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At least 19 recordsLinked to original sources

PubMed1989

Directionality of DnaA protein/DNA interaction. Active orientation of the DnaA protein/dnaA box complex in transcription termination.

The complex of DnaA protein with its 9 bp consensus binding site, the dnaA box 5'-TT(A/T)T(A/C)CA(A/C)A, blocks transcribing RNA polymerase. In a model system, the rate of transcription was monitored distal to the dnaA box 5'-TTTTCCACA by the expression of a reporter gene. DnaA-dependent transcription termination occurred irrespective of whether the dnaA box region was or was not translated. Only the dnaA box orientation 5'-TTTTCCACA on the non-coding strand, but not the reverse orientation, was active in termination. This suggests that DnaA protein contacts only one strand of the DNA duplex. Oligonucleotide-directed mutation of a dnaA box present within the dnaA coding region resulted in increased expression of dnaA. This demonstrates that DnaA protein-directed transcription termination is an element of the autoregulation of the dnaA gene.

Amino Acid Sequence↗

PubMed1992

DnaA dependent replication of plasmid R1 occurs in the presence of point mutations that disrupt the dnaA box of oriR.

We have found that DnaA dependent replication of R1 still occurred when 5 of the 9 bases in the dnaA box present in oriR were changed by site directed mutagenesis although the replication efficiency decreased to 20% and 70% of the wild-type origin in vitro and in vivo respectively. Additional mutation of a second dnaA box, 28 bp upstream oriR, that differs in only one base from the consensus sequence, did not affect the level of replication whereas polyclonal antibodies against DnaA totally abolished in vitro replication in the absence of the dnaA box. Wild-type RepA as well as a RepA mutant, RepA2623, that binds to oriR but that is inactive in promoting in vitro replication of plasmid R1, induce efficient binding of DnaA to the dnaA box. However, specific binding of DnaA to oriR was not detected by DNase I protection experiments in the absence of the dnaA box. These results suggest that the entrance of the DnaA protein in oriR is promoted initially by interactions with a RepA-oriR pre-initiation complex and that, in the absence of the dnaA box, these interactions can support, with reduced efficiency, DnaA dependent replication of plasmid R1.

Bacterial Proteins↗

PubMed1990

Regulation of expression of the dnaA gene in Escherichia coli: role of the two promoters and the DnaA box.

The dnaA gene of Escherichia coli specifies a product that is a key element in the initiation of DNA replication. Expression of dnaA occurs from two promoters, 1P and 2P, which flank a DnaA protein binding site (DnaA box). In this paper we describe the effects of DnaA box mutations on transcription from the two promoters, measured with the use of dnaA-galK gene fusions. All of the mutations examined led to a five-fold increase in expression from the upstream promoter, 1P, suggesting that expression from this promoter is negatively autoregulated by binding of DnaA protein to the box. The same mutations led to a decrease in expression from promoter 2P, suggesting that the DnaA box plays a role in activating promoter 2P under normal conditions. Overproduction of DnaA protein, above normal physiological levels, led to decreased transcription from both promoters. We present evidence from analysis of both dnaA mRNA and DnaA protein that promoter 2P is subject to growth rate-dependent control and that this promoter, but not promoter 1P, is completely shut off in the stationary phase of growth.

Bacterial Proteins↗

PubMed1987

Integrative suppression of dnaA(Ts) mutations mediated by plasmid F in Escherichia coli is a DnaA-dependent process.

The thermosensitivity of dnaA(Ts) mutations can be suppressed by integration of plasmid F (integrative suppression). In the light of the recent finding that F requires DnaA protein for both establishment and maintenance, integrative suppression of 11 dnaA(Ts) mutations by a mini-F, pML31, integrated near oriC was examined. The plating efficiency of integratively suppressed strains was dnaA(Ts) allele-dependent and medium-dependent. The initiation capability of suppressed dnaA(Ts) strains lacking the oriC site and their F- counterparts was determined at various temperatures between 30 degrees C and 42 degrees C. The degree of integrative suppression measured by the initiation capability varied in a dnaA(Ts) allele-dependent manner. F-directed DNA replication was most affected by the dnaA(Ts) mutations mapping in the middle of the gene whereas oriC-dependent replication was most thermosensitive in strains carrying mutations mapping in the carboxy-terminal half of the gene. The results indicated that the integrative suppression by F plasmid is a DnaA-dependent process and suggested that the requirements for DnaA protein in the oriC-dependent replication and F replication processes are qualitatively different.

