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Expression of the Escherichia coli dnaN-encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess β clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we obtained eight mutant clamps with an inability to impede growth and measured their ability to stimulate Pol III replication in vitro. Compared with the wild-type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the βE202K mutant that bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαeθ), suggesting that it could still sequester Pol III effectively. Of interest, βE202K supported in vitro DNA replication by Pol II and Pol IV but was defective with Pol III. Genetic experiments indicated that the dnaNE202K strain remained proficient in DNA damage-induced mutagenesis but was induced modestly for SOS and displayed sensitivity to UV light and methyl methanesulfonate. These results correlate an impaired ability of the mutant βE202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for the existence of an additional mechanism that contributes to lethality, and (iii) suggest that physical and functional interactions of the β clamp with Pol III are more extensive than appreciated currently. IMPORTANCE The β clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the β clamp impedes Escherichia coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant β clamps that fail to inhibit growth. Their analysis revealed that βE202K is unique among them. Our work offers new insights into how the β clamp interacts with and manages the actions of E. coli DNA polymerases II, III, and IV.

Expression of the Escherichia coli dnaN-encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. A mutant clamp (β(E202K) bearing a glutamic acid-to-lysine substitution at residue 202) binds to DNA polymerase III (Pol III) with higher affinity than the wild-type clamp, suggesting that its failure to impede growth is independent of its ability to sequester Pol III away from the replication fork. Our results demonstrate that the dnaN(E202K) strain underinitiates DNA replication due to insufficient levels of DnaA-ATP and expresses several DnaA-regulated genes at altered levels, including nrdAB, that encode the class 1a ribonucleotide reductase (RNR). Elevated expression of nrdAB was dependent on hda function. As the β clamp-Hda complex regulates the activity of DnaA by stimulating its intrinsic ATPase activity, this finding suggests that the dnaN(E202K) allele supports an elevated level of Hda activity in vivo compared with the wild-type strain. In contrast, using an in vitro assay reconstituted with purified components the β(E202K) and wild-type clamp proteins supported comparable levels of Hda activity. Nevertheless, co-overexpression of the nrdAB-encoded RNR relieved the growth defect caused by elevated levels of the β clamp. These results support a model in which increased cellular levels of DNA precursors relieve the ability of elevated β clamp levels to impede growth and suggest either that multiple effects stemming from the dnaN(E202K) mutation contribute to elevated nrdAB levels or that Hda plays a noncatalytic role in regulating DnaA-ATP by sequestering it to reduce its availability. IMPORTANCE DnaA bound to ATP acts in initiation of DNA replication and regulates the expression of several genes whose products act in DNA metabolism. The state of the ATP bound to DnaA is regulated in part by the β clamp-Hda complex. The dnaN(E202K) allele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While the dnaN(E202K) strain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and β(E202K) clamp proteins support equivalent levels of Hda activity in vitro. Taken together, these results suggest that β(E202K)-Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed.

Expression of the Escherichia coli dnaN-encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess β clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we obtained eight mutant clamps with an inability to impede growth and measured their ability to stimulate Pol III replication in vitro. Compared with the wild-type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the β(E202K) mutant that bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still sequester Pol III effectively. Of interest, β(E202K) supported in vitro DNA replication by Pol II and Pol IV but was defective with Pol III. Genetic experiments indicated that the dnaN(E202K) strain remained proficient in DNA damage-induced mutagenesis but was induced modestly for SOS and displayed sensitivity to UV light and methyl methanesulfonate. These results correlate an impaired ability of the mutant β(E202K) clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for the existence of an additional mechanism that contributes to lethality, and (iii) suggest that physical and functional interactions of the β clamp with Pol III are more extensive than appreciated currently. IMPORTANCE The β clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the β clamp impedes Escherichia coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant β clamps that fail to inhibit growth. Their analysis revealed that β(E202K) is unique among them. Our work offers new insights into how the β clamp interacts with and manages the actions of E. coli DNA polymerases II, III, and IV.

Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda–sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda–β clamp complex. This complex contains two pairs of Hda dimers sandwiched between two β clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the β clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda–β clamp complex indicate that the interaction of the β clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda–β clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function.

