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The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E.coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E.coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E.coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method enables the pathogenicity analysis and vaccine development using genetically modified viruses, and it is expected to allow for faster analysis of newly emerging variants than ever before. Furthermore, its application to other RNA viruses is also expected., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tanaka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recent studies have shown that the reconstituted cell-free DNA replisome and in vitro transcription and translation systems from Escherichia coli are highly important in applied and synthetic biology. To date, no attempt has been made to combine those two systems. Here, we study the performance of the mixed two separately exploited systems commercially available as RCR and PURE systems. Regarding the genetic information flow from DNA to proteins, mixtures with various ratios of RCR/PURE gave low protein expression, possibly due to the well-known conflict between replication and transcription or inappropriate buffer conditions. To further increase the compatibility of the two systems, rationally designed reaction buffers with a lower concentration of nucleoside triphosphates in 50 mM HEPES (pH7.6) were evaluated, showing increased performance from RCR/PURE (85%/15%) in a time-dependent manner. The compatibility was also validated in compartmentalized cell-sized droplets encapsulating the same RCR/PURE soup. Our findings can help to better fine-tune the reaction conditions of RCR-PURE systems and provide new avenues for rewiring the central dogma of molecular biology as self-sustaining systems in synthetic cell models. KEY POINTS: • Commercial reconstituted DNA amplification (RCR) and transcription and translation (PURE) systems hamper each other upon mixing. • A newly optimized buffer with a low bias for PURE was formulated in the RCR-PURE mixture. • The performance and dynamics of RCR-PURE were investigated in either bulk or compartmentalized droplets., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The physical stability of bacterial chromosomes is important for their in vitro manipulation, while genetic stability is important in vivo. However, extracted naked chromosomes in the open circular form are fragile due to nicks and gaps. Using a nick/gap repair and negative supercoiling reaction (named SCR), we first achieved the negative supercoiling of the whole genomes extracted from Escherichia coli and Vibrio natriegens cells. Supercoiled chromosomes of 0.2-4.6 megabase (Mb) were separated by size using a conventional agarose gel electrophoresis and served as DNA size markers. We also achieved the enzymatic replication of 1-2 Mb chromosomes using the reconstituted E. coli replication-cycle reaction (RCR). Electroporation-ready 1 Mb chromosomes were prepared by a modified SCR performed at a low salt concentration (L-SCR) and directly introduced into commercial electrocompetent E. coli cells. Since successful electroporation relies on the genetic stability of a chromosome in cells, genetically stable 1 Mb chromosomes were developed according to a portable chromosome format (PCF). Using physically and genetically stabilized chromosomes, the democratization of genome synthetic biology will be greatly accelerated.

DNA amplification is a fundamental technique in molecular biology. The replication cycle reaction is a new method for amplification of large circular DNA having oriC sequences, which is a replication initiation site of the Escherichia coli chromosome. We here developed a replication cycle reaction-based method useful for amplification of various circular DNAs lacking oriC , even in the absence of any sequence information, via transposon-mediated oriC insertion to the circular DNA template. A 15-kb non- oriC plasmid was amplified from a very small amount of starting DNA (50 fg, 1 fM). The method was also applicable to GC-rich plasmid (69%) or large F-plasmid (230 kb). This method thus provides a powerful tool to amplify various environmental circular DNAs.

Reconstitution of the DNA amplification system in microcompartments is the primary step toward artificial cell construction through a bottom-up approach. However, amplification of >100 kbp DNA in micrometer-sized reactors has not yet been achieved. Here, implementing a fully reconstituted replisome of Escherichia coli in micrometer-sized water-in-oil droplets, we developed the in-droplet replication cycle reaction (RCR) system. For a 16 kbp template DNA, the in-droplet RCR system yielded positive RCR signals with a high success rate (82%) for the amplification from single molecule template DNA. The success rate for a 208 kbp template DNA was evidently lower (23%). This study establishes a platform for genome-sized DNA amplification from a single copy of template DNA with the potential to build more complex artificial cell systems comprising a large number of genes.