Bacterial Proteins↗

PubMed1988

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.

Bacillus subtilis↗

PubMed1988

Requirement of the Escherichia coli dnaA gene function for integrative suppression of dnaA mutations by plasmid R 100-1.

The phenotype of Escherichia coli dnaA missense and nonsense mutations was integratively suppressed by plasmid R100-1. The suppressed strains, however, could not survive when the dnaA function was totally inactivated. This was demonstrated by the inability of replacing the dnaA allele in the suppressed strain by a dnaA::Tn10 insertion using phage P1-mediated transduction. When the intact dnaA+ allele was additionally supplied by a specialized transducing phage, lambda imm21 dnaA+, which integrated at the att lambda site on the E. coli chromosome, then the dnaA::Tn10 insertion, together with a delta oriC deletion, were able to be introduced into the suppressed strain. Thus, the mechanisms of dnaA function for oriC and for the replication origin of R100-1 may not be quite the same.

Bacterial Proteins↗

PubMed1992

Structure of the dnaA and DnaA-box region in the Mycoplasma capricolum chromosome: conservation and variations in the course of evolution.

We have previously shown that the dnaA gene and the DnaA-box region were conserved in bacteria representative of all three major branches of the eubacterial phylogenic tree: high G + C Gram+, low-G + C Gram+ and Gram-. In the present work, we determined the structure of the dnaA region of Mycoplasma capricolum and found that the dnaA gene and at least two other genes, rpmH and dnaN, were conserved in this bacterium. An unusually high level of amino acid (aa) substitutions was observed in M. capricolum DnaA. It was the case even in those aa which were well conserved in other bacterial species. The nontranslatable region upstream from the dnaA gene was also conserved in this bacterium, as it was universally found in both Gram+ and Gram- bacteria. An additional nontranslatable region downstream from the dnaA gene, which is common to Gram+ bacteria, was also found in M. capricolum, consistent with the proposal that M. capricolum is Gram+ in origin. These regions were rich in A + T and contained ten DnaA-box-like sequences (9-mers that differ from TTATCCACA by one or two bases).

Amino Acid Sequence↗

PubMed1991

Structure and function of DnaA and the DnaA-box in eubacteria: evolutionary relationships of bacterial replication origins.

DnaA protein (a trans-acting element) and its binding sequence, DnaA-box: (a cis-acting element) are two elements essential for the initiation of chromosomal replication in Escherichia coli and other enteric bacteria. Recently these two elements have been found to be conserved in three Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus and Mycoplasma capricolum) as well as in Gram-negative pseudomonads. DnaA protein was also found to be essential in the initiation of the replication of the B. subtilis chromosome, and regions containing multiple repeats of DnaA-box (DnaA-box region) are found to be active as autonomously replicating elements both in B. subtilis and pseudomonads. In this MicroReview we compare first the structures of these DnaA-box regions and their locations on the chromosome and then functional aspects of DnaA protein and DnaA-box regions in the initiation and regulation of chromosomal replication. From these observations we propose evolutionary relationships between replication origins of eubacteria.

Amino Acid Sequence↗

PubMed1987

Transcriptional repression of the dnaA gene of Escherichia coli by dnaA protein.

The promoter region of the dnaA gene and of a gene which encodes a 16 kDa protein contain sites which are recognized and bound by dnaA protein. Using assays of run-off transcription of restriction fragments, purified dnaA protein specifically repressed transcription from both dnaA promoters and from the promoter for the 16 KD gene to almost undetectable levels. This repressive effect was observed at levels of dnaA protein required for specific binding of dnaA protein to restriction fragments containing the promoters for these genes. These results indicate that transcription of these genes is regulated by binding of dnaA protein to the promoter regions of these genes.

Bacterial Proteins↗

PubMed1990

Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells' initiation potential.

The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.

Bacillus subtilis↗

PubMed1979

Characterization of the dnaA, gyrB and other genes in the dnaA region of the Escherichia coli chromosome on specialized transducing phages lambda tna.