The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature.

Helicase loading at a DNA replication origin often requires the dynamic interactions between the DNA helicase and an accessory protein. In E. coli, the DNA helicase is DnaB and DnaC is its loading partner. We used the method of hydrogen/deuterium exchange mass spectrometry to address the importance of DnaB–DnaC complex formation as a prerequisite for helicase loading. Our results show that the DnaB ring opens and closes, and that specific amino acids near the N-terminus of DnaC interact with a site in DnaB's C-terminal domain to trap it as an open ring. This event correlates with conformational changes of the RecA fold of DnaB that is involved in nucleotide binding, and of the AAA+ domain of DnaC. DnaC also causes an alteration of the helical hairpins in the N-terminal domain of DnaB, presumably occluding this region from interacting with primase. Hence, DnaC controls the access of DnaB by primase.

Former studies relying on hydrogen/deuterium exchange analysis suggest that DnaC bound to DnaB alters the conformation of the N-terminal domain (NTD) of DnaB to impair the ability of this DNA helicase to interact with primase. Supporting this idea, the work described herein based on biosensor experiments and enzyme-linked immunosorbent assays shows that the DnaB-DnaC complex binds poorly to primase in comparison with DnaB alone. Using a structural model of DnaB complexed with the C-terminal domain of primase, we found that Ile-85 is located at the interface in the NTD of DnaB that contacts primase. An alanine substitution for Ile-85 specifically interfered with this interaction and impeded DnaB function in DNA replication, but not its activity as a DNA helicase or its ability to bind to ssDNA. By comparison, substitutions of Asn for Ile-136 (I136N) and Thr for Ile-142 (I142T) in a subdomain previously named the helical hairpin in the NTD of DnaB altered the conformation of the helical hairpin and/or compromised its pairwise arrangement with the companion subdomain in each brace of protomers of the DnaB hexamer. In contrast with the I85A mutant, the latter were defective in DNA replication due to impaired binding to both ssDNA and primase. In view of these findings, we propose that DnaC controls the ability of DnaB to interact with primase by modifying the conformation of the NTD of DnaB.

The initiation of chromosomal DNA replication starts at a replication origin, which in bacteria is a discrete locus that contains DNA sequence motifs recognized by an initiator protein whose role is to assemble the replication fork machinery at this site. In bacteria with a single chromosome, DnaA is the initiator and is highly conserved in all bacteria. As an adenine nucleotide binding protein, DnaA bound to ATP is active in the assembly of a DnaA oligomer onto these sites. Other proteins modulate DnaA oligomerization via their interaction with the N-terminal region of DnaA. Following the DnaA-dependent unwinding of an AT-rich region within the replication origin, DnaA then mediates the binding of DnaB, the replicative DNA helicase, in a complex with DnaC to form an intermediate named the prepriming complex. In the formation of this intermediate, the helicase is loaded onto the unwound region within the replication origin. As DnaC bound to DnaB inhibits its activity as a DNA helicase, DnaC must dissociate to activate DnaB. Apparently, the interaction of DnaB with primase (DnaG) and primer formation leads to the release of DnaC from DnaB, which is coordinated with or followed by translocation of DnaB to the junction of the replication fork. There, DnaB is able to coordinate its activity as a DNA helicase with the cellular replicase, DNA polymerase III holoenzyme, which uses the primers made by primase for leading strand DNA synthesis.

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.

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.

An AAA+ ATPase, DnaC, delivers DnaB helicase at the E. coli chromosomal origin by a poorly understood process. This report shows that mutant proteins bearing alanine substitutions for two conserved arginines in a motif named box VII are defective in DNA replication, but this deficiency does not arise from impaired interactions with ATP, DnaB, or single-stranded DNA. Despite their ability to deliver DnaB to the chromosomal origin to form the prepriming complex, this intermediate is inactive. Quantitative analysis of the prepriming complex suggests that the DnaB-DnaC complex contains three DnaC monomers per DnaB hexamer and that the interaction of primase with DnaB and primer formation triggers the release of DnaC, but not the mutants, from DnaB. The interaction of primase with DnaB and the release of DnaC mark discrete events in the transition from initiation to the elongation stage of DNA replication.

