The DNA-membrane fraction of Pneumococcus contains a DNA replication complex.
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Arginine↗
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Origin of replication of Mycoplasma genitalium.
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Base Composition↗
Requirements for replication restart proteins during constitutive stable DNA replication in Escherichia coli K-12.
Constitutive stable DNA replication (cSDR) is a mechanism for replisome loading in Escherichia coli K-12. This occurs in a dnaA-independent fashion in an rnhA mutant. cSDR is dependent on recA, priA, and transcription. In this report, it is shown that dnaA rnhA mutants using cSDR for initiation of their DNA replication additionally require priB, but not priC, for viability. Two subtle priA missense mutations either eliminated the ability to grow using cSDR (priA301 C479Y) or resulted in very small colonies (priA300 K230R). DnaC809, a priA suppressor, failed to allow priA or priB mutants to grow using cSDR to initiate DNA replication. Furthermore, unlike dnaC(+) strains, dnaC809 strains require priC for cSDR. DnaC809,820, a priC-independent suppressor of priA2::kan phenotypes, allowed priA and priC (but not priB) mutants to grow using cSDR to initiate DNA replication. It is also shown that rep and rnhA mutations are synthetically lethal. DnaC809 and dnaC809,820 mutations suppress this lethality. Rep is further shown to be required for cSDR in a dnaC809 strain. A model whereby these different sets of replication restart proteins interact preferentially with substrates associated with either RecA or SSB during replication restart and cSDR, respectively, is proposed.
Bacterial Proteins↗
DnaA, the initiator of Escherichia coli chromosomal replication, is located at the cell membrane.
Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.
Animals↗
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↗
The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12.
The sdrA224 mutants of Escherichia coli K-12, capable of continued DNA replication in the absence of protein synthesis (stable DNA replication), tolerate inactivation of the dnaA gene by insertion of transposon Tn10. Furthermore, oriC, the origin of E. coli chromosome replication, can be deleted from the chromosome of sdrA mutants without loss of viability. The results suggest the presence of a second, normally repressed, initiation system for chromosome replication alternative to the 'normal' dnaA+ oriC+-dependent initiation mechanism.
Bacterial Proteins↗
Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication.
The first step in conversion of varphiX174 singlestranded DNA to the duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate [Weiner, J. H., McMacken, R. & Kornberg, A. (1976) Proc. Natl. Acad. Sci. USA 73, 752-756]. We now demonstrate that dnaB protein (approximately one molecule per DNA circle) is an essential component of the intermediate and retains its ATPase activity. Synthesis of RNA primers, dependent on dnaG protein (primase), occurred only on DNA that had been converted to the intermediate form. In a coupled RNA priming-DNA replication reaction the first primer synthesized was extended by DNA polymerase III holoenzyme into full-length complementary strand DNA. In RNA priming uncoupled from replication, multiple RNA primers were initiated on a varphiX174 circle. The single dnaB protein molecule present on each DNA circle participated in initiation of each of the RNA primers, which appear to be aligned at regular intervals along the template strand. We propose that dnaB protein, once bound to the template, migrates in a processive fashion along the DNA strand, perhaps utilizing energy released by hydrolysis of ATP for propulsion; in this scheme the actively moving dnaB protein acts as a "mobile promoter" signal for dnaG protein (primase) to produce many RNA primers. Schemes are proposed for participation of dnaB protein both in the initiation of replication at the origin of the Escherichia coli chromosome and in the initiation of primers for nascent (Okazaki) fragments at a replication fork.
Adenosine Triphosphatases↗
TrfA dimers play a role in copy-number control of RK2 replication.