Specialized transducing phages lambda tna (tryptophanase) harboring chromosomal DNA and genetic markers from the dnaA region of the Escherichia coli chromosome were isolated. Transductional analysis showed that some of these tnaA transducing phages carry two genes important in DNA replication, namely the dnaA gene (initiation of chromosome replication) and the gyrB gene (subunit B of DNA gyrase), formerly designated couR. The following clockwise order of genetic markers was found: uhp, gyrB, dnaA, rimA, tnaA, bglB. The gene-protein relationship was established by the determination of the gene products encoded on the chromosomal DNA of the different lambda tna. A 54 kD and a 91 kD polypeptide appear to be coded for by the dnaA and gyrB genes, respectively; the 91 kD protein is encoded on a region in which coumermycin sensitivity maps and is with respect to electrophoretic behavior identical to subunit B of DNA gyrase. The 54 kD protein is encoded on the region in which different independently isolated dnaA(Ts) mutations (dnaA5, dnaA46, dnaA167, dnaA203, dnaA204, dnaA205, dnaA211, dnaA508) are located. Additional genes which code for polypeptides with hitherto unknown functions were identified and mapped. The acriflavin sensitivity mutation acrB1 was found to be an allele of the gyrB gene (see "Note Added in Proof").

Bacteriophage lambda↗

PubMed1990

The ABC-primosome. A novel priming system employing dnaA, dnaB, dnaC, and primase on a hairpin containing a dnaA box sequence.

A priming mechanism requiring dnaA, dnaB, and dnaC proteins operates on a single-stranded DNA coated with single-stranded DNA-binding protein. This novel priming, referred to as "ABC-priming," requires a specific hairpin structure whose stem carries a dnaA protein recognition sequence (dnaA box). In conjunction with primase and DNA polymerase III holoenzyme, ABC-priming can efficiently convert single-stranded DNA into the duplex replicative form. dnaA protein specifically recognizes and binds the single-stranded hairpin and permits the loading of dnaB protein to form a prepriming protein complex containing dnaA and dnaB proteins which can be physically isolated. ABC-priming can replace phi X174 type priming on the lagging strand template of pBR322 in vitro, suggesting a possible function of ABC-priming for the lagging strand synthesis and duplex unwinding. Similar to the phi X174 type priming, a mobile nature of ABC-priming was indicated by helicase activity in the presence of ATP of a prepriming protein complex formed at the hairpin. The implications of this novel priming in initiation of replication at the chromosomal origin, oriC, and in its contribution to the replication fork are discussed.

Bacterial Proteins↗

PubMed1977

Dominance of dnaA+ to dnaA in Escherichia coli.

The dominance of dnaA+ to the dnaA508 mutation was complete and was unaffected by the presence of a copy of the chromosomal replication origin on the episome. These results prove that the dnaA gene of Escherichia coli produces a diffusible product.

Alleles↗

PubMed1992

Expression and regulation of a dnaA homologue isolated from Pseudomonas putida.

A gene homologous to the Escherichia coli dnaA gene was isolated from Pseudomonas putida and its transcription was investigated in E. coli as well as in P. putida. In both species the P. putida dnaA gene is transcribed from two promoters, one of which shows strong homology to promoters recognized by the sigma 54 factor found in both bacteria. In E. coli transcription of the P. putida dnaA gene can be repressed by overproduction of E. coli DnaA protein, presumably due to the presence of several DnaA-box-like sequences found in the promoter region. Likewise the P. putida DnaA protein is able to regulate expression of the E. coli dnaA gene but we failed to demonstrate autoregulation of the P. putida dnaA gene. A point mutation was introduced into the P. putida dnaA gene, equivalent to the ATP binding site mutation present in E. coli dnaA5 and dnaA46 mutants, and this alteration abolished the ability of the protein to repress the expression of the E. coli dnaA gene. These results indicate that DnaA proteins from other species than E. coli have maintained the ability to recognize the DnaA box sequence and that the conservation between the DnaA proteins reflects functionally similar domains.

Alleles↗

PubMed1985

Autoregulation of the DNA replication gene dnaA in E. coli K-12.