Summary Mutants of dnaAcos are inviable at 30°C because DnaAcos hyperinitiates, leading to new replication forks that apparently collide from behind with stalled forks, thereby generating lethal double-strand breaks. By comparison, an elevated level of DnaA also induces extra initiations, but lethality occurs only in strains defective in repairing double-strand breaks. To explore the model that the chromosomal level of DnaAcos, or the increased abundance of DnaA, increases initiation frequency by, escaping or overcoming pathways that control initiation, respectively, we developed a genetic selection and identified seqA, datA, dnaN and hda, which function in pathways that either act at oriC or modulate DnaA activity. To assess each pathway's relative effectiveness, we used genetically inactivated strains, and quantified initiation frequency after elevating the level of DnaA. The results indicate that the hda-dependent pathway has a stronger effect on initiation than pathways involving seqA and datA. Testing the model that DnaAcos overinitiates because it fails to respond to one or more regulatory mechanisms, we show that dnaAcos is unresponsive to hda and dnaN, which encodes the β clamp, and also datA, a locus proposed to titer excess DnaA. These results explain how DnaAcos hyperinitiates to interfere with viability.

During exponential growth, the level of Dps transiently increases in response to oxidative stress to sequester and oxidize Fe2+, which would otherwise lead to hydroxyl radicals that damage the bacterial chromosome. We report that Dps specifically interacts with DnaA protein by affinity chromatography and a solid phase binding assay, requiring the N-terminal region of DnaA to interact. In vitro, Dps inhibits DnaA function in initiation by interfering with strand opening of the replication origin. Comparing isogenic dps+ and dps::kan strains by flow cytometry and by quantitative polymerase chain reaction assays at either the chromosomally encoded level, or at an elevated level encoded by an inducible plasmid, we show that Dps causes less frequent initiations. Results from genetic experiments support this conclusion. We suggest that Dps acts as a checkpoint during oxidative stress to reduce initiations, providing an opportunity for mechanisms to repair oxidative DNA damage. Because Dps does not block initiations absolutely, duplication of the damaged DNA is expected to increase the genetic variation of a population, and the probability that genetic adaptation leads to survival under conditions of oxidative stress.

Summary Escherichia coli HU protein is a dimer encoded by two closely related genes whose expression is growth phase-dependent. As a major component of the bac- terial nucleoid, HU binds to DNA non-specifically, but acts at the chromosomal origin (oriC) during initiation by stimulating strand opening in vitro. We show that the a dimer of HU is more active than other forms of HU in initiation of an oriC-containing plasmid because it more effectively promotes strand opening of oriC. Other results demonstrate that HU stabilizes the DnaA oligomer bound to oriC, and that the a subunit of HU interacts with the N-terminal region of DnaA. These observations support a model whereby DnaA interacts with the a dimer or the ab heterodimer, depending on their cellular abundance, to recruit the respective form of HU to oriC. The greater activity of the a dimer of HU at oriC may stimulate initiation during early log phase compared with the lesser activity of the ab heterodimer or the b dimer.

In the initiation of bacterial DNA replication, DnaA protein recruits DnaB helicase to the chromosomal origin, oriC, leading to the assemble of the replication fork machinery at this site. Because a region near the N terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC, we asked if these functions are separable or interdependent by substituting many conserved amino acids in this region with alanine to identify essential residues. We show that alanine substitutions of leucine 3, phenylalanine 46, and leucine 62 do not affect DnaA function in initiation. In contrast, we find on characterization of a mutant DnaA that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogates self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase.

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.

Summary 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 molecular engine that drives bidirectional replication fork movement from the Escherichia coli replication origin (oriC) is the replicative helicase, DnaB. At oriC, two and only two helicase molecules are loaded, one for each replication fork. DnaA participates in helicase loading; DnaC is also involved, because it must be in a complex with DnaB for delivery of the helicase. Since DnaA induces a local unwinding of oriC, one model is that the limited availability of single-stranded DNA at oriC restricts the number of DnaB molecules that can bind. In this report, we determined that one DnaB helicase or one DnaB-DnaC complex is bound to a single-stranded DNA in a biologically relevant DNA replication system. These results indicate that the availability of single-stranded DNA is not a limiting factor and support a model in which the site of entry for DnaB is altered so that it cannot be reused. We also show that 2-4 DnaA monomers are bound on the single-stranded DNA at a specific site that carries a DnaA box sequence in a hairpin structure.

