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.     

New non-detrimental DNA-binding mutants of the Escherichia coli initiator protein DnaA

The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino acid residues are located between positions 30 and 80 of the 94 residue domain. Residues where substitutions with non-closely related amino acids have very little effect on protein function are located primarily on the periphery of the 3D structure. By comparison of the effect of substitutions on the activity for initiation of replication with the activity for repression of the mioC promoter, we identified residues that might be involved specifically in the cooperative interaction with ATP-DnaA boxes.     

Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP. ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear. We constructed, by site-directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo. The ATPase activity of purified mutated DnaA protein (Glu204-->Gln) decreased to one-third that of the wild-type DnaA protein. The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP. The mutant dnaA gene showed lethality in wild-type cells but not in cells growing independently of the function of oriC. Induction of the mutated DnaA protein in wild-type cells caused an overinitiation of DNA replication. Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal DNA replication in E. coli cells.     

Site-directed mutational analysis of DnaA protein, the initiator of chromosomal DNA replication in E. coli

DnaA protein, the initiator for chromosomal DNA replication in Escherichia coli, has various activities, such as oligomerization (DnaA-DnaA interaction), ATP-binding, ATPase activity and membrane-binding. Site-directed mutational analyses have revealed not only the amino acid residues that are essential for these activities but also the functions of these activities. Following is a summary of the functions and regulatory mechanisms of DnaA protein in the initiation of chromosomal DNA replication. ATP-bound DnaA protein, but not other forms of the protein binds to the origin of DNA replication and forms oligomers to open-up the duplex DNA. This oligomerization is mediated by a DnaA-DnaA interaction through the N-terminal region of the protein. After initiation of DNA replication, the ATPase activity of DnaA protein is stimulated and DnaA protein is inactivated to the ADP-bound form to suppress the re-initiation of DNA replication. DnaA protein binds to acidic phospholipids through an ionic interaction between basic amino acid residues of the protein and acidic residues of phospholipids. This interaction seems to be involved in the re-activation of DnaA protein (from the ADP-bound form to the ATP-bound form) to initiate DNA replication after the appropriate interval.     

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.     

Electrostatic interactions during acidic phospholipid reactivation of DnaA protein, the Escherichia coli initiator of chromosomal replication.

The initiation of Escherichia coli chromosomal replication by DnaA protein is strongly influenced by the tight binding of the nucleotides ATP and ADP. Anionic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA protein to replicatively active ATP-DnaA protein in vitro, and thus likely play a key role in regulating DnaA activity. Previous studies have revealed that, during this reactivation, a specific region of DnaA protein inserts into the hydrophobic portion of the lipid bilayer in an acidic phospholipid-dependent manner. To elucidate the requirement for acidic phospholipids in the reactivation process, the contribution of electrostatic forces in the interaction of DnaA and lipid was examined. DnaA-lipid binding required anionic phospholipids, and DnaA-lipid binding as well as lipid-mediated release of DnaA-bound nucleotide were inhibited by increased ionic strength, suggesting the involvement of electrostatic interactions in these processes. As the vesicular content of acidic phospholipids was increased, both nucleotide release and DnaA-lipid binding increased in a linear, parallel manner. Given that DnaA-membrane binding, the insertion of DnaA into the membrane, and the consequent nucleotide release all require anionic phospholipids, the acidic headgroup may be necessary to recruit DnaA protein to the membrane for insertion and subsequent reactivation for replication.     

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.     

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.     

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.     

Analysis of the DNA-binding domain of Escherichia coli DnaA protein

The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374-467). The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC:lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC:lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.     

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.     

The N-terminus promotes oligomerization of the Escherichia coli initiator protein DnaA

Initiation of chromosome replication in Escherichia coli is governed by the interaction of the initiator protein DnaA with the replication origin oriC. Here we present evidence that homo-oligomerization of DnaA via its N-terminus (amino acid residues 1-86) is also essential for initiation. Results from solid-phase protein-binding assays indicate that residues 1-86 (or 1-77) of DnaA are necessary and sufficient for self interaction. Using a 'one-hybrid-system' we found that the DnaA N-terminus can functionally replace the dimerization domain of coliphage lambda cl repressor: a lambdacl-DnaA chimeric protein inhibits lambda plasmid replication as efficiently as lambdacI repressor. DnaA derivatives with deletions in the N-terminus are incapable of supporting chromosome replication from oriC, and, conversely, overexpression of the DnaA N-terminus inhibits initiation in vivo. Together, these results indicate that (i) oligomerization of DnaA N-termini is essential for protein function during initiation, and (ii) oligomerization does not require intramolecular cross-talk with the nucleotide-binding domain III or the DNA-binding domain IV. We propose that E. coli DnaA is composed of largely independent domains - or modules - each contributing a partial, though essential, function to the proper functioning of the 'holoprotein'.     

