) that helps reprogram cellular physiology, including inhibiting new rounds of DNA replication. While ppGpp has been known to block replication initiation in Escherichia coli for decades, the mechanism responsible was unknown. Early work suggested that ppGpp drives a decrease in levels of the replication initiator protein DnaA. However, we found that this decrease is not necessary to block replication initiation. Instead, we demonstrate that ppGpp leads to a change in DNA topology that prevents initiation. ppGpp is known to inhibit bulk transcription, which normally introduces negative supercoils into the chromosome, and negative supercoils near the origin of replication help drive its unwinding, leading to replication initiation. Thus, the accumulation of ppGpp prevents replication initiation by blocking the introduction of initiation-promoting negative supercoils. This mechanism is likely conserved throughout proteobacteria.

Minichromosome maintenance 3 promotes hepatocellular carcinoma radioresistance by activating the NF-\u03baB pathway

BackgroundHepatocellular carcinoma (HCC) is the most common tumors in the worldwide, it develops resistance to radiotherapy during treatment, understanding the regulatory mechanisms of radioresistance generation is the urgent need for HCC therapy.MethodsqRT-PCR, western blot and immunohistochemistry were used to examine MCM3 expression. MTT assay, colony formation assay, terminal deoxynucleotidyl transferase nick end labeling assay and In vivo xenograft assay were used to determine the effect of MCM3 on radioresistance. Gene set enrichment analysis, luciferase reporter assay, western blot and qRT-PCR were used to examine the effect of MCM3 on NF-κB pathway.ResultsWe found DNA replication initiation protein Minichromosome Maintenance 3 (MCM3) was upregulated in HCC tissues and cells, patients with high MCM3 expression had poor outcome, it was an independent prognostic factor for HCC. Cells with high MCM3 expression or MCM3 overexpression increased the radioresistance determined by MTT assay, colony formation assay, TUNEL assay and orthotopic transplantation mouse model, while cells with low MCM3 expression or MCM3 knockdown reduced the radioresistance. Mechanism analysis showed MCM3 activated NF-κB pathway, characterized by increasing the nuclear translocation of p65, the expression of the downstream genes NF-κB pathway and the phosphorylation of IKK-β and IκBα. Inhibition of NF-κB in MCM3 overexpressing cells using small molecular inhibitor reduced the radioresistance, suggesting MCM3 increased radioresistance through activating NF-κB pathway. Moreover, we found MCM3 expression positively correlated with NF-κB pathway in clinic.ConclusionsOur findings revealed that MCM3 promoted radioresistance through activating NF-κB pathway, strengthening the role of MCM subunits in the tumor progression and providing a new target for HCC therapy.

Transcriptional Control and mRNA Capping by the GDP Polyribonucleotidyltransferase Domain of the Rabies Virus Large Protein.

Rabies virus (RABV) is a causative agent of a fatal neurological disease in humans and animals. The large (L) protein of RABV is a multifunctional RNA-dependent RNA polymerase, which is one of the most attractive targets for developing antiviral agents. A remarkable homology of the RABV L protein to a counterpart in vesicular stomatitis virus, a well-characterized rhabdovirus, suggests that it catalyzes mRNA processing reactions, such as 5′-capping, cap methylation, and 3′-polyadenylation, in addition to RNA synthesis. Recent breakthroughs in developing in vitro RNA synthesis and capping systems with a recombinant form of the RABV L protein have led to significant progress in our understanding of the molecular mechanisms of RABV RNA biogenesis. This review summarizes functions of RABV replication proteins in transcription and replication, and highlights new insights into roles of an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase, domain of the RABV L protein in mRNA capping and transcription initiation.