[39] DNA synthesis initiated at oriC:In Vitro replication reactions

doi:10.1016/0076-6879(95)62041-9

Methods in Enzymology

Volume 262, 1995, Pages 500-506

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In vitro replication initiated at the Escherichia coli chromosomal origin (oriC) can occur in a precisely prepared cell extract or it can be reconstituted with purified proteins. The development of these systems was possible following the identification of oriC and the construction of oriC-containing plasmids. In vivo, these plasmids act as minichromosomes in that their replication is initiated synchronously with that of the cell chromosome and with similar requirements. Behaving as such, these plasmids can serve as templates in vitro to study replication at oriC. Another major advance in the development of systems to replicate oriC in vitro is the preparation of an extract containing all of the required factors and lacking inhibitors. Combined with knowledge gained from studying replication in other systems such as bacteriophage φX174, replication in the crude extract permitted the identification, isolation, and characterization of the numerous components necessary to reconstitute oriC DNA replication. Replication in cell extracts and in systems of defined components has many physiological features. It requires the addition of template DNA, which contains the oriC sequence, and replication is initiated at or near oriC. It is dependent on certain replication proteins that are identified as important for chromosomal replication in vivo. Included in these is DnaA, a protein that is implicated genetically as playing a central role in initiating replication at oriC.

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Cited by (20)

