dnaA Gene Research Articles

Phenotypes of dnaA Mutants of Escherichia coli Sensitive to Detergents and Organic Solvents

We examined growth sensitivities of dnaA mutants of Escherichia coli to detergents (deoxycholic acid, cholic acid) and organic solvents (ethanol, glycerol). The dnaA602, dnaA604, dnaA5, and dnaA46 mutants with mutations in the putative ATP binding site of DnaA protein showed higher sensitivities to the detergents than did the wild-type strain. The dnaA508 and dnaA167 mutants with mutations in the N-terminal region of DnaA protein, however, showed higher sensitivities to organic solvents. The dnaA204 and dnaA205 mutants with a mutation in the C-terminal region of DnaA protein showed similar sensitivity as did the wild-type strain to reagents. Complementation analysis with a plasmid carrying the wild-type dnaA gene confirmed that dnaA mutations are responsible for these phenotypes that are sensitive to the reagents.

Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bové, and F. Laigret, Curr. Microbiol. 29:23-29, 1994). In the present studies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia coli replicon and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.

Transcription termination in the Escherichia coli dnaA gene is not mediated by the internal DnaA box.

DnaA protein is a DNA-binding protein which recognizes a 9-bp consensus sequence called the DnaA box. By binding to DnaA boxes, DnaA protein regulates initiation of chromosomal replication and transcription of several genes. The dnaA gene contains two DnaA boxes, one located in the regulatory region and one within the structural gene. In this paper, we explore the role of the internal DnaA box in dnaA expression because it has been proposed that the DnaA box-DnaA protein complex can block transcribing RNA polymerase. Firstly, we analyzed the degree of derepression of the dnaA gene, measured as beta-galactosidase activity of a dnaA-lacZ fusion inserted onto the bacterial chromosome, produced by an extra copy number of the dnaA DnaA boxes carried by multicopy plasmids. Secondly, we analyzed repression produced by elevated levels of DnaA protein on single-copy dnaA-lacZ fusions containing or not containing the internal DnaA box. Our results indicate that the internal DnaA box does not play a regulatory role in dnaA expression.

Initiation of chromosome replication after induction of DnaA protein synthesis in a dnaA(null) rhn mutant of Escherichia coli

The kinetics of initiation of chromosome replication after induction of DnaA protein synthesis was studied in a dnaA(nuII) rnh mutant of Escherichia coli. DnaA protein synthesis was induced to different extents using the wild-type dnaA gene controlled by a lac promoter. Initiation of chromosome replication from oriC, measured as an increase in origin to terminus ratio, took place at different times after addition of an inducer dependent on the DnaA protein synthesis rate. The first initiations always occurred when DnaA protein had accumulated approximately to the average wild-type concentration (24 ng of DnaA protein per ml cells at OD450 = 1.0). At a low DnaA protein accumulation rate one synchronous round of replication was obtained after 30 min of induction. The initiation kinetics obtained when DnaA protein accumulated rapidly was complicated and indicated that other factors might also be involved.

Gene organization in the dnaA-gyrA region of the Streptomyces coelicolor chromosome

The gene organization has been determined for an 8-kb portion of the Streptomyces coelicolor chromosome close to the origin of DNA replication (oriC). Hybridization and DNA sequence analyses revealed the presence of five open reading frames (ORFs) oriented in the same direction as the proximal dnaA and dnaN genes. The deduced products of three of the ORFs have been identified as the S. coelicolor homologs of RecF, GyrB and GyrA. These genes are usually clustered in the dnaA region of bacterial chromosomes. In S. coelicolor however, the usual gene arrangement is altered. The recF gene is flanked by two ORFs, one of which encodes a protein with significant similarity to 6-phosphogluconate dehydrogenases (6PGDH), an enzyme that is not immediately linked to DNA metabolism.

Spontaneous mutation in Escherichia coli containing the dnaE911 DNA polymerase antimutator allele.

