dnaA Gene Research Articles

Interactions of the Streptomyces lividans initiator protein DnaA with its target.

The Streptomyces lividans DnaA protein (73 kDa) consists, like other bacterial DnaA proteins, of four domains; it binds to 19 DnaA boxes in the complex oriC region. The S. lividans DnaA protein differs from others in that it contains an additional stretch of 120 predominantly acidic amino acids within domain II. Interactions between the DnaA protein and the two DnaA boxes derived from the promoter region of the S. lividans dnaA gene were analysed in vitro using three independent methods: Dnase-I-footprinting experiments, mobility-shift assay and surface plasmon resonance (SPR). The Dnase-I-footprinting analysis showed that the wild-type DnaA protein binds to both DnaA boxes. Thus, as in Escherichia coli and Bacillus subtilis, the S. lividans dnaA gene may be autoregulated. SPR analysis showed that the affinity of the DnaA protein for a DNA fragment containing both DnaA boxes from the dnaA promoter region (KD = 1.25 nM) is 10 times higher than its affinity for the single 'strong' DnaA box (KD = 12.0 nM). The mobility-shift assay suggests the presence of at least two classes of complex containing different numbers of bound DnaA molecules. The above data reveal that the DnaA protein binds to the two DnaA boxes in a cooperative manner. To deduce structural features of the Streptomyces domain II of DnaA protein, the amino acid DnaA sequences of three Streptomyces species were compared. However, according to the secondary structure prediction, Streptomyces domain II does not contain any common relevant secondary structural element(s). It can be assumed that domain II of DnaA protein can play a role as a flexible protein spacer between the N-terminal domain I and the highly conserved C-terminal part of DnaA protein containing ATP-binding domain III and DNA-binding domain IV.

Replication origin region of the chromosome of alkaliphilic Bacillus halodurans C-125.

An 18.5-kb DNA fragment containing the oriC region of the chromosome of the alkaliphilic Bacillus halodurans C-125 was obtained by PCR and sequenced. Sixteen open reading frames (ORFs) were identified in this region. A sequencing similarity search using the BSORF database found that ORF1 to 13 all had significant similarities to gene products of Bacillus subtilis. Three other ORFs (ORF14-16) of unknown function were positioned down-stream of gyrB instead of rrnO, which is found in the same region in the case of B. subtilis. The ORF organization from gidA to gyrA was the same as that of B. subtilis. The gene organization and the location of the DnaA-box region were also similar to those of the chromosomes of other bacteria, such as Escherichia coli and Pseudomonas putida. There were two DnaA-box clusters (Box-region C and R) with a consensus sequence TTATCCACA on both sides of the dnaA gene but another DnaA box cluster (Box-region L) which is found in the region between thdF and jag in B. subtilis was not found in the corresponding region in the case of alkaliphilic Bacillus halodurans C-125.

Chitobiase, a new reporter enzyme.

N,N'-diacetylchitobiase (chitobiase) from the marine organism Vibrio harveyi is a highly stable reporter enzyme for gene fusions. This enzyme hydrolyzes the disaccharide chitobiose to N-acetyl glucosamine. The advantages of the reporter gene encoding chitobiase (chb) are: (i) that chitobiase and N-acetyl-beta-D-glucosaminidase activities are missing in E. coli strains, (ii) chitobiase can be monitored using blue/white colony indicator plates and (iii) convenient substrates for this enzyme are commercially available. The use of chitobiase as a reporter enzyme is generally applicable to the study of gene expression in those bacteria that do not contain N-acetyl-beta-D-glucosaminidases. We constructed plasmid vectors containing a multiple cloning site for producing in-frame fusions to chitobiase, the attP of lambda phase for movement into the bacterial chromosome for single-copy analysis, the gene encoding chloramphenicol acetyltransferase (cat), the pACYC184 origin of replication and the rrnBt1t2 terminator region upstream of the chb gene to prevent read-through from other promoters. In-frame fusions between the dnaA gene and chb were moved to the chromosome by site-specific recombination with the chromosomal attB site. These single-copy fusions were assayed for chitobiase to examine the effects of a deletion in the dnaA regulatory region.

Escherichia coli dnaA gene function and bacteriophage lambda replication.