Escherichia coli DnaC protein bound to ATP forms a complex with DnaB protein. To identify the domain of DnaC that interacts with DnaB, a genetic selection was used based on the lethal effect of induced dnaC expression and a model that inviability arises by the binding of DnaC to DnaB to inhibit replication fork movement. The analysis of dnaC alleles that preserved viability under elevated expression revealed an N-terminal domain of DnaC involved in binding to DnaB. Mutant proteins bearing single amino acid substitutions (R10P, L11Q, L29Q, S41P, W32G, and L44P) that reside in regions of predicted secondary structure were inert in DNA replication activity because of their inability to bind to DnaB, but they retained ATP binding activity, as indicated by UV cross-linking to [alpha-(32)P]ATP. These alleles also failed to complement a dnaC28 mutant. Other selected mutations that map to regions carrying Walker A and B boxes are expected to be defective in ATP binding, a required step in DnaB-DnaC complex formation. Lastly, we found that the sixth codon from the N terminus encodes aspartate, resolving a reported discrepancy between the predicted amino acid sequence based on DNA sequencing data and the results from N-terminal amino acid sequencing (Nakayama, N., Bond, M. W., Miyajima, A., Kobori, J., and Arai, K. (1987) J. Biol. Chem. 262, 10475-10480).

Initiation of DNA replication at the Escherichia coli chromosomal origin occurs through an ordered series of events that depends first on the binding of DnaA protein, the replication initiator, to DnaA box sequences followed by unwinding of an AT-rich region. A step that follows is the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We show that DnaA protein actively mediates the entry of DnaB at oriC. One region (amino acids 111-148) transiently binds to DnaB as determined by surface plasmon resonance. A second functional domain, possibly involving formation of a unique nucleoprotein structure, promotes the stable binding of DnaB during the initiation process and is inactivated in forming an intermediate termed the prepriming complex by removal of the N-terminal 62 residues. Based on similarities in the replication process between prokaryotes and eukaryotes, these results suggest that a similar mechanism may load the eukaryotic replicative helicase.

The Escherichia coli chromosomal origin contains several bindings sites for factor for inversion stimulation (FIS), a protein originally identified to be required for DNA inversion by the Hin and Gin recombinases. The primary FIS binding site is close to two central DnaA boxes that are bound by DnaA protein to initiate chromosomal replication. Because of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1, 10-phenanthroline-copper of complexes formed with FIS and DnaA protein that were separated by native gel electrophoresis. These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA boxes R2 and R3. Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a restriction fragment containing oriC . Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems. Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of the negative superhelicity of the oriC plasmid that is essential for the initiation process.

The Escherichia coli DnaA protein is a sequence-specific DNA binding protein that promotes the initiation of replication of the bacterial chromosome, and of several plasmids including pSC101. Twenty-eight novel missense mutations of the E. coli dnaA gene were isolated by selecting for their inability to replicate a derivative of pSC101 when contained in a lambda vector. Characterization of these as well as seven novel nonsense mutations and one in-frame deletion mutation are described here. Results suggest that E. coli DnaA protein contains four functional domains. Mutations that affect residues in the P-loop or Walker A motif thought to be involved in ATP binding identify one domain. The second domain maps to a region near the C terminus and is involved in DNA binding. The function of the third domain that maps near the N terminus is unknown but may be involved in the ability of DnaA protein to oligomerize. Two alleles encoding different truncated gene products retained the ability to promote replication from the pSC101 origin but not oriC, identifying a fourth domain dispensable for replication of pSC101 but essential for replication from the bacterial chromosomal origin, oriC.

