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1.
In bacteria, initiation of DNA replication requires the DnaA protein. Regulation of DnaA association and activity at the origin of replication, oriC, is the predominant mechanism of replication initiation control. One key feature known to be generally important for replication is DNA topology. Although there have been some suggestions that topology may impact replication initiation, whether this mechanism regulates DnaA‐mediated replication initiation is unclear. We found that the essential topoisomerase, DNA gyrase, is required for both proper binding of DnaA to oriC as well as control of initiation frequency in Bacillus subtilis. Furthermore, we found that the regulatory activity of gyrase in initiation is specific to DnaA and oriC. Cells initiating replication from a DnaA‐independent origin, oriN, are largely resistant to gyrase inhibition by novobiocin, even at concentrations that compromise survival by up to four orders of magnitude in oriC cells. Furthermore, inhibition of gyrase does not impact initiation frequency in oriN cells. Additionally, deletion or overexpression of the DnaA regulator, YabA, significantly modulates sensitivity to gyrase inhibition, but only in oriC and not oriN cells. We propose that gyrase is a negative regulator of DnaA‐dependent replication initiation from oriC, and that this regulatory mechanism is required for cell survival. 相似文献
2.
Replication of the Escherichia coli chromosome is initiated synchronously from all origins (oriC) present in a cell at a fixed time in the cell cycle under given steady state culture conditions. A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which titrates exceptionally large amounts of the bacterial initiator protein, DnaA, to prevent overinitiation. Deletion of the datA locus results in extra initiations and altered temporal control of replication. There are many other sites on the E. coli chromosome that can bind DnaA protein, but the contribution of these sites to the control of replication initiation has not been investigated. In the present study, seven major DnaA binding sites other than datA have been examined for their influence on the timing of replication initiation. Disruption of these seven major binding sites, either individually or together, had no effect on the timing of initiation of replication. Thus, datA seems to be a unique site that adjusts the balance between free and bound DnaA to ensure that there is only a single initiation event in each bacterial cell cycle. Mutation either in the second or the third DnaA box (a 9 basepair DnaA-binding sequence) in datA was enough to induce asynchronous and extra initiations of replication to a similar extent as that observed with the datA-deleted strain. These DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region. 相似文献
3.
Control of DNA replication initiation is essential for normal cell growth. A unifying characteristic of DNA replication initiator proteins across the kingdoms of life is their distinctive AAA+ nucleotide-binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains, that in vitro stretches DNA to promote replication origin opening. The Bacillus subtilis protein Soj/ParA has previously been shown to regulate DnaA-dependent DNA replication initiation; however, the mechanism underlying this control was unknown. Here, we report that Soj directly interacts with the AAA+ domain of DnaA and specifically regulates DnaA helix assembly. We also provide critical biochemical evidence indicating that DnaA assembles into a helical oligomer in vivo and that the frequency of replication initiation correlates with the extent of DnaA oligomer formation. This work defines a significant new regulatory mechanism for the control of DNA replication initiation in bacteria. 相似文献
4.
Mizushima T 《Journal of biochemistry》2000,127(1):1-7
DnaA protein, the initiator for chromosomal DNA replication in Escherichia coli, has various activities, such as oligomerization (DnaA-DnaA interaction), ATP-binding, ATPase activity and membrane-binding. Site-directed mutational analyses have revealed not only the amino acid residues that are essential for these activities but also the functions of these activities. Following is a summary of the functions and regulatory mechanisms of DnaA protein in the initiation of chromosomal DNA replication. ATP-bound DnaA protein, but not other forms of the protein binds to the origin of DNA replication and forms oligomers to open-up the duplex DNA. This oligomerization is mediated by a DnaA-DnaA interaction through the N-terminal region of the protein. After initiation of DNA replication, the ATPase activity of DnaA protein is stimulated and DnaA protein is inactivated to the ADP-bound form to suppress the re-initiation of DNA replication. DnaA protein binds to acidic phospholipids through an ionic interaction between basic amino acid residues of the protein and acidic residues of phospholipids. This interaction seems to be involved in the re-activation of DnaA protein (from the ADP-bound form to the ATP-bound form) to initiate DNA replication after the appropriate interval. 相似文献
5.