Allele specificity of the Escherichia coli dnaA gene function in the replication of plasmids derived from bacteriophage lambda has been demonstrated previously. Here, using a series of dnaA temperature-sensitive mutants, we investigated dnaA allele specificity of the replication of phages lambda P+ and lambda Pts 1 pi A66. We found that phage lambda P+ produces its progeny efficiently at 43 degrees C irrespective of the dnaA allele, whereas lambda Pts 1 pi A66, which is unable to develop lytically in the dnaA+ host at this temperature, can replicate with different efficiency in certain dnaA mutants. Since the main role of DnaA in lambda development seems to be stimulation of transcription from the pR promoter, we measured the activity of this promoter (using a pR-lacZ fusion) and the abundance of pR-derived transcripts (by Northern blotting analysis) in dnaA+ host and dnaA(ts) mutants at 30 and 43 degrees C. We found significant differences in the activity of pR in various dnaA(ts) mutants at 30 degrees C, which indicate different levels of stimulation of this promoter by products of particular dnaA alleles at permissive temperature. Differential levels of DnaA-mediated stimulation of pR in various dnaA(ts) mutants were also found at 43 degrees C. Stimulation of the pR promoter by DnaA is necessary for both efficient production of the lambda replication proteins, O and P, and effective transcriptional activation of ori lambda. The differences in the efficiency of pR activation observed in dnaA mutants at 30 and 43 degrees C can explain the mechanisms of allele specificity of dnaA gene function in the replication of bacteriophage lambda and plasmids derived from this phage.

Escherichia coli dnaA gene function and bacteriophage λ replication

Allele specificity of the Escherichia coli dnaA gene function in the replication of plasmids derived from bacteriophage λ has been demonstrated previously. Here, using a series of dnaA temperature-sensitive mutants, we investigated dnaA allele specificity of the replication of phages λ P + and λ Pts1πA66. We found that phage λ P + produces its progeny efficiently at 43°C irrespective of the dnaA allele, whereas λ Pts1πA66, which is unable to develop lytically in the dnaA + host at this temperature, can replicate with different efficiency in certain dnaA mutants. Since the main role of DnaA in λ development seems to be stimulation of transcription from the p R promoter, we measured the activity of this promoter (using a p R- lacZ fusion) and the abundance of p R-derived transcripts (by Northern blotting analysis) in dnaA + host and dnaA(ts) mutants at 30 and 43°C. We found significant differences in the activity of p R in various dnaA(ts) mutants at 30°C, which indicate different levels of stimulation of this promoter by products of particular dnaA alleles at permissive temperature. Differential levels of DnaA-mediated stimulation of p R in various dnaA(ts) mutants were also found at 43°C. Stimulation of the p R promoter by DnaA is necessary for both efficient production of the λ replication proteins, O and P, and effective transcriptional activation of oriλ. The differences in the efficiency of p R activation observed in dnaA mutants at 30 and 43°C can explain the mechanisms of allele specificity of dnaA gene function in the replication of bacteriophage λ and plasmids derived from this phage.

Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803.

A split intein capable of protein trans-splicing is identified in a DnaE protein of the cyanobacterium Synechocystis sp. strain PCC6803. The N- and C-terminal halves of DnaE (catalytic subunit alpha of DNA polymerase III) are encoded by two separate genes, dnaE-n and dnaE-c, respectively. These two genes are located 745,226 bp apart in the genome and on opposite DNA strands. The dnaE-n product consists of a N-extein sequence followed by a 123-aa intein sequence, whereas the dnaE-c product consists of a 36-aa intein sequence followed by a C-extein sequence. The N- and C-extein sequences together reconstitute a complete DnaE sequence that is interrupted by the intein sequences inside the beta- and tau-binding domains. The two intein sequences together reconstitute a split mini-intein that not only has intein-like sequence features but also exhibited protein trans-splicing activity when tested in Escherichia coli cells.

Effect of Escherichia coli dnaE antimutator mutants on mutagenesis by the base analog N4-aminocytidine

Previous studies in our laboratory have identified a set of mutations in the Escherichia coli dnaE gene that confer increased accuracy of DNA replication (antimutators). The dnaE gene encodes the polymerase subunit of DNA polymerase III holoenzyme that replicates the E. coli chromosome. Here, we have investigated their effect on mutagenesis by the base analog N 4-aminocytidine (4AC). For three different mutational markers, rifampicin resistance, nalidixic acid resistance and lacI forward mutagenesis, the dnaE911 allele reduced 4AC-induced mutagenesis by approximately 2.5-fold, while the dnaE915 allele reduced it by 2.5-, 3.5- and 6.5-fold, respectively. We also investigated the dependence of 4AC mutagenesis on mutations in the MutHLS mismatch repair system and the UvrABC nucleotide excision repair system. The results show that mutagenesis by 4AC is unaffected by defects in either system. The combined results point to the critical role of the DNA polymerase in preventing mutations by base analogs.

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium.

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinus dnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them.