In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Although Escherichia coli has been a popular tool for plasmid construction, this bacterium was believed to be "unsuitable" for constructing a large plasmid whose size exceeds 500 kilobases. We assumed that traditional plasmid vectors may lack some regulatory DNA elements required for the stable replication and segregation of such a large plasmid. In addition, the use of a few site-specific recombination systems may facilitate cloning of large DNA segments. Here we show two strategies for constructing 1-megabase (1-Mb) secondary chromosomes by using new bacterial artificial chromosome (BAC) vectors. First, the 3-Mb genome of a genome-reduced E. coli strain was split into two chromosomes (2-Mb and 1-Mb), of which the smaller one has the origin of replication and the partitioning locus of the Vibrio tubiashii secondary chromosome. This chromosome fission method (Flp-POP cloning) works via flippase-mediated excision, which coincides with the reassembly of a split chloramphenicol resistance gene, allowing chloramphenicol selection. Next, we developed a new cloning method ( oriT -POP cloning) and a fully equipped BAC vector (pMegaBAC1H) for developing a 1-Mb plasmid. Two 0.5-Mb genomic regions were sequentially transferred from two donor strains to a recipient strain via conjugation and captured by pMegaBAC1H in the recipient strain to produce a 1-Mb plasmid. This 1-Mb plasmid was transmissible to another E. coli strain via conjugation. Furthermore, these 1-Mb secondary chromosomes were amplifiable in vitro by using the reconstituted E. coli chromosome replication cycle reaction (RCR). These strategies and technologies would make popular E. coli cells a productive factory for designer chromosome engineering.

The highly efficient genetic transformation of cells is essential for synthetic biology procedures, especially for the transformation of large gene clusters. In this technical note, we present a novel cell-penetrating peptide (CPP)-mediated large-sized plasmid DNA transformation system for Escherichia coli. A large plasmid (pMSR227, 205 kb) was complexed with cationic peptides containing a CPP motif and was successfully transformed into E. coli cells. The transformants containing the plasmid DNA exhibited expression of a reporter gene encoding a red fluorescent protein. The transformation efficiency was significantly higher than that obtained using the heat-shock method and was similar to that of electroporation. This technique can be used as a platform for the simple and highly efficient transformation of large DNA molecules under mild conditions without causing significant damage to DNA, accelerating synthetic biology investigations for the design of genetically engineered microorganisms for industrial purposes.

Propagation of genetic information is a fundamental prerequisite for living cells. We recently developed the replication cycle reaction (RCR), an in vitro reaction for circular DNA propagation, by reconstitution of the replication cycle of the Escherichia coli chromosome. In RCR, two replication forks proceed bidirectionally from the replication origin, oriC , and meet at a region opposite oriC , yielding two copies of circular DNA. Although RCR essentially propagates supercoiled monomers, concatemer byproducts are also produced due to inefficient termination of the replication fork progression. Here, we examined the effect of the Tus -ter replication fork trap in RCR. Unexpectedly, when the fork traps were placed opposite oriC , mimicking their arrangement on the chromosome, the propagation of circular DNA was inhibited. On the other hand, fork traps flanking oriC allowed efficient propagation of circular DNA and repressed concatemer production. These findings suggest that collision of the two convergence forks through the fork trap is detrimental to repetition of the replication cycle. We further demonstrate that this detrimental effect was rescued by the UvrD helicase. These results provide insights into the way in which circular DNA monomers replicate repetitively without generating concatemers.

Mismatch repair (MMR) systems based on MutS eliminate mismatches originating from replication errors. Despite extensive conservation of mutS homologues throughout the three domains of life, Actinobacteria and some archaea do not have genes homologous to mutS. Here, we report that EndoMS/NucS of Corynebacterium glutamicum is the mismatch-specific endonuclease that functions cooperatively with a sliding clamp. EndoMS/NucS function in MMR was fully dependent on physical interaction between EndoMS/NucS and sliding clamp. A combination of endoMS/nucS gene disruption and a mutation in dnaE, which reduced the fidelity of DNA polymerase, increased the mutation rate synergistically and confirmed the participation of EndoMS in replication error correction. EndoMS specifically cleaved G/T, G/G and T/T mismatches in vitro, and such substrate specificity was consistent with the mutation spectrum observed in genome-wide analyses. The observed substrate specificity of EndoMS, together with the effects of endoMS gene disruption, led us to speculate that the MMR system, regardless of the types of proteins in the system, evolved to address asymmetrically occurring replication errors in which G/T mismatches occur much more frequently than C/A mismatches.