Madiraju MV Moomey M Neuenschwander PF Muniruzzaman S Yamamoto K Grimwade JE Rajagopalan M 《Molecular microbiology》2006,59(6):1876-1890
Oligomerization of the initiator protein, DnaA, on the origin of replication (oriC) is crucial for initiation of DNA replication. Studies in Escherichia coli (Gram-negative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram-positive), we investigated interactions of M. tuberculosis DnaA (DnaA(TB)) with oriC using surface plasmon resonance in the presence of ATP and ADP. We provide evidence that, in contrast to what is observed in E. coli, ATPase activity of DnaA(TB) promoted rapid oligomerization on oriC. In support, we found that a recombinant mutant DnaA(TB) proficient in binding to ATP, but deficient in ATPase activity, did not oligomerize as rapidly. The corresponding mutation in the dnaA gene of M. tuberculosis resulted in non-viability, presumably due to a defect in oriC-DnaA interactions. Dimethy sulphate (DMS) footprinting experiments revealed that DnaA(TB) bound to DnaA boxes similarly with ATP or ADP. DnaA(TB) binding to individual DnaA boxes revealed that rapid oligomerization on oriC is triggered only after the initial interaction of DnaA with individual DnaA boxes. We propose that ATPase activity enables the DnaA protomers on oriC to rapidly form oligomeric complexes competent for replication initiation. 相似文献
6.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2017,1861(9):2165-2174
DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~ 15 Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication. 相似文献
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The replication origin of the broad-host-range plasmid RK2, oriV, contains four DnaA boxes, which bind the DnaA protein isolated from Escherichia coli. Using a transformation assay, mutational analysis of these boxes showed a differential requirement for replication in different Gram-negative bacteria. DnaA boxes 3 and 4 were required in E. coli and Pseudomonas putidabut not as strictly in Azotobacter vinelandii and not at all in P. aeruginosa. In vitro replication results using an extract prepared from E. coli demonstrated that the activity of origin derivatives containing mutations in boxes 3 or 4 or a deletion of all four DnaA boxes could be restored by the addition of increasing amounts of purified DnaA protein. High levels of DnaA protein in the presence of the TrfA protein also resulted in the stimulation of open complex formation and DnaB helicase loading on oriV, even in the absence of the four DnaA boxes. These observations at least raise the possibility that an alternative mechanism of initiation of oriV is being used in the absence of the four DnaA boxes and that this mechanism may be similar to that used in P. aeruginosa, which does not require these four DnaA boxes for replication. 相似文献
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Bacteria regulate the frequency and timing of DNA replication initiation by controlling the activity of the replication initiator protein DnaA. SirA is a recently discovered regulator of DnaA in Bacillus subtilis whose synthesis is turned on at the start of sporulation. Here, we demonstrate that SirA contacts DnaA at a patch of 3 residues located on the surface of domain I of the replication initiator protein, corresponding to the binding site used by two unrelated regulators of DnaA found in other bacteria. We show that the interaction of SirA with domain I inhibits the ability of DnaA to bind to the origin of replication. DnaA mutants containing amino acid substitutions of the 3 residues are functional in replication initiation but are immune to inhibition by SirA. 相似文献
12.