We have previously isolated mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants have a mutation in the dnaE gene, encoding the alpha subunit of DNA polymerase III. They were isolated in a mismatch-repair-defective mutL background, in which mutations can be considered to represent uncorrected DNA replication errors. In the present study we analyze the effect of one of these alleles, dnaE911, on spontaneous mutagenesis in a mismatch-repair-proficient background. In this background, spontaneous mutations may be the sum of uncorrected replication errors and mutations resulting from other pathways. Hence, the effect of the dnaE allele may provide insights into the contribution of uncorrected DNA replication errors to spontaneous mutation. The data show that dnaE911 decreases the level of Rifr, lacI and galK mutations in this background by 1.5-2-fold. DNA sequencing of 748 forward mutants in the lacI gene reveals that this effect has a clear specificity. Transversions are decreased by approximately 3-fold, whereas transitions, frameshifts, deletions and duplications remain essentially unchanged. Among the transversions, A.T-->T.A are affected most strongly (approximately 6-fold). In addition to this effect on transversions within the lacI gene, one previously recognized A.T-->G.C base-pair substitution hotspot in the lac operator is also reduced (approximately 5-fold). The data are discussed in the light of the role of DNA replication errors in spontaneous mutation, as well as other possible explanations for the observed antimutator effects.

An unusual gene containing a dnaJ N-terminal box flanks the putative origin of replication of Mycoplasma genitalium.

Origins of replication are known to be highly conserved among widely divergent microbial species, with the gene order in those regions being dnaA-dnaN-recF-gyrB. On the basis of sequence identities to entries in GenBank, the gene order of a 6-kb fragment of Mycoplasma genitalium DNA was determined to be dnaN-orf311-gyrB-gyrA-serS, which is structurally similar to the ancestral origin of replication. We have directly linked the dnaN gene to the M. genitalium dnaA gene by PCR amplification. However, we found a novel open reading frame, designated orf311, in place of an expected sequence encoding recF. Orf311 contains a DnaJ box motif at its N terminus, but it has no overall homology to any other protein or sequence in the database. We are unable to detect any recF homolog in M. genitalium by hybridization or during a random sequencing survey of the genome.

An improved vector system for constructing transcriptional lacZ fusions: analysis of regulation of the dnaA, dnaN, recF and gyrB genes of Escherichia coli.

We describe a new vector system for the in vitro construction of transcriptional fusions to the lacZ gene, which is expressed from the translational start signals of galK. The galK ribosome-binding site (RBS) and its natural preceding region ensure a constant efficiency for lacZ translation and, thus, the beta-galactosidase (beta Gal) production of a given fusion is directly proportional to the in vivo transcriptional activity of the inserted DNA fragment. Single-copy lambda prophage versions of multicopy constructs can be made by in vivo recombination. We use this system to compare the transcriptional activities of the promoters present in the dnaA-dnaN-recF-gyrB cluster. The order of strength of these promoters is gyrB > dnaA > recF > dnaN. It is assumed that gyrB belongs to the dnaA-dnaN-recF operon, because the short recF-gyrB intercistronic region does not contain a terminator. By using this new vector system, we have detected strong termination signals within recF that are functional even when recF is translated at its normal rate. The low level of transcription coming to the end of recF, and the highest activity of the gyrB promoter, as well as results obtained with several gyrB::lacZ translational fusions, support the conclusion that gyrB is predominantly expressed from its own promoter under standard growth conditions. Finally, we have found that transcription from the dnaA promoters is constant at different growth rates. This supports the idea that autoregulation of the dnaA gene is responsible for the coupling of the DnaA protein synthesis to cell mass increase, and accumulation of DnaA protein governs the initiation of chromosome replication.

Cloning and sequencing of the replication origin (oriC) of the Spiroplasma citri chromosome and construction of autonomously replicating artificial plasmids.

A 5.6-kbp fragment of Spiroplasma citri DNA containing the dnaA gene has been cloned and sequenced. Nucleotide sequence analysis shows that this fragment harbors the genes for the replication initiator protein (dnaA), the beta subunit of DNA polymerase III (dnaN), and the DNA gyrase subunits A and B (gyrA and gyrB). The arrangement of these genes, dnaA-dnaN-gyrB-gyrA, is similar to that found in all Gram-positive bacterial genomes studied so far, except that no recF gene was found between dnaN and gyrB. Several DnaA-box consensus sequences were found upstream of dnaA and in the dnaA-dnaN intergenic region. The dnaA region with the flanking DnaA-boxes and the tetracycline resistance determinant, tetM, were linked into a circular recombinant DNA. This DNA was able to replicate autonomously when introduced by electroporation into S. citri cells. These experiments show that the dnaA region with the DnaA-boxes is the origin of replication of S. citri and can be used to construct gene vectors.