WalRK is an essential two-component signal transduction system that plays a central role in coordinating cell wall synthesis and cell growth in Bacillus subtilis. However, the physiological role of WalRK and its essentiality for growth have not been elucidated. We investigated the behaviour of WalRK during heat stress and its essentiality for cell proliferation. We determined that the inactivation of the walHI genes which encode the negative modulator of WalK, resulted in growth defects and eventual cell lysis at high temperatures. Screening of suppressor mutations revealed that the inactivation of LytE, an dl-endopeptidase, restored the growth of the ΔwalHI mutant at high temperatures. Suppressor mutations that reduced heat induction arising from the walRK regulon were also mapped to the walK ORF. Therefore, we hypothesized that overactivation of LytE affects the phenotype of the ΔwalHI mutant. This hypothesis was corroborated by the overexpression of the negative regulator of LytE, IseA and PdaC, which rescued the growth of the ΔwalHI mutant at high temperatures. Elucidating the cause of the temperature sensitivity of the ΔwalHI mutant could explain the essentiality of WalRK. We proved that the constitutive expression of lytE or cwlO using a synthetic promoter uncouples these expressions from WalRK, and renders WalRK nonessential in the pdaC and iseA mutant backgrounds. We propose that the essentiality of WalRK is derived from the coordination of cell wall metabolism with cell growth by regulating dl-endopeptidase activity under various growth conditions.

Propagation of genetic information is a fundamental property of living organisms. Escherichia coli has a 4.6 Mb circular chromosome with a replication origin, oriC. While the oriC replication has been reconstituted in vitro more than 30 years ago, continuous repetition of the replication cycle has not yet been achieved. Here, we reconstituted the entire replication cycle with 14 purified enzymes (25 polypeptides) that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation and decatenation of the replicated circular products. Because decatenation provides covalently closed supercoiled monomers that are competent for the next round of replication initiation, the replication cycle repeats autonomously and continuously in an isothermal condition. This replication-cycle reaction (RCR) propagates ∼10 kb circular DNA exponentially as intact covalently closed molecules, even from a single DNA molecule, with a doubling time of ∼8 min and extremely high fidelity. Very large DNA up to 0.2 Mb is successfully propagated within 3 h. We further demonstrate a cell-free cloning in which RCR selectively propagates circular molecules constructed by a multi-fragment assembly reaction. Our results define the minimum element necessary for the repetition of the chromosome-replication cycle, and also provide a powerful in vitro tool to generate large circular DNA molecules without relying on conventional biological cloning., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The SMC-ScpAB complex plays a crucial role in chromosome organization and segregation in many bacteria. It is composed of a V-shaped SMC dimer and an ScpAB subcomplex that bridges the two Structural Maintenance of Chromosomes (SMC) head domains. Despite its functional significance, the mechanistic details of SMC-ScpAB remain obscure. Here we provide evidence that ATP-dependent head-head engagement induces a lever movement of the SMC neck region, which might help to separate juxtaposed coiled-coil arms. Binding of the ScpA N-terminal domain (NTD) to the SMC neck region is negatively regulated by the ScpB C-terminal domain. Mutations in the ScpA NTD compromise this regulation and profoundly affect the overall shape of the complex. The SMC hinge domain is structurally relaxed when free from coiled-coil juxtaposition. Taken together, we propose that the structural parts of SMC-ScpAB are subjected to the balance between constraint and relaxation, cooperating to modulate dynamic conformational changes of the whole complex., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The sliding clamp is an essential component of the replisome required for processivity of DNA synthesis and several other aspects of chromosome metabolism. However, the in vivo dynamics of the clamp are poorly understood. We have used various biochemical and cell biological methods to study the dynamics of clamp association with the replisome in Bacillus subtilis cells. We find that clamps form large assemblies on DNA, called "clamp zones." Loading depends on DnaG primase and is probably driven by Okazaki fragment initiation on the lagging strand. Unloading, which is probably regulated, only occurs after many clamps have accumulated on the DNA. On/off cycling allows chromosomal zones of about 200 accumulated clamps to follow the replisome. Since we also show that clamp zones recruit proteins bearing a clamp-binding sequence to replication foci, the results highlight the clamp as a central organizer in the structure and function of replication foci., (Copyright © 2011 Elsevier Inc. All rights reserved.)