The Escherichia coli dnaA gene is required for replication of the bacterial chromosome. To identify residues critical for its replication activity, a method to select novel mutations was developed that relied on lytic growth of λ from an inserted pSC101 replication origin. Replication from the λ origin was inhibited by lysogen-encoded c I repressor. Replication from the pSC101 origin that resulted in lytic growth was dependent on active DnaA protein encoded by a plasmid in a host strain lacking the chromosomal dnaA gene. With this approach, a large collection of missense, nonsense, and a few internal deletion mutations were obtained. Nucleotide sequence analysis of the missense mutations indicated that 28 of 50 were unique. Of these, one was identical to the dnaA205 allele whereas the remainder are novel. These missense mutations were clustered into three regions, suggesting three functional domains of DnaA protein required for its replication activity. Many of the missense mutations mapping to the C-terminal 61 residues were inactive for replication from the pSC101 origin. These are defective in DNA binding. Mutations that mapped elsewhere were temperature-sensitive.

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.

In free-living organisms, including Escherichia coli , DNA replication starts at replication origins. E. coli contains a single replication origin ( oriC ), which serves as the site for assembly of the replisome, a multi-protein molecular machine that duplicates the circular duplex genome. Replisome assembly is a highly regulated event that is strictly coordinated with the cell cycle. This article reviews the molecular mechanism of replisome assembly at the E. coli replication origin.

DnaA protein of Escherichia coli acts in initiation of chromosomal DNA replication by binding specific sequences, termed DnaA boxes in the chromosomal origin, oriC. On binding, it induces a localized unwinding to create a structure recognized by other replication proteins that act subsequently in the initiation process. In this report, we examined the binding of DnaA protein to each of the DnaA boxes in oriC. By gel mobility shift assays, DnaA protein formed at least six discrete complexes. ATP or ADP included in the reaction mixture prior to electrophoresis was required. Chemical cleavage of isolated complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in an ordered manner to the DnaA boxes in oriC. Preferential binding to one DnaA box (R4) was confirmed by demonstration that a DNA fragment containing it was bound with greater affinity than another DnaA box sequence (R1). In vitro replication activity correlated with a complex formed at a ratio of 30 DnaA monomers/oriC in which all DnaA boxes are occupied. The last site bound is DnaA box R3. This event may be critical in promoting initiation from oriC as it correlates with in vivo observations that binding of DnaA protein to box R3 occurs at the time of initiation of chromosomal replication, whereas other DnaA boxes are bound by DnaA protein throughout the cell cycle (Cassler, M. R., Grimwade, J. E., and Leonard, A. C. (1995) EMBO J. 14, 5833-5841).

The temperature-sensitive dnaA5 and dnaA46 alleles each contain two missense mutations. These mutations have been separated and the resulting mutant proteins studied with regard to their role in initiation of DNA replication in vitro. Whereas the His-252 to tyrosine substitution (H252Y) unique to the dnaA46 allele did not affect the activities of DnaA protein, the unique substitution of the dnaA5 allele, Gly-426 to serine (G426S), was reduced in its DNA-binding affinity for oriC, the chromosomal origin. This suggests that the C-terminal region of the DnaA protein is involved in DNA binding. The alanine-to-valine substitution at amino acid 184 (A184V) that is common to both of the alleles is responsible for the thermolabile defect and lag in DNA synthesis of these mutants. Mutant proteins bearing the common substitution were defective in ATP binding and were inactive in a replication system reconstituted with purified proteins. DnaK and GrpE protein activated these mutant proteins for replication and ATP binding; the latter was measured indirectly by the ATP-dependent formation of a trypsin-resistant peptide. However, with this assay, the ATP-binding affinity appeared to be reduced relative to wild-type DnaA protein. Activation was by conversion of a self-aggregate to the monomer, and also by a conformational alteration that correlated with ATP binding.

Five novel alleles of the Escherichia coli dnaA gene that were temperature sensitive in maintenance of pSC101, a plasmid that is dependent on this gene for replication, were isolated. Nucleotide sequence analysis revealed that four of the five alleles arose from single base substitutions, whereas the fifth contained three base substitutions, two of which were silent. Whereas all five alleles were temperature sensitive in vivo for pSC101 maintenance, genetic and biochemical characterization indicated that only two were defective in replication from the chromosomal origin, oriC. As previously characterized mutations are defective in replication for both pSC101 and oriC, the dnaA mutations specifically defective in pSC101 maintenance represent a novel class. We speculate that one or more of these pSC101-specific mutants are defective in interaction with pSC101 RepA protein, which is also required for initiation of plasmid DNA replication.