Fate of the DnaA initiator protein in replication at the origin of the Escherichia coli chromosome in vitro

The dnaA initiator protein binds specific sequences in the 245-base pair Escherichia coli origin (oriC) to form a series of complexes which eventually are opened enough to admit dnaB helicase into a prepriming complex (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). ATP bound to a high-affinity site on dnaA protein is the preferred form for one or more of the early stages, but an elevated level of ATP is needed for a later stage; further evidence for a low-affinity site has now been obtained. We find that at limiting levels of dnaA protein only the ATP form produces an active initial complex; neither the ADP nor the non-nucleotide forms are effective. Augmentation of the activity of a limiting level of the ATP form of dnaA protein by the otherwise inert ADP form implies that at some stage of initiation both forms are active. The dnaA protein is essential up to the stage of forming the prepriming complex; upon salt dissociation from an oriC complex, the protein can be recycled to function at a fresh origin. Distinctive conformational states of the ATP form are implied by interactions with oriC DNA, by the influence of phospholipids on accelerating nucleotide exchange, and by the susceptibility to proteolytic cleavage.     

DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori

The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized. The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane. The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies. DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes. In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments. As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM). Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity. Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all.     

Inactivation of Escherichia coli DnaA protein by DNA polymerase III and negative regulations for initiation of chromosomal replication

Genetic and biochemical evidence indicates that initiation of chromosomal replication in Escherichia coli occurs in a nucleoprotein complex at the replication origin (oriC) formed with DnaA protein. The frequency of initiation at oriC is tightly regulated to only once per chromosome per cell cycle. To prevent untimely, extra initiations, negative control for initiation is indispensable. Recently, we found that the function of the initiator protein, DnaA, is controlled by DNA polymerase III holoenzyme, the replicase of the chromosome. The ATP-bound form of DnaA protein, an active form for initiation, is efficiently converted to the ADP bound form, an inactive form, since a subunit of the polymerase loaded on DNA (beta subunit sliding clamp) stimulates hydrolysis of ATP bound to DnaA protein. Comparison of this system, RIDA (regulatory inactivation of DnaA), with other systems for negative regulation of initiation is included in this review, and the roles of these systems for concerted control for initiation during the cell cycle are discussed.     

Mutant DnaA proteins defective in duplex opening of oriC, the origin of chromosomal DNA replication in Escherichia coli

We characterized three mutant DnaA proteins with an amino acid substitution of R334H, R342H and E361G that renders chromosomal replication cold (20 degrees C) sensitive. Each mutant DnaA protein was highly purified from overproducers, and replication activities were assayed in in vitro oriC replication systems. At 30 degrees C, all three mutant proteins exhibited specific activity similar to that seen with the wild-type protein, whereas at 20 degrees C, there was much less activity in a replication system using a crude replicative extract. Regarding the affinity for ATP, the dissociation rate of bound ATP and binding to oriC DNA, the three mutant DnaA proteins showed a capacity indistinguishable from that of the wild-type DnaA protein. Activity for oriC DNA unwinding of the two mutant DnaA proteins, R334H and R342H, was more sensitive to low temperature than that of the wild-type DnaA protein. We propose that R334H and R342H have a defect in their potential to unwind oriC DNA at low temperatures, the result being the cold-sensitive phenotype in oriC DNA replication. The two amino acid residues of DnaA protein, located in a motif homologous to that of NtrC protein, may play a role in the formation of the open complex. The E361 residue may be related to interaction with another protein present in a crude cell extract.     

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.     

Intermediates of chromosomal DNA replication in Escherichia coli

The product of bacteriophage T4 gene 63 has two activities, one which catalyzes the attachment of tail fibers to base plates during morphogenesis (TFA) and one which catalyzes the joining of single-stranded polynucleotides (RNA ligase). The only phenotype attributed to mutations in gene 63 is a defect in attachment of tail fibers leading to fiberless T4 particles. However, it is suspected that TFA and RNA ligase are unrelated activities of the same protein since they have very different requirements in vitro.We have isolated new mutants which have lost the RNA ligase but have retained the TFA activity of the product of gene 63. These mutants exhibit defects in T4 DNA replication and late gene expression in some strains of Escherichia coli. This work allows us to draw three conclusions: (1) the TFA and RNA ligase activities are unrelated functions of the gene 63 product making this the prototype for a protein which has more than one unrelated function; (2) the RNA ligase is probably involved in DNA metabolism rather than RNA processing as has been proposed: (3) the RNA ligase and polynucleotide 5′ kinase 3′ phosphatase of T4 perform intimately related functions.