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.     

Quantitative analysis of the mechanism of DNA binding by Bacillus DnaA protein

DnaA protein has the sole responsibility of initiating a new round of DNA replication in prokaryotic organisms. It recognizes the origin of DNA replication, and initiates chromosomal DNA replication in the bacterial genome. In Gram-negative Escherichia coli, a large number of DnaA molecules bind to specific DNA sequences (known as DnaA boxes) in the origin of DNA replication, oriC, leading to the activation of the origin. We have cloned, expressed, and purified full-length DnaA protein in large quantity from Gram-positive pathogen Bacillus anthracis (DnaA_BA). DnaA_BA was a highly soluble monomeric protein making it amenable to quantitative analysis of its origin recognition mechanisms. DnaA_BA bound DnaA boxes with widely divergent affinities in sequence and ATP-dependent manner. In the presence of ATP, the K_D ranged from 3.8 × 10⁻⁸ M for a specific DnaA box sequence to 4.1 × 10⁻⁷ M for a non-specific DNA sequence and decreased significantly in the presence of ADP. Thermodynamic analyses of temperature and salt dependence of DNA binding indicated that hydrophobic (entropic) and ionic bonds contributed to the DnaA_BA·DNA complex formation. DnaA_BA had a DNA-dependent ATPase activity. DNA sequences acted as positive effectors and modulated the rate (V_max) of ATP hydrolysis without any significant change in ATP binding affinity.     

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.     

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. This protein was stable as a monomer and maintained DnaA box-specific binding activity. We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments. We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six alpha-helices and no beta-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the alpha-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins.     

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.     

Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli

The replication period of Escherichia coli cells grown in rich medium lasts longer than one generation. Initiation thus occurs in the 'mother-' or 'grandmother generation'. Sister origins in such cells were found to be colocalized for an entire generation or more, whereas sister origins in slow-growing cells were colocalized for about 0.1-0.2 generations. The role of origin inactivation (sequestration) by the SeqA protein in origin colocalization was studied by comparing sequestration-deficient mutants with wild-type cells. Cells with mutant, non-sequesterable origins showed wild-type colocalization of sister origins. In contrast, cells unable to sequester new origins due to loss of SeqA, showed aberrant localization of origins indicating a lack of organization of new origins. In these cells, aberrant replisome organization was also found. These results suggest that correct organization of sister origins and sister replisomes is dependent on the binding of SeqA protein to newly formed DNA at the replication forks, but independent of origin sequestration. In agreement, in vitro experiments indicate that SeqA is capable of pairing newly replicated DNA molecules.     

Role of the amino-terminal region of the DnaA protein in opening of the duplex DNA at the oriC region

We report in this paper that the amino acid residues Ile-26 and Leu-40 of the DnaA protein are essential for the DNA replication activity in vitro. Lines of evidence to support this conclusion are as follows. Variants of the DnaA protein containing either an Ile-26-Ser or Leu-40-Ser replacement were unable to support oriC DNA replication in vitro. Though the mutant DnaA proteins retained the capability to bind oriC DNA, they were unable to open the duplex DNA at oriC. Based on these and other results, we conclude that the N-terminal region of the DnaA protein is involved in the oligomerization of this protein, an essential step for the duplex opening activity at oriC.     

The Arg Fingers of Key DnaA Protomers Are Oriented Inward within the Replication Origin oriC and Stimulate DnaA Subcomplexes in the Initiation Complex

ATP-DnaA binds to multiple DnaA boxes in the Escherichia coli replication origin (oriC) and forms left-half and right-half subcomplexes that promote DNA unwinding and DnaB helicase loading. DnaA forms homo-oligomers in a head-to-tail manner via interactions between the bound ATP and Arg-285 of the adjacent protomer. DnaA boxes R1 and R4 reside at the outer edges of the DnaA-binding region and have opposite orientations. In this study, roles for the protomers bound at R1 and R4 were elucidated using chimeric DnaA molecules that had alternative DNA binding sequence specificity and chimeric oriC molecules bearing the alternative DnaA binding sequence at R1 or R4. In vitro, protomers at R1 and R4 promoted initiation regardless of whether the bound nucleotide was ADP or ATP. Arg-285 was shown to play an important role in the formation of subcomplexes that were active in oriC unwinding and DnaB loading. The results of in vivo analysis using the chimeric molecules were consistent with the in vitro data. Taken together, the data suggest a model in which DnaA subcomplexes form in symmetrically opposed orientations and in which the Arg-285 fingers face inward to mediate interactions with adjacent protomers. This mode is consistent with initiation regulation by ATP-DnaA and bidirectional loading of DnaB helicases.     

The initiator protein DnaA contributes to keeping new origins inactivated by promoting the presence of hemimethylated DNA

The Escherichia coli replication origin oriC and other regions with high numbers of GATC sites remain hemimethylated after replication much longer than regions with average numbers of GATC sites. The prolonged period of hemimethylation has been attributed to the presence of bound SeqA protein. Here, it was found that a GATC cluster inserted at the datA site, which binds large amounts of DnaA in vivo, did not become remethylated at all, unless the availability of the DnaA protein was severely reduced. Sequestration of oriC was also found to be affected by the availability of DnaA. The period of origin hemimethylation was reduced by approximately 30% upon a reduction in the availability of DnaA. The result shows that not only SeqA binding but also DnaA binding to newly replicated origins contributes to keeping them hemimethylated. It was also found that the number of SeqA foci increased in cells with a combination of DnaA-mediated protection and sequestration at the GATC::datA cluster.     