DnaA protein of Escherichia coli acts in the initiation of chromosomal replication to bind to sequences in the chromosomal origin. On binding, it promotes the assembly of other replication proteins that serve to prime DNA replication and assemble the replication apparatus for bidirectional replication fork movement. A collection of monoclonal antibodies to DnaA protein have been produced, one of which is described here, that interferes with the action of DnaA protein in promoting formation of a prepriming complex. On the analysis of this process, the antibody appears to interfere with the physical interaction between DnaA and DnaB protein in the DnaB.DnaC complex. Cross-linking studies confirm that DnaA and DnaB proteins interact directly. These results provide the first direct evidence that one of the roles of DnaA protein is to act as a site for binding of DnaB protein to the DNA and perhaps orients DnaB helicase to account for the directionality of replication fork movement.

DnaK protein is involved in the initiation of DNA synthesis from the Escherichia coli chromosomes as well as from the replication origins of phage λ and P1. The synthesis of dnaK mRNA and protein has been reported to vary during the cell cycle of Caulobacter crescentus (Gomes et al. , 1990). We have measured the expression of DnaK protein during the E. coli division cycle using the membrane-elution method. Cells labelled with a radioactive amino acid at different times during the division cycle were analysed for radiolabelled DnaK protein by quantitative immunoprecipitation, gel electrophoresis and autoradiography. In contrast to reports of cell-cycle-specific synthesis of DnaK protein in C. crescentus , we find the synthesis of DnaK protein to be invariant during the E. coli division cycle. Its synthesis occurs exponentially, as does the synthesis of total cell protein.

Bioinformatics has its origins in the development of DNA sequencing methods by Alan Maxam and Walter Gilbert (Maxam and Gilbert, 1977), and by Frederick Sanger and coworkers (Sanger et al., 1977). By entirely different approaches, the first genomes determined at the nucleotide sequence level were that of bacteriophage φX174, and the recombinant plasmid named pBR322 composed of about 5,400 (Sanger et al., 1977), or 4,400 base pairs (Sutcliffe, 1979), respectively. In contrast, two articles that appeared in February 2001 reported on the preliminary DNA sequence of the human genome, which corresponds to 3 billion nucleotides of DNA sequence information (Lander et al., 2001; Venter et al., 2001). Only two years later, the GenBank sequence database contained more than 29.3 billion nucleotide bases in greater than 23 million sequences. With the development of new technologies, experts predict that the cost to sequence an individual’s DNA will be about $1000. This reduction in cost suggests that efforts in the area of comparative genomics will increase substantially, leading to an enormous database that vastly exceeds the existing one. By way of comparative genomics approaches, computational methods have led to the identification of homologous genes shared among species, and their classification into superfamilies based on amino acid sequence similarity. In combination with their evolutionary relatedness, superfamily members have been clustered into clades. In addition, high throughput sequencing of small RNAs and bioinformatics analyses have contributed to the identification of regions between genes that can code small RNAs (siRNA, microRNA, and long noncoding RNA), which act during the development of an organism to modulate gene expression at the post-transcriptional level (Fire et al., 1998; Hamilton and Baulcombe, 1999) reviewed in Elbashir et al., 2001; Ghildiyal and Zamore, 2009; Christensen et al., 2010). An emerging area is functional genomics whereby gene function is deduced using largescale methods by identifying the involvement of specific genes in metabolic pathways. More recently, phenotype microarray methods have been used to correlate the functions of genes of microbes with cell phenotypes under a variety of growth conditions (Bochner, 2009). These methods contrast with the traditional approach of mapping a gene via the phenotype of a mutation, and deducing the function of the gene product based on its biochemical analysis in concert with physiological studies. Such studies have been performed to confirm the functional importance of conserved residues shared by superfamily members, and also

To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to the elongation stage of DNA replication. Freed from DnaC, DnaB helicase unwinds the parental duplex DNA while interacting the cellular replicase, DNA polymerase III holoenzyme, and primase as it intermittently forms primers that are extended by the replicase in duplicating the chromosome.

