DnaA boxes are important elements in setting the initiation mass of Escherichia coli

The binding of DnaA protein to its DNA binding sites-DnaA boxes-in the chromosomal oriC region is essential for initiation of chromosome replication. In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass. The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the oriC region into Escherichia coli and by growing the strains at different generation times to obtain different plasmid copy numbers. In fast-growing cells, where the DnaA box plasmid copy number per oriC locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass. In slowly growing cells, where the DnaA box plasmid copy number per oriC locus was higher, we observed more pronounced increases in the initiation mass. Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in E. coli cells.     

DnaA protein overproduction abolishes cell cycle specificity of DNA replication from oriC in Escherichia coli.

Initiation of DNA replication from oriC in Escherichia coli takes place at a specific time in the cell division cycle, whether the origin is located on a chromosome or a minichromosome, and requires participation of the product of the dnaA gene. The effects of overproduction of DnaA protein on the cell cycle specificity of the initiation event were determined by using minichromosome replication as the assay system. DnaA protein was overproduced by inducing the expression of plasmid-encoded dnaA genes under control of either the ptac or lambda pL promoter. Induction of DnaA protein synthesis caused a burst of minichromosome replication in cells at all ages in the division cycle. The magnitude of the burst was consistent with the initiation of one round of replication per minichromosome in all cells. The replication burst was followed by a period of reduced minichromosome replication, with the reduction being greater at 30 than at 41 degrees C. The results support the idea that the DnaA protein participates in oriC replication at a stage that is limiting for initiation. Excess DnaA protein enabled all cells to achieve the state required for initiation of DNA polymerization by either effecting or overriding the normal limiting process.     

The datA locus predominantly contributes to the initiator titration mechanism in the control of replication initiation in Escherichia coli

Replication of the Escherichia coli chromosome is initiated synchronously from all origins (oriC) present in a cell at a fixed time in the cell cycle under given steady state culture conditions. A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which titrates exceptionally large amounts of the bacterial initiator protein, DnaA, to prevent overinitiation. Deletion of the datA locus results in extra initiations and altered temporal control of replication. There are many other sites on the E. coli chromosome that can bind DnaA protein, but the contribution of these sites to the control of replication initiation has not been investigated. In the present study, seven major DnaA binding sites other than datA have been examined for their influence on the timing of replication initiation. Disruption of these seven major binding sites, either individually or together, had no effect on the timing of initiation of replication. Thus, datA seems to be a unique site that adjusts the balance between free and bound DnaA to ensure that there is only a single initiation event in each bacterial cell cycle. Mutation either in the second or the third DnaA box (a 9 basepair DnaA-binding sequence) in datA was enough to induce asynchronous and extra initiations of replication to a similar extent as that observed with the datA-deleted strain. These DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region.     

Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control

A novel method was devised to measure the number of plasmids in individual Escherichia coli cells. With this method, involving measurement of plasmid-driven expression of the green fluorescent protein gene by flow cytometry, the copy number distribution of a number of different plasmids was measured. Whereas natural plasmids had fairly narrow distributions, minichromosomes, which are plasmids replicating only from a cloned oriC copy, have a wide distribution, suggesting that there is no copy number control for minichromosomes. When the selection pressure (kanamycin concentration) for minichromosomes was increased, the copy number of minichromosomes was also increased. At up to 30 minichromosomes per host chromosome, replication and growth of the host cell was unaffected. This is evidence that there is no negative element for initiation control in oriC and that there is no incompatibility between oriC located on the chromosome and minichromosome. However, higher copy numbers led to integration of the minichromosomes at the chromosomal oriC and to initiation asynchrony of the host chromosome. At a minichromosome copy number of approximately 30, the cell's capacity for synchronous initiation is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome.     

DnaA Protein of Escherichia coli: oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N-terminal amino acids

Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At the E. coli chromosomal origin, oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation at oriC that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the non-permissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to DnaA box sequences within oriC. We show that the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Relying on this assay, leucine 5, tryptophan 6 and cysteine 9 in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. Glutamine 8 is additionally needed for oligomer formation on an oriC-containing plasmid, suggesting that the structure of the DnaA-oriC complex at the chromosomal oriC locus is similar but not identical to that assembled on a plasmid. Other evidence suggests that proline 28 of DnaA is involved in the recruitment of DnaB to oriC. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur.     

The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome

We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.     

Coupling the initiation of chromosome replication to cell size in Escherichia coli

Bacterial cells change size dramatically with change in growth rate, but the ratio between cell volume and the number of copies of the origin of chromosome replication (oriC) is roughly constant at the time of initiation of DNA replication at almost all growth rates. Recent research on the inactivation of initiator protein (DnaA) and depletion of DnaA pools by the high-affinity DnaA-binding locus datA allows us to propose a simple model to explain the long-standing question of how Escherichia coli couples DNA replication to cell size.     

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli.

An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control. The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit. The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C). In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected. Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C. The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C. It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene. However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration. This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected.     

Overexpression of the dnaA gene in Escherichia coli B/r: chromosome and minichromosome replication in the presence of rifampin.