A robust screen for novel antibiotics: specific knockout of the initiator of bacterial DNA replication

We have developed a novel type of a positive screen for the discovery of antibacterial compounds that target the Escherichia coli replication initiator protein DnaA. DnaA is an essential replication protein, conserved in (almost) all bacteria--including all human pathogens--and no existing antibiotics target the main components of the DNA replication machinery. This makes DnaA an attractive target and compounds discovered by this screen will constitute a new group of antibiotics. The conditional mutant, dnaA219, has a cold sensitive phenotype due to overreplication. In the screen, a DnaA inhibitor will reduce DnaA overactivity and thus restore growth at the nonpermissive temperature. This positive type of selection utilizes the rare phenomenon of lethal overactivity. In addition, the mutant strain has been made independent of DnaA activity by introduction of an alternative initiation pathway that allows growth under conditions of complete knockdown of DnaA. The resulting dnaA219rnhA strain is the basis of a robust, cell-based assay amenable to high-throughput screening. The screening assay has been validated against (1) a library of microbial fermentation extracts and (2) a known intracellular DnaA inhibitor.     

DNA synthesis by Escherichia coli B/r/l synchronized and grown under conditions of slow growth

Escherichia coli B/r/l was synchronized by a novel method and its growth was followed in a minimal salts medium containing glucose, acetate, aspartate or succinate as the sole carbon source. Thymine incorporation experiments showed agreement with the Cooper-Helmstetter model for DNA synthesis, during the division cycle, both in glucose grown culture with a doubling time 57.5 min and in acetate, aspartate and succinate where the doubling time was extended up to 90 min. The ratio C/C+D was identical or close to that predicted by the model. Prolonged growth of the synchronized cultures prior to each experiment was practised in order to ensure their physiological state without causing any considerable deterioration of synchrony.     

Insights into the quality of DnaA boxes and their cooperativity

Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replication-inactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperativity between the DnaA boxes, and to study in vivo the in vitro-defined 9mer DnaA box consensus sequence (TT(A)/(T)TNCACA). The quality and cooperativity of the DnaA boxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box in the promoter-distal position, whereas a promoter-proximal DnaA box with the sequence TTATCCACA titrated DnaA protein and caused significant repression of the mioC promoter without a promoter-distal DnaA box. The quality of the eight different consensus DnaA boxes located in the promoter-proximal position was determined: TTATCCACA had the highest affinity for DnaA protein. In the third position, A was better than T, and the four possibilities in the fifth position could be ranked as C >A >or=G >T. Parallel in vitro experiments using a purified DNA-binding domain of DnaA protein gave the same ranking of the binding affinities of the eight DnaA boxes.     

Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli

Summary Increased synthesis of DnaA protein, obtained with plasmids carrying the dnaA gene controlled by the heat inducible pL promoter, stimulated initiation of replication from oriC about threefold. The overinitiation was determined both as an increase in copy number of a minichromosome and as an increase in chromosomal gene dosage of oriC proximal DNA. The additional replication forks which were initiated on the chromosome did not lead to an overall increase in DNA content. DNA/DNA hybridization showed an amplification encompassing less than a few hundred kilobases on each side of oriC. Kinetic studies showed that the overinitiation occurred very rapidly after the induction, and that the initiation frequency then decreased to a near normal frequency per oriC. The results indicate that the DnaA protein is one important factor in regulation of initiation of DNA replication from oriC.     

Structural and thermodynamic signatures of DNA recognition by Mycobacterium tuberculosis DnaA

An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 Å resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 Å). These structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.     

Host controlled plasmid replication: Escherichia coli minichromosomes

Escherichia coli minichromosomes are plasmids replicating exclusively from a cloned copy of oriC, the chromosomal origin of replication. They are therefore subject to the same types of replication control as imposed on the chromosome. Unlike natural plasmid replicons, minichromosomes do not adjust their replication rate to the cellular copy number and they do not contain information for active partitioning at cell division. Analysis of mutant strains where minichromosomes cannot be established suggest that their mere existence is dependent on the factors that ensure timely once per cell cycle initiation of replication. These observations indicate that replication initiation in E. coli is normally controlled in such a way that all copies of oriC contained within the cell, chromosomal and minichromosomal, are initiated within a fairly short time interval of the cell cycle. Furthermore, both replication and segregation of the bacterial chromosome seem to be controlled by sequences outside the origin itself.     

Initiation of chromosomal DNA replication which is stimulated without oversupply of DnaA protein in Escherichia coli

Summary The temperature-sensitive dnaA46 mutation in Escherichia coli can be phenotypically suppressed at 42° C by oversupply of GroELS proteins, and the suppressed cells grow extremely slowly at 30° C. We found that the phenotype of dnaA46 showing this cold sensitivity was dominant over the phenotype of dnaA ⁺, and could not be rescued by introduction of oriC-independent replication systems. These results suggest that the cold sensitivity was not caused by a simple defect in replication. When a growing culture of a dnaA46 strain with a GroELS-overproducing plasmid was shifted from 42° to 30° C in the presence of chloramphenicol, the chromosomal DNA replicated excessively. Initiation of replication occurred at the site of oriC repeatedly four or five times during a 4 h incubation period without concomitant protein synthesis, indicating an excessive capacity for initiation. Such overreplication did not take place at 42° C in the suppressed dnaA46 strain, or at either temperature in GroELS-oversupplied dnaA ⁺ cells. No significant difference was detected between the cellular content of DnaA protein in suppressed cells where the initiation capacity was abnormally high, and that in wild-type cells in which the initiation capacity was normal. Thus, DnaA protein might function in vivo through some phase control mechanism for initiation, apart from a simple regulation by its total amount. A possible mechanism is proposed based on the participation of GroELS proteins in protein folding.A preliminary account of this work was presented at the Annual Meeting of the Molecular Biology Society of Japan in 1989.