Overinitiation of chromosome replication in the Escherichia coli dnaAcos mutant depends on activation of oriC function by the dam gene product

The activity of DnaA protein, the initiator of chromosome replication in Escherichia coli , is regulated by adenine nucleotide binding; the ATP-bound form, not the ADP-bound form, is active. DnaAcos is a mutant protein that is insensitive to negative regulation by ADP. Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30°C, a restrictive temperature for growth. To determine the control factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn 5 insertion mutagenesis. Three of the suppressors carried Tn 5 in the aroK or aroB gene, the first two cistrons in the dam operon. Complementation tests revealed that the dam gene is responsible for the suppression. Over-replication of the chromosome was inhibited in the dnaAcos aroK ::Tn 5 double mutant, and initiation of chromosome replication in the dnaA ⁺ aroK ::Tn 5 mutant was partially inhibited. The aroK  (or B  )::Tn 5 cells contained DnaA molecules at a level similar to that in the parental aroBK  ⁺ strain. Moreover, dnaAcos suppression depended on the function of the seqA gene. Thus, Dam activity positively regulates initiation of chromosome replication in vivo . SeqA function seems to be distinguished from the control of DnaA protein by adenine nucleotide binding.     

The rcbA gene product reduces spontaneous and induced chromosome breaks in Escherichia coli

Elevated levels of DnaA cause excessive initiation, which leads to an increased level of double-strand breaks that are proposed to arise when newly formed replication forks collide from behind with stalled or collapsed forks. These double-strand breaks are toxic in mutants that are unable to repair them. Using a multicopy suppressor assay to identify genes that suppress this toxicity, we isolated a plasmid carrying a gene whose function had been unknown. This gene, carried by the cryptic rac prophage, has been named rcbA for its ability to reduce the frequency of chromosome breaks. Our study shows that the colony formation of strains bearing mutations in rep, recG, and rcbA, like recA and recB mutants, is inhibited by an oversupply of DnaA and that a multicopy plasmid carrying rcbA neutralizes this inhibition. These and other results suggest that rcbA helps to maintain the integrity of the bacterial chromosome by lowering the steady-state level of double-strand breaks.     

Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability

Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell.     

Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins

Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30°C. Whereas purified wild-type DnaA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen with the wild-type protein. In a system reconstituted with purified proteins at 30°C, the mutant protein initiated replication of single-stranded DNA that contains a DnaA-binding hairpin structure. Thus, DnaAcos protein basically sustains affinity to a DnaA-binding sequence and functions in the loading of DnaB helicase onto single-stranded DNA. Thermal stabilities of wild-type DnaA and DnaAcos activities were comparable. Unlike wild-type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP-bound form of DnaA protein is required for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.     

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.     

Suppressors of DnaA(ATP) imposed overinitiation in Escherichia coli

Chromosome replication in Escherichia coli is limited by the supply of DnaA associated with ATP. Cells deficient in RIDA (Regulatory Inactivation of DnaA) due to a deletion of the hda gene accumulate suppressor mutations (hsm) to counteract the overinitiation caused by an elevated DnaA(ATP) level. Eight spontaneous hda suppressor mutations were identified by whole-genome sequencing, and three of these were analysed further. Two mutations (hsm-2 and hsm-4) mapped in the dnaA gene and led to a reduced ability to initiate replication from oriC. One mutation (hsm-1) mapped to the seqA promoter and increased the SeqA protein level in the cell. hsm-1 cells had prolonged origin sequestration, reduced DnaA protein level and reduced DnaA-Reactivating Sequence (DARS)-mediated rejuvenation of DnaA(ADP) to DnaA(ATP) , all of which could contribute to the suppression of RIDA deficiency. Despite of these defects hsm-1 cells were quite similar to wild type with respect to cell cycle parameters. We speculate that since SeqA binding sites might overlap with DnaA binding sites spread throughout the chromosome, excess SeqA could interfere with DnaA titration and thereby increase free DnaA level. Thus, in spite of reduction in total DnaA, the amount of DnaA molecules available for initiation may not be reduced.     

Mutant DnaAs of Escherichia coli that are refractory to negative control

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.     

Escherichia coli cells with increased levels of DnaA and deficient in recombinational repair have decreased viability

The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, beta clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, beta clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the beta clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 micro m) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional approximately 100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.     

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.     

Regulation of chromosomal replication by DnaA protein availability in Escherichia coli: effects of the datA region.

Initiation of chromosomal replication in Escherichia coli is dependent on availability of the initiator protein DnaA. We have introduced into E. coli cells plasmids carrying the chromosomal locus datA, which has a high affinity for DnaA. To be able to monitor oriC initiation as a function of datA copy number, we introduced a minichromosome which only replicates from oriC, using a host cell which replicates its chromosome independently of oriC. Our data show that a moderate increase in datA copy number is accompanied by increased DnaA protein synthesis that allows oriC initiation to occur normally, as measured by minichromosome copy number. As datA gene dosage is increased dnaA expression cannot be further derepressed, and the minichromosome copy number is dramatically reduced. Under these conditions the minichromosome was maintained by integration into the chromosome. These findings suggest that the datA locus plays a significant role in regulating oriC initiation, by its capacity to bind DnaA. They also suggest that auto regulation of the dnaA gene is of minor importance in regulation of chromosome initiation.     