Different effects of ppGpp on Escherichia coli DNA replication in vivo and in vitro
2013, FEBS Open Bio
Citation Excerpt :
In this report, we have demonstrated for the first time that ppGpp can directly inhibit E. coli DNA replication in vitro. Although Fraction II, used in our experimental system, contains some RNA polymerase molecules, indirect effects of ppGpp on in vitro DNA replication, through modulating transcription efficiency, are unlikely since neither gene expression nor transcriptional activation of oriC are required under these conditions [24]. Therefore, we suggest that the observed impairment of DNA synthesis in the presence of ppGpp, as shown in Fig. 1, may be caused by depression of the DnaG primase activity (Fig. 2).
Inhibition of Escherichia coli DNA replication by guanosine tetraphosphate (ppGpp) is demonstrated in vitro. This finding is compatible with impairment of the DnaG primase activity by this nucleotide. However, in agreement to previous reports, we were not able to detect a rapid inhibition of DNA synthesis in E. coli cells under the stringent control conditions, when intracellular ppGpp levels increase dramatically. We suggest that the process of ppGpp-mediated inhibition of DnaG activity may be masked in E. coli cells, which could provide a rationale for explanation of differences between ppGpp effects on DNA replication in E. coli and Bacillus subtilis.
Remodeling of nucleoprotein complexes is independent of the nucleotide state of a mutant AAA+ protein
2011, Journal of Biological Chemistry
Citation Excerpt :
Protein concentrations were determined according to Bradford (38). In vitro DNA replication and ATP binding activities of DnaA protein were determined as described elsewhere (30, 37, 39). DnaA footprinting was performed as described previously (12, 15, 40).
DnaA protein, a member of the AAA+ (ATPase associated with various cellular activities) family, initiates DNA synthesis at the chromosomal origin of replication (oriC) and regulates the transcription of several genes, including its own. The assembly of DnaA complexes at chromosomal recognition sequences is affected by the tight binding of ATP or ADP by DnaA. DnaA with a point mutation in its membrane-binding amphipathic helix, DnaA(L366K), previously described for its ability to support growth in cells with altered phospholipid content, has biochemical characteristics similar to those of the wild-type protein. Yet DnaA(L366K) fails to initiate in vitro or in vivo replication from oriC. We found here, through in vitro dimethyl sulfate footprinting and gel mobility shift assays, that DnaA(L366K) in either nucleotide state was unable to assemble into productive prereplication complexes. In contrast, at the dnaA promoter, both the ATP and the ADP form of DnaA(L366K) generated active nucleoprotein complexes that efficiently repressed transcription in a manner similar to wild-type ATP-DnaA. Thus, it appears that unlike wild-type DnaA protein DnaA(L366K) can adopt architectures that are independent of its bound nucleotide, and instead the locus determines the functionality of the higher order DnaA(L366K)-DNA complexes.
Restoration of growth to acidic phospholipid-deficient cells by DnaA(L366K) is independent of its capacity for nucleotide binding and exchange and requires DnaA
2005, Journal of Biological Chemistry
Citation Excerpt :
Nucleotides incorporated into acid-insoluble materials were retained on GF/C filters (Millipore) and measured by liquid scintillation counting. DNA Replication with Defined Components—In vitro replication of pBSoriC, dependent on eight purified replication proteins, was largely performed as described (30). Reactions (25 μl) contained: 30 mm Tricine·KOH (pH 8.25); 7 mm magnesium acetate; 2 mm ATP; 0.5 mm each GTP, CTP, and UTP; 0.1 mm each dATP, dGTP, dCTP, and [α-32P]TTP (∼250 mCi/mmol); 200 ng (600 pmol as nucleotide) pBSoriC; 450 ng of single strand binding protein (SSB); 10 ng of HU; 400 ng of gyrase A subunit; 180 ng of gyrase B subunit; 150 ng of DnaB·DnaC complex; 12 ng of DnaG primase; 500 ng of DNA polymerase III*; 25 ng of DNA polymerase III holoenzyme β-subunit; and the indicated amounts of DnaA proteins.
In the absence of adequate levels of cellular acidic phospholipids, Escherichia coli remain viable but are arrested for growth. Expression of a DnaA protein that contains a single amino acid substitution in the membrane-binding domain, DnaA(L366K), in concert with expression of wild-type DnaA protein, restores growth. DnaA protein has high affinity for ATP and ADP, and in vitro lipid bilayers that are fluid and contain acidic phospholipids reactivate inert ADP-DnaA by promoting an exchange of ATP for ADP. Here, nucleotide and lipid interactions and replication activity of purified DnaA(L366K) were examined to gain insight into the mechanism of how it restores growth to cells lacking acidic phospholipids. DnaA(L366K) behaved like wild-type DnaA with respect to nucleotide binding affinities and hydrolysis properties, specificity of acidic phospholipids for nucleotide release, and origin binding. Yet, DnaA(L366K) was feeble at initiating replication from oriC unless augmented with a limiting quantity of wild-type DnaA, reflecting the in vivo requirement that both wild-type and a mutant form of DnaA must be expressed and act together to restore growth to acidic phospholipid deficient cells.
Reconstitution of F Factor DNA Replication in Vitro with Purified Proteins
2004, Journal of Biological Chemistry
Citation Excerpt :
The DNA synthesis was quantified by measuring the total picomoles of acid precipitable radioactivity in the reaction product. OriC Replication—Replication in vitro of oriC (cloned in pUC18) template was carried out as described (17, 24, 25). The oriC replication was used as a system to check for optimizing the stoichiometry of the various components needed for maximal synthesis.
Jacob, Brenner, and Cuzin pioneered the development of the F plasmid as a model system to study replication control, and these investigations led to the development of the “replicon model” (Jacob, F., Brenner, S., and Cuzin, F. (1964) Cold Spring Harbor Symp. Quant. Biol. 28, 329–348). To elucidate further the mechanism of initiation of replication of this plasmid and its control, we have reconstituted its replication in vitro with 21 purified host-encoded proteins and the plasmid-encoded initiator RepE. The replication in vitro was specifically initiated at the F ori (oriV) and required both the bacterial initiator protein DnaA and the plasmid-encoded initiator RepE. The wild type dimeric RepE was inactive in catalyzing replication, whereas a monomeric mutant form called RepE* (R118P) was capable of catalyzing vigorous replication. The replication topology was mostly of the Cairns form, and the fork movement was unidirectional and mostly from right to left. The replication was dependent on the HU protein, and the structurally and functionally related DNA bending protein IHF could not efficiently substitute for HU. The priming was dependent on DnaG primase. Many of the characteristics of the in vitro replication closely mimicked those of in vivo replication. We believe that the in vitro system should be very useful in unraveling the mechanism of replication initiation and its control.
Reconstitution of R6K DNA Replication in Vitro Using 22 Purified Proteins
2003, Journal of Biological Chemistry
Citation Excerpt :
The precipitate was trapped on GFC glass fiber filters, dried, and counted with liquid scintillation counter. OriC Replication—Replication in vitro of the pUC19-OriC template was carried out as described previously (38, 48, 49). Origin Mapping by DideoxyNTP Incorporation—The reactions were carried out as described above but with [α-32P]dATP and [α-32P]dCTP and 0, 40, 60, and 80 μm ddTTP.
We have reconstituted a multiprotein system consisting of 22 purified proteins that catalyzed the initiation of replication specifically at ori γ of R6K, elongation of the forks, and their termination at specific replication terminators. The initiation was strictly dependent on the plasmid-encoded initiator protein π and on the host-encoded initiator DnaA. The wild type π was almost inert, whereas a mutant form containing 3 amino acid substitutions that tended to monomerize the protein was effective in initiating replication. The replication in vitro was primed by DnaG primase, whereas in a crude extract system that had not been fractionated, it was dependent on RNA polymerase. The DNA-bending protein IHF was needed for optimal replication and its substitution by HU, unlike in the oriC system, was less effective in promoting optimal replication. In contrast, wild type π-mediated replication in vivo requires IHF. Using a template that contained ori γ flanked by two asymmetrically placed Ter sites in the blocking orientation, replication proceeded in the Cairns type mode and generated the expected types of termination products. A majority of the molecules progressed counterclockwise from the ori, in the same direction that has been observed in vivo. Many features of replication in the reconstituted system appeared to mimic those of in vivo replication. The system developed here is an important milestone in continuing biochemical analysis of this interesting replicon.
DnaA protein Lys-415 is close to the ATP-binding site: ATP-pyridoxal affinity labeling
2001, Biochemical and Biophysical Research Communications
Binding of ATP, but not of ADP, activates Escherichia coli DnaA protein for replicational initiation of the chromosome. To elucidate this switching mechanism, we used the affinity-labeling agent ATP-pyridoxal, which forms a covalent bond with the Lys residue located at or near the γ-phosphate of ATP. ATP-pyridoxal inhibited the ATP binding for DnaA protein, with a competitive mode. Binding stoichiometry was 0.28 ATP-pyridoxal/DnaA molecule, a value consistent with that of ATP. Thus, ATP-pyridoxal was a potent antagonist for the DnaA ATP-binding site. The labeled DnaA protein was inactive for minichromosome replication in vitro, suggesting that conformation of the region is important for DnaA activity. Isolation of the labeled, tryptic fragment and the Edman degradation revealed that ATP-pyridoxal modified Lys-415. Thus, this residue is likely close to the bound ATP. Since Lys-415 is located in the DNA-binding domain, these findings imply internal interaction between the domains for ATP binding and DNA binding.

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[39] DNA synthesis initiated at oriC:In Vitro replication reactions

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Cell

J. Biol. Chem.

Cell

BBA

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Cell

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J. Biol. Chem.

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