Kedar GC Ozcan F Guzmán EC Smith DW Newman VG Zyskind JW 《Journal of molecular microbiology and biotechnology》2000,2(3):301-310
DnaA protein is required for the initiation of DNA replication at the bacterial chromosomal origin, oriC, and at the origins of many plasmids. The concentration of DnaA protein is an important factor in determining when initiation occurs during the cell cycle. Methylation of GATC sites in the dnaAp2 promoter, two of which are in the -35 and -10 sequences, has been predicted to play an important role in regulating dnaA gene expression during the cell cycle because the promoter is sequestered from methylation immediately following replication. Mutations that eliminate these two GATC sites but do not substantially change the activity of the promoter were introduced into a reporter gene fusion and into the chromosome. The chromosomal mutants are able to initiate DNA replication synchronously at both moderately slow and fast growth rates, demonstrating that GATC methylation at these two sites is not directly involved in providing the necessary amount of DnaA for precise timing of initiation during the cell cycle. Either sequestration does not involve these GATC sites, or cell cycle control of DnaA expression is not required to supply the concentration necessary for correct timing of initiation. 相似文献
13.
In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes. 相似文献
14.
Nishida S Fujimitsu K Sekimizu K Ohmura T Ueda T Katayama T 《The Journal of biological chemistry》2002,277(17):14986-14995
The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp- and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides. 相似文献
15.
The purified DnaA protein has a high affinity for cyclic AMP (cAMP). Using equilibrium dialysis, we determined the K(A) value for cAMP as 0.819 muM(-1). The number of cAMP binding sites per DnaA protein molecule was calculated to be 1.04. This binding was quite specific for cAMP. ATP was also bound by DnaA protein and inhibited cAMP binding. This inhibition was non-competitive in nature with an inhibition constant (K(i)) of about 8.25 muM. However, in vivo we have found not only that the DnaA protein level is reduced in a cyclase deletion mutant strain, Delta++ cya, but also that DnaA protein is not degraded. The Delta cya mutants of E. coli are unable to continue DNA synthesis in the absence of de novo protein synthesis and the initiation of DNA replication in these mutants takes place from oriC. 相似文献
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Christoph Weigel rea Schmidt Harald Seitz Doreen Tüngler Michaela Welzeck & Walter Messer 《Molecular microbiology》1999,34(1):53-66
Initiation of chromosome replication in Escherichia coli is governed by the interaction of the initiator protein DnaA with the replication origin oriC. Here we present evidence that homo-oligomerization of DnaA via its N-terminus (amino acid residues 1-86) is also essential for initiation. Results from solid-phase protein-binding assays indicate that residues 1-86 (or 1-77) of DnaA are necessary and sufficient for self interaction. Using a 'one-hybrid-system' we found that the DnaA N-terminus can functionally replace the dimerization domain of coliphage lambda cl repressor: a lambdacl-DnaA chimeric protein inhibits lambda plasmid replication as efficiently as lambdacI repressor. DnaA derivatives with deletions in the N-terminus are incapable of supporting chromosome replication from oriC, and, conversely, overexpression of the DnaA N-terminus inhibits initiation in vivo. Together, these results indicate that (i) oligomerization of DnaA N-termini is essential for protein function during initiation, and (ii) oligomerization does not require intramolecular cross-talk with the nucleotide-binding domain III or the DNA-binding domain IV. We propose that E. coli DnaA is composed of largely independent domains - or modules - each contributing a partial, though essential, function to the proper functioning of the 'holoprotein'. 相似文献
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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. 相似文献
20.
The dnaA gene function, indispensable for the initiation of Escherichia coli replication from oriC is not essential for the growth of phage lambda. The in-vitro replication of plasmids derived from phage lambda does not seem to require DnaA protein either. However, we present evidence that in vivo the normal replication of lambda plasmids is dnaA-dependent. After inactivating the dnaA gene function, half of the plasmid molecules may enter a single round of replication. Rifampicin sensitivity of this abortive, as well as normal, replication indicates involvement of RNA polymerase. The rifampicin resistance of the normal replication of lambda plasmids in E. coli carrying the dnaAts46 or dnaAts5, but not the dnaAts204 allele at 30 degrees C implies the interaction of DnaA protein and RNA polymerase in this process. We propose that DnaA protein co-operates with RNA polymerase in the initiation of replication at ori lambda. The dispensability of DnaA in the growth of phage lambda and in lambda plasmid replication in vitro is discussed. 相似文献