Replication of P1 plasmid requires both the plasmid-specific initiator, RepA, and the host initiator, DnaA. Here we show that DnaA can make the P1 origin reactive to the single-strand specific reagents KMnO4 and mung bean nuclease. Addition of RepA further increased the KMnO4 reactivity of the origin, although RepA alone did not influence the reaction. The increased reactivity implies that the two initiators interact in some way to alter the origin conformation. The KMnO4 reactivity was restricted to one strand of the origin. We suggest that the roles of DnaA in P1 plasmid and bacterial replication are similar: origin opening and loading of the DnaB helicase. The strand-bias in chemical reactivity at the P1 origin most likely indicates that only one of the strands is used for the loading of DnaB, a scenario consistent with the unidirectional replication of the plasmid.

Mutant forms of DnaA protein, inert in a replication system composed of other purified proteins, are "activated" by DnaK and GrpE heat shock proteins (Hupp, T. R., and Kaguni, J. M. (1993) J. Biol. Chem. 268, 13137-13142). The effect of these heat shock proteins on DnaA5 and DnaA46 protein was separated from the event of DNA synthesis by incubation in two stages. Components necessary during the first stage for "activation" included GrpE and DnaK proteins, ATP at 0.2 mM or greater, and polyvinyl alcohol (8%) or glycerol, optimal at concentrations between 20 and 30%. An ATP regenerating system provided by creatine kinase and creatine phosphate was stimulatory. Addition of the activated form of DnaA5 or DnaA46 protein to a reconstituted system containing other purified replication proteins during the second stage of incubation resulted in DNA replication. Activation of DnaA5 or DnaA46 protein by heat shock proteins was thermolabile, suggesting that the temperature sensitivity of dnaA5 and dnaA46 mutants is related to this thermolabile interaction. A third heat shock protein, DnaJ protein, interfered with the activation of DnaA5 protein if present during the first stage of incubation. This inhibitory effect was less striking if included during the second stage of incubation. These results suggest a mechanism for regulation of the activity of DnaA protein.

A mutant form of DnaA protein encoded by the dnaA5 allele has been purified and compared biochemically to its wild type counterpart. Biochemical properties of DnaA5 protein include: 1) thermolabile activity in an oriC plasmid replication system dependent on a crude enzyme fraction, 2) comparable affinity relative to DnaA protein in binding to restriction fragments containing either the Escherichia coli chromosomal origin, oriC, or the dnaA promoter, 3) formation of a nucleoprotein complex similar to DnaA protein at the DnaA boxes within the dnaA promoter as detected by protection from DNase I cleavage, 4) formation of an altered nucleoprotein complex with oriC as judged by DNase I protection experiments, 5) inactivity in unwinding of oriC, 6) inactivity in oriC plasmid replication systems dependent on purified enzymes, and 7) inhibition of DnaA protein by addition of DnaA5 protein in assays of replication and of unwinding of oriC, suggesting that mixed complexes formed between wild type and mutant proteins are inactive.

DnaA5 protein, inactive in replication systems dependent on purified enzymes, was activated by addition of a crude enzyme fraction. Based on this assay, two proteins in the crude enzyme fraction were identified that confer replication activity upon DnaA5 protein in a purified enzyme system. That one is DnaK protein was suggested initially from studies in which DnaK protein stimulated another mutant form of DnaA protein in replication assays dependent on a crude enzyme fraction (Hwang, D. S., and Kaguni, J. M. (1991) J. Biol. Chem. 266, 7537-7555). The identification of DnaK protein as a required protein for activation of DnaA5 protein, and its inclusion in a reconstituted enzyme system of purified replication proteins, allowed for the partial purification of the second activating protein. GrpE protein was deduced to be the second activating protein based on three criteria. First, activity in partially purified fractions correlated with a protein similar in size to GrpE protein on a sodium dodecyl sulfate-polyacrylamide gel. Second, activity in partially purified fractions correlated with a protein that reacted with anti-GrpE antibody. Third, purified GrpE protein functionally replaced the second protein in activation of DnaA5 protein.