Replication of the linear chromosomal DNA from the centrally located oriC of Streptomyces ambofaciens revealed by PFGE gene dosage analysis

From a cosmid clone of Streptomyces ambofaciens containing the dnaA and gyrAB genes, a 2.7-kb self-replicating DNA fragment containing the chromosome replication origin oriC was isolated. This cosmid was previously mapped physically to a region near the middle of the 8-Mb linear chromosomal DNA. A pulsed-field gel electrophoresis time-course analysis revealed that sequences flanking oriC were overrepresented relative to the rest of the chromosomal DNA during rapid growth, indicating that this origin is active. In addition, the terminal regions of the chromosomal DNA showed a slight overrepresentation at the onset of stationary phase.

Organization around the dnaA gene of Streptococcus pneumoniae

The dnaA gene region of Streptococcus pneumoniae was cloned and sequenced. A tRNA gene, seven ORFs and three DnaA box clusters were identified. The order of the genes and intergene regions found was tRNA(Arg)-orf1-DnaA box cluster 3-htrA-spoOJ-DnaA box cluster 2-dnaA-DnaA box cluster 1-dnaN-orfX-orfY. Five ORFs are homologous to known bacterial genes. The tRNA(Arg) gene and orf1, also called orfL, have already been described in pneumococci and have been reported to be preceded by the competence regulation locus comCDE. In Escherichia coli, htrA encodes a serine protease. In Bacillus subtilis, spoOJ plays a role in sporulation and partition. dnaA encodes an initiator replication protein, very well conserved in several bacteria and dnaN encodes the beta subunit of DNA polymerase III in E. coli. The function of orfX is unknown. The N-terminal part of another reading frame, orfY, revealed high homology with a GTP-binding protein, DnaA box clusters were found upstream and downstream from dnaA. The presence of two such clusters suggests that the chromosomal origin of S. pneumoniae is located within this region. The position of dnaA, and therefore the putative origin of replication, were localized on the physical map of S. pneumoniae.

Guanosine tetraphosphate (ppGpp)-mediated inhibition of the activity of the bacteriophage lambda pR promoter in Escherichia coli.

It was previously demonstrated that the activity of bacteriophage lambda promoter pR is decreased in wild-type Escherichia coli cells starved for amino acids (during the stringent response). Since pR activity is necessary for the transcriptional activation of ori lambda, this leads to inhibition of the replication of plasmids derived from phage lambda. These results led to the proposal that the pR promoter susceptible to control by the stringent response. However, subsequent studies demonstrated that this promoter is activated by the host dnaA gene product and since the dnaA promoter was reported to be controlled by the stringent response, it is possible that the inhibition of pR activity in amino acid-starved cells is indirect, and results from the impairment of DnaA-mediated transcriptional activation. Here we present evidence that pR is negatively regulated by ppGpp, even when DnaA protein is provided in excess as well as in cells devoid of DnaA function. We have checked that the level of ppGpp is increased during prolonged (up to 4 h) starvation for isoleucine in relA+ cells but not in the relA- mutant. At the same time we observed inhibition of lambda plasmid replication during the stringent, but not relaxed, response, even when DnaA was overproduced. Finally, we found that the activity of a pR-lacZ fusion is inhibited after gratuitously induced overproduction of ppGpp in unstarved cells, irrespective of the status of the dnaA gene product. We conclude that the activity of the pR promoter is inhibited directly by ppGpp.

Alteration in levels of unsaturated fatty acids in mutants of Escherichia coli defective in DNA replication.

We previously reported that mutations in the dnaA gene which encodes the initiator of chromosomal DNA replication in Escherichia coli caused an alteration in the levels of unsaturated fatty acids of phospholipids in membranes. In this study, we examined fatty acid compositions in other mutants which are defective in DNA replication. As in the case of temperature-sensitive dnaA mutants, temperature-sensitive dnaC and dnaE mutants, which have defects in initiation and elongation, respectively, of DNA replication showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) compared with the wild-type strain, especially at high temperatures. On the other hand, temperature-sensitive mutants defective in cellular processes other than DNA replication, such as RNA synthesis and cell division, did not show a lower level of unsaturation of fatty acids compared with the wild-type strain. These results suggest that the inhibition of DNA replication causes a lower level of unsaturation of fatty acids in Escherichia coli cells.

