Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

Summary Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA ⁺ gene. When the dnaA gene was induced from either the plac or the pL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter pL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.     

Interactions of Escherichia coli molecular chaperone HtpG with DnaA replication initiator DNA

The bacterial chaperone high-temperature protein G (HtpG), a member of the Hsp90 protein family, is involved in the protection of cells against a variety of environmental stresses. The ability of HtpG to form complexes with other bacterial proteins, especially those involved in fundamental functions, is indicative of its cellular role. An interaction between HtpG and DnaA, the main initiator of DNA replication, was studied both in vivo, using a bacterial two-hybrid system, and in vitro with a modified pull-down assay and by chemical cross-linking. In vivo, this interaction was demonstrated only when htpG was expressed from a high copy number plasmid. Both in vitro assays confirmed HtpG–DnaA interactions.     

DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect.

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.     

The box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E. coli replication origin

Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Structure-function studies indicate that the replication initiator comprises four domains. Based on the structural similarity of Aquifex aeolicus DnaA to other AAA+ proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. (2002) EMBO J. 21, 4763-4773). Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif. All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo. Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication. Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC. Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase. Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB.     

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. 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.     

Sequence-independent DNA binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli

The DnaA protein specifically binds to the origin of chromosomal DNA replication and initiates DNA synthesis. In addition to this sequence-specific DNA binding, DnaA protein binds to DNA in a sequence-independent manner. We here compared the two DNA binding activities. Binding of ATP and ADP to DnaA inhibited the sequence-independent DNA binding, but not sequence-specific binding. Sequence-independent DNA binding, but not sequence-specific binding, required incubation at high temperatures. Mutations in the C-terminal domain affected the sequence-independent DNA binding activity less drastically than they did the sequence-specific binding. On the other hand, the mutant DnaA433, which has mutations in a membrane-binding domain (K327 to I344) was inert for sequence-independent binding, but could bind specifically to DNA. These results suggest that the two DNA binding activities involve different domains and perform different functions from each other in Escherichia coli cells.     

Chromosome replication in Escherichia coli induced by oversupply of DnaA

Summary Overexpression of DnaA protein from a multicopy plasmid accompanied by a shift to 42°C causes initiation of one extra round of replication in a dnaA ⁺ strain grown in glycerol minimal medium. This extra round of replication does not lead to an extra cell division, such that cells contain twice the normal number of chromosomes.     

DnaA, the initiator of Escherichia coli chromosomal replication, is located at the cell membrane

Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.     

Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein

Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.     

Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli

The bacterial DnaA protein binds to the chromosomal origin of replication to trigger a series of initiation reactions, which leads to the loading of DNA polymerase III. In Escherichia coli, once this polymerase initiates DNA synthesis, ATP bound to DnaA is efficiently hydrolyzed to yield the ADP-bound inactivated form. This negative regulation of DnaA, which occurs through interaction with the beta-subunit sliding clamp configuration of the polymerase, functions in the temporal blocking of re-initiation. Here we show that the novel DnaA-related protein, Hda, from E.coli is essential for this regulatory inactivation of DnaA in vitro and in vivo. Our results indicate that the hda gene is required to prevent over-initiation of chromosomal replication and for cell viability. Hda belongs to the chaperone-like ATPase family, AAA(+), as do DnaA and certain eukaryotic proteins essential for the initiation of DNA replication. We propose that the once-per-cell-cycle rule of replication depends on the timely interaction of AAA(+) proteins that comprise the apparatus regulating the activity of the initiator of replication.     

DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.     

Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication

We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.     

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 bacteriophage lambda DNA replication protein P inhibits the oriC DNA- and ATP-binding functions of the DNA replication initiator protein DnaA of Escherichia coli

Under the condition of expression of lambda P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the lambda P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the lambda P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of lambda P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.