Replication of a Bacillus subtilis oriC plasmid in vitro

We constructed an in vitro replication system specific for a Bacillus subtilis oriC plasmid using a soluble fraction derived from cell extracts of B. subtilis. DNA polymerase III and two initiation proteins, DnaA and DnaB, were required for in vitro replication as observed in vivo. Both upstream and downstream DnaA box regions of the dnaA gene were required as cis-elements for in vitro synthesis of the B. subtilis oriC plasmid as well as for in vivo activity. The replication was semi-conservative and only one round of replication occurred within 15 min. These results indicate that in vitro replication faithfully reproduced in vivo replication. To elucidate the site of initiation and the direction of replication, we analysed replicative intermediates generated in vitro in the presence of various concentrations of ddGTP by two methods. First, analysis of restriction fragments around the dnaA gene showed a high level of incorporation of the radioactive substrate, indicating that replication began within the vicinity of the dnaA gene. Second, using 2-dimensional gel electrophoresis, bubble arcs were detected only on fragments containing the DnaA box region downstream of the dnaA gene, indicating that the initiation site resided within this region. The distribution of the bubble arcs suggested that both bidirectional and undirectional replication occurred in vitro.

Inducible transcription of the dnaA gene from Streptomyces lividans 66

The dnaA gene of Streptomyces lividans was cloned using the Escherichia coli medium-copy-number vector pSU18 and E. coli strain TC1963, which can by-pass the requirement for the DnaA protein. Its regulatory region was subcloned in the Streptomyces probe vector pIJ4083. Primer extension and S1 mapping studies allowed the identification of a class I Streptomyces promotor (P2). An additional, previously unknown promoter type (P1) was found by S1 mapping. The presence of two DnaA box motifs between P1 and P2 suggests that the transcriptions of the S. lividans dnaA gene is autoregulated by its gene product. It was shown that the transcription of the dnaA gene is significantly induced by mitomycin C, an agent known to inhibit DNA replication. The data suggest that, as in E. coli, one of the regulatory mechanisms governing the transcription of the dnaA gene in S. lividans is probably related to the SOS response network.

Expression of Caulobacter dnaA as a function of the cell cycle

The initiation of DNA replication is under differential control in Caulobacter crescentus. Following cell division, only the chromosome in the progeny stalked cell is able to initiate DNA replication, while the chromosome in the progeny swarmer cell does not replicate until later in the cell cycle. We have isolated the dnaA gene in order to determine whether this essential and ubiquitous replication initiation protein also contributes to differential replication control in C. crescentus. Analysis of the cloned C. crescentus dnaA gene has shown that the deduced amino acid sequence can encode a 486-amino-acid protein that is 37% identical to the DnaA protein of Escherichia coli. The gene is located 2 kb from the origin of replication. Primer extension analysis revealed a single transcript originating from a sigma 70-type promoter. Immunoprecipitation of a DnaA'-beta-lactamase fusion protein showed that although expression occurs throughout the cell cycle, there is a doubling in the rate of expression just prior to the initiation of replication.

Comparison of the conserved region in the dnaA gene from three mollicute species

Comparison of the conserved region in the dnaA gene from three mollicute species

Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli

Transcriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication. The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique. In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation. Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA. It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin. It is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription. Conversely, the active transcription of gidA and dnaA prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation.

Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli

The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located on a plasmid. In these cells with different DnaA protein levels, we measured several parameters: dnaA gene expression; cell size, amount of DNA per cell, and number of origins per cell by flow cytometry; and origin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to higher levels of DnaA protein could be divided into three states. From the normal level to a level 1.5-fold higher, DnaA protein had little effect on dnaA gene expression and the rate of DNA replication but led to nearly proportional increases in DNA and origin concentrations. Between 1.5- and 3-fold, the normal DnaA protein concentration, dnaA gene expression was gradually decreased. In this interval, the origin concentration increased significantly; however, the replication rate was severely affected, becoming slower--especially near the origin--the higher the DnaA protein concentration, and as a result, the DNA concentration was constant. Further increases in the DnaA protein concentration did not lead to an increased origin concentration. Thus, the initiation mass was set by the DnaA protein from the normal level to an at least twofold-increased level, but the increased initiation did not lead to a large increase in the amount of DNA per unit of mass because of the inhibition of replication fork velocity.

