In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system.

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.

Optimization and compartmentalization of a cell-free mixture of DNA amplification and protein translation.

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.

Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation.

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 and cells.

amplification of whole large plasmids via transposon-mediated insertion.

DNA amplification is a fundamental technique in molecular biology. The replication cycle reaction is a new method for amplification of large circular DNA having sequences, which is a replication initiation site of the chromosome. We here developed a replication cycle reaction-based method useful for amplification of various circular DNAs lacking , even in the absence of any sequence information, via transposon-mediated insertion to the circular DNA template.

Amplification of over 100 kbp DNA from Single Template Molecules in Femtoliter Droplets.

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 in micrometer-sized water-in-oil droplets, we developed the replication cycle reaction (RCR) system.

Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes.

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.

Overcoming the Challenges of Megabase-Sized Plasmid Construction in .

Although 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.

Cell-Penetrating Peptide-Mediated Transformation of Large Plasmid DNA into Escherichia coli.

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.

Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction.

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 chromosome. In RCR, two replication forks proceed bidirectionally from the replication origin, , and meet at a region opposite , yielding two copies of circular DNA.

Bacterial EndoMS/NucS acts as a clamp-mediated mismatch endonuclease to prevent asymmetric accumulation of replication errors.

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.

Essentiality of WalRK for growth in Bacillus subtilis and its role during heat stress.

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.

Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle.

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.

Overall Shapes of the SMC-ScpAB Complex Are Determined by Balance between Constraint and Relaxation of Its Structural Parts.

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.

The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA.

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.

The replicase sliding clamp dynamically accumulates behind progressing replication forks in Bacillus subtilis cells.

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.

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

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.

Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli.

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.

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP DnaA-specific initiation complexes.

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.

An isolated Hda-clamp complex is functional in the regulatory inactivation of DnaA and DNA replication.

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.

Involvement of the Escherichia coli folate-binding protein YgfZ in RNA modification and regulation of chromosomal replication initiation.

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.

Protein associations in DnaA-ATP hydrolysis mediated by the Hda-replicase clamp complex.

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.

Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.

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.

Transcriptional control for initiation of chromosomal replication in Escherichia coli: fluctuation of the level of origin transcription ensures timely initiation.

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.

Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain.

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.