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 A resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP-DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP-DnaA, and stimulated the assembly of inactive ADP-DnaA-oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP-DnaA-specific-oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP-DnaA-oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner.

Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.

In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10-100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda-clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III*). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III* and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.

The Escherichia coli hda gene codes for a DnaA-related protein that is essential for the regulatory inactivation of DnaA (RIDA), a system that controls the initiation of chromosomal replication. We have identified the ygfZ gene, which encodes a folate-binding protein, as a suppressor of hda mutations. The ygfZ null mutation suppresses an hda null mutation. The over-initiation and abortive elongation phenotypes conferred by the hda mutations are partially suppressed in an hda ygfZ background. The accumulation of the active form of DnaA, ATP-DnaA, in the hda mutant is suppressed by the disruption of the ygfZ gene, indicating that YgfZ is involved in regulating the level of ATP-DnaA. Although ygfZ is not an essential gene, the ygfZ disruptant grows slowly, especially at low temperature, demonstrating that this gene is important for cellular proliferation. We have identified mnmE (trmE) as a suppressor of the ygfZ disruption. This gene encodes a GTPase involved in tRNA modification. Examination of RNA modification in the ygfZ mutant reveals reduced levels of 2-methylthio N(6)-isopentenyladenosine [corrected] indicating that YgfZ participates in the methylthio-group formation of this modified nucleoside in some tRNAs. These results suggest that YgfZ is a key factor in regulatory networks that act via tRNA modification.

In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the beta-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg(168) in the AAA(+) Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain approximately 50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA(+) proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA(+) domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding., (Copyright Blackwell Publishing Limited)

Background: During the cell cycle, the initiation of chromosomal replication is strictly controlled. In Escherichia coli, the initiator DnaA and the replication origin oriC are major targets for this regulation. Here, we assessed the role of transcription of the mioC gene, which reads through the adjacent oriC region. This mioC-oriC transcription is regulated in coordination with the replication cycle so that it is activated after initiation and repressed before initiation., Results: We isolated a strain bearing a mioC promoter mutation that causes constitutive mioC-oriC transcription from the chromosome. A quantitative S1 nuclease assay indicated that in this mutant, the level of transcription does not fluctuate. Introduction of this mutation suppressed the growth defect of an overinitiation-type dnaAcos mutant, and severely inhibited the growth of initiation-defective dnaA mutants at semipermissive temperatures in a dnaA allele-specific manner. These results suggest that mioC-oriC transcription inhibits initiation at oriC. Indeed, flow cytometry analysis and quantification of DNA replication in synchronized cultures revealed that the mioC promoter mutation alters the control of the initiation of chromosomal replication, for instance by delaying replication within the cell cycle., Conclusions: These results suggest that the transcriptional regulation of the mioC gene is required for cell cycle-coordinated initiation of chromosomal replication.

DnaA protein binds specifically to a group of binding sites collectively called as DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. The DNA-binding domain of DnaA, domain IV, comprises the C-terminal 94 amino acid residues of the protein. We overproduced and purified a protein containing only this domain plus a methionine residue. This protein was stable as a monomer and maintained DnaA box-specific binding activity. We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments. We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six alpha-helices and no beta-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the alpha-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins.

The DnaA protein, which initiates chromosomal replication in Escherichia coli, is negatively regulated by both the sliding clamp of DNA polymerase III holoenzyme and the IdaB protein. We have found that, when the amount of minichromosome is limited in an in vitro replication system, minichromosomal replication-stimulated hydrolysis of DnaA-bound ATP yields the ADP-bound inactive form. The number of sliding clamps formed during replication was at least five per minichromosome, which is 2.7-fold higher than the number formed during incubation without replication. These results support the notion that coupling of DnaA-ATP hydrolysis to DNA replication is the outcome of enhanced clamp formation. We have also found that the amino acid substitution R334H in DnaA severely inhibits the hydrolysis of bound ATP in vitro. Whereas ATP bound to wild-type DnaA is hydrolysed in a DNA-dependent intrinsic manner or in a sliding clamp-dependent manner, ATP bound to DnaA R334H protein was resistant to hydrolysis under the same conditions. This arginine residue may be located in the vicinity where ATP binds, and therefore may play an essential role in ATP hydrolysis. This residue is highly conserved among DnaA homologues and also in the Box VIII motif of the AAA+ protein family.