Cooperation of the prs and dnaA gene products for initiation of chromosome replication in Escherichia coli.

A new Escherichia coli mutant allele, named dnaR, that causes thermosensitive initiation of chromosome replication has been identified to be an allele of the prs gene, the gene for phosphoribosylpyrophosphate synthetase (Y. Sakakibara, J. Mol. Biol. 226:979-987, 1992; Y. Sakakibara, J. Mol. Biol. 226:989-996, 1992). The dnaR mutant became temperature resistant by acquisition of a mutation in the dnaA gene that did not affect the intrinsic activity for the initiation of replication. The suppressor mutant was capable of initiating replication from oriC at a high temperature restrictive for the dnaR single mutant. The thermoresistant DNA synthesis was inhibited by the presence of the wild-type dnaA allele at a high but not a low copy number. The synthesis was also inhibited by an elevated dose of a mutant dnaR allele retaining dnaR activity. Therefore, thermoresistant DNA synthesis in the suppressor mutant was dependent on both the dnaA and the dnaR functions. On the basis of these results, I conclude that the initiation of chromosome replication requires cooperation of the prs and dnaA products.

The mutational specificity of two Escherichia coli dnaE antimutator alleles as determined from lacI mutation spectra.

In a companion study we have described the isolation of a series of mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants carry a mutation in the dnaE gene, encoding the alpha (polymerase) subunit of DNA polymerase III holoenzyme, which is responsible for the faithful replication of the bacterial chromosome. The mutants were detected as suppressors of the high mutability of a mutL strain (defective in postreplicative mismatch correction), in which mutations may be considered to arise predominantly from errors of DNA replication. To investigate the specificity of these antimutator effects, we have analyzed spectra of forward mutations in the N-terminal part of the lacI gene (i-d mutations) for two of the mutL dnaE derivatives (dnaE911 and dnaE915), as well as the control mutL strain. DNA sequencing of over 600 mutants revealed that in the mutL background both antimutator alleles reduce specifically transition mutations (A.T-->G.C and G.C-->A.T). However, the two alleles behave differently in this respect. dnaE911 reduces A.T-->G.C more strongly than it does G.C-->A.T, whereas the reverse is true for dnaE915. Second, dnaE911 does not appear to affect either transversion or frameshift mutations, whereas dnaE915 displays a distinct mutator effect for both. This mutator effect of dnaE915 for frameshift mutations was confirmed by the frequency of reversion of the trpE9777 frameshift mutation. The discovery that dnaE antimutator alleles possess distinct specificities supports the notion that DNA polymerases discriminate against errors along multiple pathways and that these pathways can be influenced independently.

Antimutator mutations in the alpha subunit of Escherichia coli DNA polymerase III: identification of the responsible mutations and alignment with other DNA polymerases.

The dnaE gene of Escherichia coli encodes the DNA polymerase (alpha subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.

Unique genetic arrangement in the dnaA region of the Borrelia burgdorferi linear chromosome: Nucleotide sequence of the dnaA gene

Unique genetic arrangement in the dnaA region of the Borrelia burgdorferi linear chromosome: Nucleotide sequence of the dnaA gene

Localizing the replication origin region on the physical map of the Mycoplasma capricolum genome

Four lines of evidence argue that the replication origin of the Mycoplasma capricolum genome lies within the 46-kb BamHI fragment bordered by two BamHI sites of the total of nine BamHI sites that have been located on the physical map (M. Miyata, L. Wang, and T. Fukumura, FEMS Microbiol. Lett. 79:329-334, 1991). First, this fragment lost its labeling in preference to other fragments when log-phase cultures were incubated in the presence of chloramphenicol for various times to inhibit the initiation of new rounds of replication and then further incubated with radioactive dTMP to allow DNA elongation to continue. Second, the relative frequencies of various restriction fragments of the genome DNA from exponentially growing cells decreased with increasing distance from the putative origin. Third, preferential labeling occurred when radioactive dTMP was added to cultures of a DNA elongation-defective, temperature-sensitive mutant with a simultaneous temperature downshift. Fourth, the M. capricolum homolog of the dnaA gene, which is located near the replication origin in many other bacteria, was found in the 46-kb fragment.