Copy-number regulation of the broad-host-range plasmid RK2 is dependent on the plasmid-encoded initiator protein, TrfA, and the RK2 origin of replication. The handcuffing model for copy-number control proposes that TrfA-bound oris reversibly couple to prevent the further initiation of plasmid replication when the copy number in vivo is at or above the replicon-specific copy number. TrfA mutants have been isolated which allow for oriV replication at elevated copy numbers. To better understand the mechanism of 'handcuffing', the copy-up TrfA(G254D/S267L) mutant was characterized further. In the present study we show by size exclusion chromatography and native gel electrophoresis that unlike wt TrfA which is largely dimeric, purified His6-TrfA(G254D/S267L) is primarily monomeric. In vivo, TrfA33(G254D/S267L) supports replication of an RK2 ori plasmid in trans at a greatly elevated copy number, while in cis the plasmid exhibits runaway replication. However, expression of either of two previously isolated DNA-binding defective TrfA mutants, TrfA33(P151S) or TrfA33(S257F), in a cell transformed with a mini-RK2 replicon encoding TrfA33(G254D/S267L) results in suppression of the runaway phenotype. His6-TrfA(P151S) and His6-TrfA(S257F) purify as dimers, and when expressed in vivo are incapable of supporting RK2 plasmid replication. In contrast, combination of the trfA(P151S) or trfA(S257F) mutation with the trfA(G254D/S267L) mutations results in the expression of mutant TrfA proteins which are mainly monomers and which can no longer restore copy control to replication directed by TrfA33(G254D/S267L) in vivo. On the basis of these findings a handcuffing model is proposed, whereby oriV-bound TrfA monomers are coupled by dimeric TrfA molecules.
Bacterial Proteins↗
Participation of hup gene product in ori2-dependent replication of fertility plasmid F.
The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42 degrees C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.
Bacterial Proteins↗
Molecular design of inhibitors of in vitro oriC DNA replication based on the potential to block the ATP binding of DnaA protein.
DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by binding to ATP. We earlier reported that 3-acetoxy-2,2'-bi-1H-indol inhibited the ATP binding to DnaA protein (Sasaki, S., Mizushima, T., Hashimoto, T., Maeda, M., and Sekimizu, K. (1994) Bioorg. Med. Chem. Lett. 4, 1771-1774). In the present study, derivatives of 3-acetoxy-2,2'-bi-1H-indol with different lengths of aliphatic chains at the 3-O position were synthesized, and their potential to inhibit the ATP binding to DnaA protein was examined. Elongation of the aliphatic chain resulted in inhibition of the ATP binding to DnaA protein at lower concentrations. Among the derivatives, 3-[N-(11-carboxyundecyl)]carbamoylmethoxy-2,2'-bi-1H-indol (structure 7 (3-CUCM-BI)) exhibited the most potent inhibition with an IC50 value of 7 microM. The mode of the inhibition was competitive. We further demonstrated that structure 7 (3-CUCM-BI) inhibited DNA replication of the oriC plasmid in a system reconstituted from purified proteins. This inhibition was specific for the initiation of DNA replication rather than for the elongation. The inhibition was overcome by preincubation of DnaA protein with ATP. Furthermore, structure 7 (3-CUCM-BI) showed little inhibition on DNA synthesis in the ABC primosome system. We propose that structure 7 (3-CUCM-BI) functions in the in vitro oriC DNA replication by inhibiting the ATP binding to DnaA protein.
Adenosine Triphosphate↗
Prophage P1, and extrachromosomal replication unit.
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Centrifugation, Density Gradient↗
recF and recR are required for the resumption of replication at DNA replication forks in Escherichia coli.
Escherichia coli containing a mutation in recF are hypersensitive to UV. However, they exhibit normal levels of conjugational or transductional recombination unless the major pathway (recBC) is defective. This implies that the UV sensitivity of recF mutants is not due to a defect in recombination such as occurs during conjugation or transduction. Here, we show that when replication is disrupted, at least two genes in the recF pathway, recF and recR, are required for the resumption of replication at DNA replication forks, and that in their absence, localized degradation occurs at the replication forks. Our observations support a model in which recF and recR are required to reassemble a replication holoenzyme at the site of a DNA replication fork. These results, when taken together with previous literature, suggest that the UV hypersensitivity of recF cells is due to an inability to resume replication at disrupted replication forks rather than to a defect in recombination. Current biochemical and genetic data on the conditions under which recF-mediated recombination occurs suggest that the recombinational intermediate also may mimic the structure of a disrupted replication fork.