Complexes at the replication origin of Bacillus subtilis with homologous and heterologous DnaA protein

The initial steps in the formation of the initiation complex at oriC of Bacillus subtilis were analyzed with special emphasis on the exchangeability of B. subtilis DnaA protein by DnaA of Escherichia coli. The DNA binding domain of B. subtilis DnaA protein was localized in the 93 C-terminal amino acids. Formation of the “initial complex”, as analyzed by electron microscopy, was indistinguishable with B. subtilis DnaA protein or with E. coli DnaA. Similarly, both proteins were able to form loops by interaction of DnaA proteins bound to the DnaA box regions upstream and downstream of the dnaA gene in B. subtilis oriC. The region of local unwinding in the “open complex” was precisely defined. It is located at one side of a region of helical instability, a DNA unwinding element (DUE). Unwinding in oriC could only be catalyzed by the homologous DnaA protein.

The Escherichia coli dnaA gene: four functional domains

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.

Characterization of the oriC region of Mycobacterium smegmatis.

A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M. Rajagopalan et al., J. Bacteriol. 177:6527-6535, 1995). This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions. Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity. The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity. The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences [TT(G/C)TCCACA] and a single 11-nucleotide AT-rich cluster. Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity. Mutations at other positions in two of the DnaA boxes also decreased oriC activity. Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity. These data indicate that the designated DnaA boxes and the AT-rich cluster of the M. smegmatis dnaA-dnaN intergenic region are essential for oriC activity. We suggest that M. smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.

Novel alleles of the Escherichia coli dnaA gene

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.

The binding of two dimers of IciA protein to the dnaA promoter 1P element enhances the binding of RNA polymerase to the dnaA promoter 1P.

Transcription of the dnaA gene from the promoter 1P has been shown to be activated in vitro and in vivo by the binding of IciA protein to two sites on the dnaA promoter region [Lee, Y. S., Kim, H., and Hwang, D. S. (1996) Mol. Microbiol . 19, 389-396; Lee, Y. S., and Hwang, D. S. (1997) J. Biol. Chem. 272, 83-88]. In vitro transcription assays using DNA fragments carrying variable combinations of two IciA binding sites revealed that IciA binding site I (IciA I site), which is located upstream of the promoter 1P, is responsible for the transcriptional activation. Binding of one dimeric IciA protein to the IciA I site is followed by binding of the second dimer. Two dimers of IciA protein, rather than one dimer, on the IciA I site appeared to enhance the binding of RNA polymerase to the promoter 1P, resulting in the activation of transcription from the promoter 1P.

Inhibition of Thymidine Transport in dnaA Mutants of Escherichia coli

DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. We report here our evidence that thymidine transport across cytoplasmic membranes in temperature-sensitive dnaA mutants is greatly decreased at a permissive temperature for growth of the mutants. Complementation analysis with a plasmid containing the wild type dnaA gene and P1 phage-mediated transduction confirmed that mutations in the dnaA gene were responsible for the phenotype. A low level of nucleoside transport in the dnaA mutant was specific for thymidine; transport activities for other nucleosides were much the same as those in wild type cells. Membrane vesicles prepared from the dnaA mutant showed much the same activity of thymidine transport as did those from the wild type cells. No significant difference in the activity of thymidine kinase, which is known to facilitate thymidine transport, was seen between the mutant and the wild type cells. An increase in the pool of dTTP, a negative regulator for thymidine kinase, was observed in the dnaA mutant. Based on these observations, we suggest that inhibition of thymidine transport in dnaA mutants is caused by increases in the dTTP pool.

Asymmetrical progression of replication forks just after initiation on Mycoplasma capricolum chromosome revealed by two-dimensional gel electrophoresis

Previously, we mapped the replication initiation site of the Mycoplasma capricolum chromosome into a region containing the dnaA gene [M. Miyata et al., 1993a. Nucleic Acids Res. 21, 4816–4823]. In this study, various regions including this functional domain were analyzed by two complementary two-dimensional (2D) gel electrophoretic methods. Sizes of nascent strands in a 10.7-kb and a 5.6-kb region were examined by a neutral/alkaline (N/A) method. The shortest nascent strand was detected in an 875-bp region composed of the 3′ end of the dnaA gene and its downstream non-coding sequence. The shortest nascent strand detected became longer in an asymmetrical manner as position of the probe became further from the putative initiation site in both directions. The intermediate forms of eight regions restricted at different sites were examined by a neutral/neutral (N/N) method. Bubble arcs were observed in four regions including the 875-bp region. The region containing the 875-bp region at about its center showed an asymmetrical arc, although that containing the 875-bp region at its end showed a symmetrical arc. These results show that the replication forks develop in the 875-bp region and proceed bidirectionally in an asymmetrical manner around the initiation site. The results of N/A analysis of the 5.6-kb region showed a shift of intensity in the nascent strand signal, which suggests an upshift of fork progression velocity.

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

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