SeqA limits DnaA activity in replication from oriC in Escherichia coli

A mutant Escherichia coli that transforms minichromosomes with high efficiency in the absence of Dam methylation has been Isolated and the mutation mapped to 16.25 min on the E. coli map. The mutant strain containing seqA2 is defective for growth in rich medium but not in minimal medium. A similar mutation In this gene, named seqA1, has also been isolated. Here we show that the product of the seqA gene, SeqA, normally acts as an inhibitor of chromosomal initiation. In the seqA2-containing mutant, the frequency of initiation increases by a factor of three. Introduction of the wild-type seqA gene on a low-copy plasmid suppresses the cold sensitivity of a dnaAcos mutant known to overinitiate at temperatures below 39°C. In addition, the seqA2 mutation is a suppressor of several dnaA (Ts) alleles. The seqA2 mutant overinitiates replication from oriC and displays the asynchronous initiation phenotype. Also the seqA2 mutant has an elevated level of DnaA protein (twofold). The introduction of minichromosomes or a low-copy-number plasmid carrying five DnaA-boxes from the oriC region increases the growth rate of the seqA2 mutant in rich medium to the wild-type level, reduces overinitiation but does not restore synchrony. We propose that the role of SeqA is to limit the activity level of the E. coli regulator of chromosome initiation, DnaA.     

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.     

The G157C mutation in the Escherichia coli sliding clamp specifically affects initiation of replication

Escherichia coli cells with a point mutation in the dnaN gene causing the amino acid change Gly157 to Cys, were found to underinitiate replication and grow with a reduced origin and DNA concentration. The mutant β clamp also caused excessive conversion of ATP-DnaA to ADP-DnaA. The DnaA protein was, however, not the element limiting initiation of replication. Overproduction of DnaA protein, which in wild-type cells leads to over-replication, had no effect in the dnaN(G157C) mutant. Origins already opened by DnaA seemed to remain open for a prolonged period, with a stage of initiation involving β clamp loading, presumably limiting the initiation process. The existence of opened origins led to a moderate SOS response. Lagging strand synthesis, which also requires loading of the β clamp, was apparently unaffected. The result indicates that some aspects of β clamp activity are specific to the origin. It is possible that the origin specific activities of β contribute to regulation of initiation frequency.     

The role of replication initiation control in promoting survival of replication fork damage

Dam methylase mutants were recovered in a screen for mutants sensitive to UV irradiation or mild inhibition of replication elongation. Dam's role in tolerance of DNA damage is to provide binding sites for SeqA, because seqA mutants showed similar sensitivity that was genetically epistatic to dam. The sensitivity of seqA mutants to UV irradiation and to the replication inhibitors hydroxyurea (HU) and azidothymidine (AZT) was suppressed by alleles of dnaA that reduce the efficiency of replication initiation. These results suggest that for survival of replication fork damage, SeqA's repression of replication initiation is more important than its effects on nucleoid organization. Convergence of forks upon DNA damage is a likely explanation for seqA mutant sensitivity, because its poor survival of UV was suppressed by reducing secondary initiation through minimal medium growth. Surprisingly, growth in minimal medium reduced the ability of seqA+ strains to form colonies in the presence of low levels of AZT. Double dnaA seqA mutants exhibited plating efficiencies much superior to wild-type strains during chronic low-level AZT exposure in minimal medium. This suggests that mild inhibition of replication fork progression may actively restrain initiation such that seqA+ strains fail to recover initiation capacity after sustained conditions of replication arrest.     

Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.     

Association of the chromosome replication initiator DnaA with the Escherichia coli inner membrane in vivo: quantity and mode of binding

DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein's domain III, rather than confined to a particular sequence. We propose a new binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication.     

Illegitimate Recombination Induced by Overproduction of DnaB Helicase in Escherichia coli

Illegitimate recombination that usually takes place at a low frequency is greatly enhanced by treatment with DNA-damaging agents. It is thought that DNA double-strand breaks induced by this DNA damage are important for initiation of illegitimate recombination. Here we show that illegitimate recombination is enhanced by overexpression of the DnaB protein in Escherichia coli. The recombination enhanced by DnaB overexpression occurred between short regions of homology. We propose a model for the initiation of illegitimate recombination in which DnaB overexpression may excessively unwind DNA at replication forks and induce double-strand breaks, resulting in illegitimate recombination. The defect in RecQ has a synergistic effect on the increased illegitimate recombination in cells containing the overproduced DnaB protein, implying that DnaB works in the same pathway as RecQ does but that they work at different steps.     

Oxidative DNA damage is instrumental in hyperreplication stress-induced inviability of Escherichia coli

In Escherichia coli, an increase in the ATP bound form of the DnaA initiator protein results in hyperinitiation and inviability. Here, we show that such replication stress is tolerated during anaerobic growth. In hyperinitiating cells, a shift from anaerobic to aerobic growth resulted in appearance of fragmented chromosomes and a decrease in terminus concentration, leading to a dramatic increase in ori/ter ratio and cessation of cell growth. Aerobic viability was restored by reducing the level of reactive oxygen species (ROS) or by deleting mutM (Fpg glycosylase). The double-strand breaks observed in hyperinitiating cells therefore results from replication forks encountering single-stranded DNA lesions generated while removing oxidized bases, primarily 8-oxoG, from the DNA. We conclude that there is a delicate balance between chromosome replication and ROS inflicted DNA damage so the number of replication forks can only increase when ROS formation is reduced or when the pertinent repair is compromised.