Bacterial Proteins↗
The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone.
Initiation and control of replication of the broad-host-range plasmid RK2 requires two plasmid-encoded elements, the replication origin (oriV) and the initiation protein TrfA. Purified TrfA is largely in the form of a dimer; however, only the monomeric form of the protein can bind specifically to the direct repeats (iterons) at the RK2 origin. The largely dimeric form of wild-type TrfA is inactive in the initiation of replication of RK2 in an in vitro replication system reconstituted from purified components. However, preincubation of the TrfA protein with the ClpX molecular chaperone isolated from Escherichia coli activates the initiator protein for replication in the purified system. We further observed that ClpX, in an ATP-dependent reaction, greatly increases the proportion of TrfA monomers and, therefore, the ability of this protein to bind to iterons localized within RK2 origin. Finally, a copy-up mutant of the TrfA protein which is largely in the monomer form is active in the reconstituted in vitro replication system, and its activity is not affected by ClpX.
ATPases Associated with Diverse Cellular Activitie↗
Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus.
Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.
Caulobacter crescentus↗
Control of the bacterial cell cycle.
New insights into the control of DNA replication through growth, hemimethylated DNA and DnaA protein have been described. Fundamental shifts in thinking have resulted in the identification of new cell cycle genes with potential roles in initiation of DNA replication, chromosomal segregation and division. Excitingly, this trend may also narrow the apparent differences between the prokaryotic and eukaryotic cell cycles.
Cell Cycle↗
Mutational analysis of conserved hydrophobic amino acid residues in the N-terminal region of DnaA protein.
DnaA is the initiator of chromosomal DNA replication in E. coli. We previously reported that conserved hydrophobic amino acid residues in the N-terminal region of DnaA (I26 and L40) are essential for DNA replication in vivo and in vitro using mutant DnaA proteins (DnaAI26S and DnaAL40S). In this study, we introduced further random mutations to find intragenic suppressors for dnaAI26S or dnaAL40S. By direct DNA sequence, a mutation which causes substitution of the Ser (Ile, in the wild-type DnaA) with Phe (DnaAI26F or DnaAL40F) was found in all of the suppressors. Site-directed mutational analysis showed that DnaAI26L, and DnaAL40I, but not DnaAI26S or DnaAL40S, were active for oriC DNA replication in cells. Furthermore, purified DnaAI26F but not DnaAI26S was active for oriC DNA replication in a crude extract. These results strongly suggest that hydrophobic amino acid residues in these positions of DnaA (I26 and L40) are important for the function of this protein as an initiator of DNA replication both in vivo and in vitro.
Amino Acid Sequence↗
Plasmid R1 DNA replication dependent on protein synthesis in cell-free extracts of E. coli.
Bacterial plasmids serve as model systems for studying the regulation of DNA replication. Elucidation of the molecular mechanisms involved in plasmid DNA synthesis requires the development of efficient cell-free plasmid replication systems. Such in vitro systems have previously only been described for Col E1-type plasmids and for the R6K plasmid. Here we report that extracts of Escherichia coli can carry out the complete replication of miniplasmids derived from the antibiotic-resistance plasmid R1. This R1 replication system differs from the previously described ColE1 and R6K systems in its strict dependence on DNA-directed protein synthesis. We believe this to be the first report of the functional coupling of the three fundamental reactions of genetic information transfer (transcription, translation and replication) in a cell-free system.
Bacterial Proteins↗
Mutants of Staphylococcus aureus with increased sensitivity to ultraviolet radiation.
Nitrosoguanidine (NG) mutagenesis of Staphylococcus aureus resulted in the isolation of eight mutants exhibiting 3 to 28 times greater sensitivity to ultraviolet (UV) radiation. These mutants were further characterized by their ability to repair UV-irradiated bacteriophage, to act as recipients in the transduction of antibiotic resistance, and their sensitivity to NG. Based on the available data, six of these mutants are reduced in their ability to perform host-cell reactivation. One of the remaining two mutants may be deficient in post-replication repair.
Anti-Bacterial Agents↗