The dnaA gene in E. coli K-12 is required for the initiation of DNA replication. Although the specific function of the dnaA protein is unknown, it has been suggested that it is a regulator of the frequency of initiation. In this paper we report that the expression of both a dnaA-lacZ translational fusion and a dnaA-trpA-lacZ transcriptional fusion in vivo are sensitive to changes in the level of functional dnaA protein. Overproduction of the dnaA gene product leads to a reduction in expression from both fusions while introduction of dnaA- alleles results in an increased expression. Results from a deletion analysis of the dnaA promoter/regulatory region suggest that both dnaA promoters are regulated by the dnaA gene product and that a site between the two promoters is responsible for the regulation. DNAase protection experiments showed that the dnaA protein binds to DNA in the region of the two dnaA promoters. Our results indicate that the dnaA gene product regulates its own synthesis by inhibiting transcription from both of its promoters.

Bacterial Proteins↗

PubMed1990

Purification and characterization of an initiation protein for chromosomal replication, DnaA, in Bacillus subtilis.

Bacillus subtilis DnaA protein was overproduced by a recombinant plasmid containing B. subtilis dnaA gene in a mutant Escherichia coli strain which is deficient in its own DnaA and RNaseH. The protein was purified to near homogeneity as judged by SDS-PAGE analysis. The purified protein binds preferentially to DNA fragments which are derived from flanking regions of the B. subtilis dnaA gene and contain various numbers of the repeat of 9 nucleotides, TTATCCACA, and closely related sequences. The purified protein binds ATP with high affinity (Kd = 0.02 microM) and ADP with less affinity, but does not bind cAMP. ATP stimulates the binding of the DnaA protein to the repeated sequences. DNaseI footprinting experiments demonstrated that the DnaA bound first to the consensus 9-mer and then to sequences differing by one base from the consensus. Sequences differing by two bases from the consensus were bound by the DnaA only when they were located contiguous to the strong DnaA-boxes. The three DnaA-box clusters, incA, incB, and incC, derived from the replication origin region of the B. subtilis chromosome showed different levels of growth inhibition when they were introduced into B. subtilis. We demonstrated by assaying competition for DnaA-binding among the DnaA-box clusters that there is a good correlation between the degree of growth inhibition by DnaA-box clusters in vivo and their strength of binding to the DNaA protein in vitro.

Adenosine Triphosphate↗

PubMed1992

Mutations in the DnaA binding sites of the replication origin of Escherichia coli.

Mutations (base changes) were introduced into the four DnaA binding sites (DnaA boxes) of the Escherichia coli replication origin, oriC. Mutations in a single DnaA box did not impair the ability of these origins to replicate in vivo and in vitro. A combination of mutations in two DnaA boxes, R1 and R4, resulted in slower growth of the oriC plasmid-bearing host cells. DnaA protein interaction with mutant and wild-type DnaA boxes was analyzed by DNase I footprinting. Binding of DnaA protein to a mutated DnaA box R1 was not affected by a mutation in DnaA box R4 and vice versa. Mutations in DnaA boxes R1 and R4 did not modify the ability of the DnaA protein to bind to other DnaA boxes in oriC.

Bacterial Proteins↗

PubMed1992

The chromosome origin of Escherichia coli stabilizes DnaA protein during rejuvenation by phospholipids.

DnaA protein (the initiator protein) binds and clusters at the four DnaA boxes of the Escherichia coli chromosomal origin (oriC) to promote the strand opening for DNA replication. DnaA protein activity depends on the tight binding of ATP; the ADP form of DnaA protein, generated by hydrolysis of the bound ATP, is inactive. Rejuvenation of ADP-DnaA protein, by replacement with ATP, is catalyzed by acidic phospholipids in a highly fluid bilayer. We find that interaction of DnaA protein with oriC DNA is needed to stabilize DnaA protein during this rejuvenation process. Whereas DnaA protein bound to oriC DNA responds to phospholipids, free DnaA protein is inactivated by phospholipids and then fails to bind oriC. Furthermore, oriC DNA facilitates the high affinity binding of ATP to DnaA protein during treatment with phospholipids. A significant portion of the DnaA protein associated with oriC DNA can be replaced by the ADP form of the protein, suggesting that all of the DnaA protein bound to oriC DNA need not be rejuvenated between rounds of replication.

Adenosine Diphosphate↗