dnaB protein of Escherichia coli is an essential replication protein. A missense mutant has been obtained which results in replacement of an arginine residue with cysteine at position 231 of the protein (P. Shrimankar, L. Shortle, and R. Maurer, unpublished data). This mutant displays a dominant-lethal phenotype in strains that are heterodiploid for dnaB. Biochemical analysis of the altered form of dnaB protein revealed that it was inactive in replication in several purified enzyme systems which involve specific and nonspecific primer formation on single-stranded DNAs, and in replication of plasmids containing the E. coli chromosomal origin. Inactivity in replication appeared to be due to its inability to bind to single-stranded DNA. The altered dnaB protein was inhibitory to the activity of wild type dnaB protein in replication by sequestering dnaC protein which is also required for replication. By contrast, it was not inhibitory to dnaB protein in priming of single-stranded DNA by primase in the absence of single-stranded DNA binding protein. Sequestering of dnaC protein into inactive complexes may relate to the dominant-lethal phenotype of this dnaB mutant.

Two proteins have been identified which stimulate a mutant form of dnaA protein in replication of plasmids containing the chromosomal origin, oriC. One of these is dnaK protein by the criteria of (i) absence of stimulatory activity in enzyme fractions from dnaK mutants, (ii) elevated levels of stimulatory activity in fractions from a dnaK protein overproducer, (iii) comigration of the stimulatory protein with authentic dnaK protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iv) replacement of this stimulatory protein by dnaK protein in stimulation assays. The stimulatory effect of dnaK protein on dnaA46 protein in replication suggests that this interaction, occurring prior to its action in DNA replication, may regulate its activity.

The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-β- d -thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.

Escherichia coli is a model system to study the mechanism of DNA replication and its regulation during the cell cycle. One regulatory pathway ensures that initiation of DNA replication from the chromosomal origin, oriC, is synchronous and occurs at the proper time in the bacterial cell cycle. A major player in this pathway is SeqA protein and involves its ability to bind preferentially to oriC when it is hemi-methylated. The second pathway modulates DnaA activity by stimulating the hydrolysis of ATP bound to DnaA protein. The regulatory inactivation of DnaA function involves an interaction with Hda protein and the beta dimer, which functions as a sliding clamp for the replicase, DNA polymerase III holoenzyme. The datA locus represents a third mechanism, which appears to influence the availability of DnaA protein in supporting rifampicin-resistant initiations.

The Escherichia coli dnaA73, dnaA721, and dnaA71 alleles, which encode A213D, R432L, T435K substitutions, respectively, were originally isolated as extragenic suppressors of a temperature-sensitive dnaX mutant. As the A213D substitution resides in a domain that functions in ATP binding and the R432L and T435K substitutions affect residues that recognize the DnaA box motif, they might be expected to reduce ATP and specific DNA binding, respectively. Therefore, a major objective was to quantify the biochemical defects of the mutant DnaAs to understand how the altered proteins suppress the temperature-sensitive phenotype of a dnaX mutant. A second purpose was to address the paradox that mutant proteins with substitutions of amino acids essential for recognition of the DnaA box motifs within the E. coli replication origin (oriC) may well be inactive in initiation, yet chromosomal dnaA mutants expressing DnaA proteins with the R432L and T435K substitutions are viable at temperatures from 30 to 39 degrees C. We show biochemically that mutant DnaAs carrying R432L and T435K substitutions fail to bind to the DnaA box sequence. The A213D mutant is sevenfold reduced in its affinity for ATP compared to wild-type DnaA, and its affinity for the DnaA box sequence is also reduced. However, the reduced activity of the A213D mutant in oriC plasmid replication appears to arise from a defect in DnaA oligomerization. Although the T435K mutant fails to bind to the DnaA box sequence, other results suggest that DnaA oligomerization stabilizes the binding of the mutant DnaA to oriC to support its partial activity in initiation in vitro. These results support a model that suppression of dnaX occurs by reducing the frequency of initiation to a manageable level for the mutant DnaX so that viability is maintained.

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.

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.