The replication of chromosomes and minichromosomes in Escherichia coli B/r was examined under conditions in which the dnaA gene product was overproduced. Increased levels of the DnaA protein were achieved by thermoinduction of the dnaA gene, under the control of the lambda pL promoter, or by cellular maintenance of multicopy plasmids carrying the dnaA gene under the control of its own promoters. Previous work has shown that overproduction of DnaA protein stimulates replication of the chromosomal origin, oriC, but that the newly initiated forks do not progress along the length of the chromosome (T. Atlung, K. V. Rasmussen, E. Clausen, and F. G. Hansen, p. 282-297, in M. Schaechter, F. C. Neidhardt, J. L. Ingraham, and N. O. Kjeldgaard, ed., The Molecular Biology of Bacterial Growth, 1985). In the present study, it was found that overproduction of DnaA protein caused both a two- to threefold increase in the amount of residual chromosome replication and an extended synthesis of minichromosome DNA in the presence of rifampin. The amount of residual chromosome replication was consistent with the appearance of functional replication forks on the majority of the chromosomes. Since the rate of DNA accumulation and the cellular DNA/mass ratios were not increased significantly by overexpression of the dnaA gene, we concluded that the addition of rifampin either enabled stalled replication forks to proceed beyond oriC or enabled new forks to initiate on both chromosomes and minichromosomes, or both.     

Multiple pathways regulating DnaA function in Escherichia coli: distinct roles for DnaA titration by the datA locus and the regulatory inactivation of DnaA

Escherichia coli DnaA protein forms a multimeric complex at the chromosomal origin of replication (oriC), where a series of initiation reactions occurs and DNA polymerase III holoenzyme is loaded. The ATP-bound form of DnaA, which is active for initiation, is converted to the inactive ADP-bound form through interaction with the sliding clamp, the beta subunit of DNA polymerase III holoenzyme loaded on DNA. This negative regulation, termed RIDA, is required for preventing untimely initiations. Here, we asked if RIDA is functionally related to another negative regulation, DnaA titration by the datA site. The datA site can harbor hundreds of DnaA molecules, and is also required for preventing untimely initiations. We reveal here that, in growing cells of the datA(+) and datA-deleted strains, the ATP-DnaA levels were both maintained in a limited range of about 20-30% of the total ATP- plus ADP-DnaA molecules. This indicates that RIDA functions in the absence of datA. In synchronized datA-deleted cells, the ATP-DnaA level fluctuated in a manner similar to that observed in datA(+) cells. This suggests that RIDA operates independent from DnaA titration to datA. We suggest that these two mechanisms may play complementary roles during the cell cycle to prevent untimely initiations and thus ensure the scheduled initiation.     

Coordinated replication and sequestration of oriC and dnaA are required for maintaining controlled once-per-cell-cycle initiation in Escherichia coli

Escherichia coli cells were constructed in which the dnaA gene was moved to a location opposite oriC on the circular chromosome. In these cells the dnaA gene was replicated with significant delay relative to the origin. Consequently, the period where the newly replicated and hemimethylated oriC was sequestered no longer coincided with the period where the dnaA gene promoter was sequestered. DnaA protein synthesis was therefore expected to continue during origin sequestration. Despite a normal length of the sequestration period in such cells, they had increased origin content and also displayed asynchrony of initiation. This indicated that reinitiation occasionally occurred at some origins within the same cell cycle. The extra initiations took place in spite of a reduction in total DnaA protein concentration to about half of the wild-type level. We propose that this more efficient utilization of DnaA protein results from an increased availability at the end of the origin sequestration period. Therefore, coordinated sequestration of oriC and dnaA is required for maintaining controlled once-per-cell-cycle initiation.     

Regulation of the initiation of chromosomal replication in bacteria

The initiation of chromosomal replication occurs only once during the cell cycle in both prokaryotes and eukaryotes. Initiation of chromosome replication is the first and tightly controlled step of a DNA synthesis. Bacterial chromosome replication is initiated at a single origin, oriC, by the initiator protein DnaA, which specifically interacts with 9-bp non-palindromic sequences (DnaA boxes) at oriC. In Escherichia coli, a model organism used to study the mechanism of DNA replication and its regulation, the control of initiation relies on a reduction of the availability and/or activity of the two key elements, DnaA and the oriC region. This review summarizes recent research into the regulatory mechanisms of the initiation of chromosomal replication in bacteria, with emphasis on organisms other than E. coli.     

Titration of the Escherichia coli DnaA protein to excess datA sites causes destabilization of replication forks, delayed replication initiation and delayed cell division

In Escherichia coli, the level of the initiator protein DnaA is limiting for initiation of replication at oriC. A high-affinity binding site for DnaA, datA, plays an important role. Here, the effect of extra datA sites was studied. A moderate increase in datA dosage ( approximately fourfold) delayed initiation of replication and cell division, but increased the rate of replication fork movement about twofold. At a further increase in the datA gene dosage, the SOS response was induced, and incomplete rounds of chromosome replication were detected. Overexpression of DnaA protein suppressed the SOS response and restored normal replication timing and rate of fork movement. In the presence of extra datA sites, cells showed a dependency on PriA and RecA proteins, indicating instability of the replication fork. The results suggest that wild-type replication fork progression normally includes controlled pausing, and that this is a prerequisite for normal replication fork function.