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  • 1
    Electronic Resource
    Electronic Resource
    Analysis of the DNA-binding domain of Escherichia coli DnaA protein (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374–467). The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC::lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC::lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01881.x
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  • 2
    Electronic Resource
    Electronic Resource
    The Escherichia coli SeqA protein binds specifically and co-operatively to two sites in hemimethylated and fully methylated oriC (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Escherichia coli SeqA protein has been found to affect initiation of replication negatively, both in vivo and in vitro. The mechanism of inhibition is, however, not known. SeqA has been suggested to affect the formation and activity of the initiation complex at oriC, either by binding to DNA or by interacting with the DnaA protein. We have investigated the binding of SeqA to oriC by electron microscopy and found that SeqA binds specifically to two sites in oriC, one on each side of the DnaA binding site R1. Specific binding was found for fully and hemimethylated but not unmethylated oriC in good agreement with earlier mobility shift studies. The affinity of SeqA for hemimethylated oriC was higher than for fully methylated oriC. The binding was in both cases strongly co-operative. We suggest that SeqA binds to two nucleation sites in oriC, and by the aid of protein–protein interaction spreads to adjacent regions in the same oriC as well as recruiting additional oriC molecules and/or complexes into larger aggregates.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01943.x
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  • 3
    Electronic Resource
    Electronic Resource
    The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome (2001)
    Oxford, UK : Blackwell Science, Ltd
    Molecular microbiology 40 (2001), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2001.02409.x
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  • 4
    Electronic Resource
    Electronic Resource
    The interaction domains of the DnaA and DnaB replication proteins of Escherichia coli (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The initiation of chromosome replication in Escherichia coli requires the recruitment of the replicative helicase DnaB from the DnaBC complex to the unwound region within the replication origin oriC, supported by the oriC-bound initiator protein DnaA. We defined physical contacts between DnaA and DnaB that involve residues 24–86 and 130–148 of DnaA and residues 154–210 and 1–156 of DnaB respectively. We propose that contacts between DnaA and DnaB occur via two interaction sites on each of the proteins. Interaction domain 24–86 of DnaA overlaps with its N-terminal homo-oligomerization domain (residues 1–86). Interaction domain 154–210 of DnaB overlaps or is contiguous with the domains known to interact with plasmid initiator proteins. Loading of the DnaBC helicase in vivo can only be performed by DnaA derivatives containing (in addition to residues 24–86 and the DNA-binding domain 4) a structurally intact domain 3. Nucleotide binding by domain 3 is, however, not required. The parts of DnaA required for replication of pSC101 were clearly different from those used for helicase loading. Domains 1 and 4 of DnaA, but not domain 3, were found to be involved in the maintenance of plasmid pSC101.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.02096.x
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  • 5
    Electronic Resource
    Electronic Resource
    DnaA protein stimulates polA gene expression in Escherichia coli (1997)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 23 (1997), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The polA gene of Escherichia coli encodes DNA polymerase I that is involved in DNA replication and repair. Despite the wide knowledge about structure and function of DNA polymerase I, there is little insight into the regulatory mechanisms involved in polA expression. DnaA is the initiator protein for DNA replication in E. coli. There are two putative DnaA-binding sites within the extended promoter region of polA. In this work we studied the influence of altered levels of DnaA protein on polA expression. We found that DnaA overproduction increases polA expression in stationary-phase cultures. The stimulation effect was independent of rpoS, which encodes the sigma factor for stationary-phase-inducible genes. However, it was modulated by ppGpp. Comparative S1 analyses revealed that the induction was based on transcriptional stimulation. Footprint-ing experiments demonstrated that DnaA binds only to the proximal DnaA box near the polA promoter. These results suggest an additional role for DnaA as transcriptional activator of polA at least under certain physiological conditions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1997.2961658.x
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  • 6
    Electronic Resource
    Electronic Resource
    DnaA initiator—also a transcription factor (1997)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 24 (1997), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The replication-initiator protein DnaA is ubiquitous in the eubacterial world. It binds to an asymmetric 9 bp consensus DNA sequence, the DnaA box. Besides its primary function as an initiator, it acts as a transcription factor that represses or activates several genes, or terminates transcription, depending on the location and arrangement of DnaA boxes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1997.3171678.x
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  • 7
    Electronic Resource
    Electronic Resource
    The N-terminus promotes oligomerization of the Escherichia coli initiator protein DnaA (1999)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 34 (1999), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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 cI repressor: a λcI-DnaA chimeric protein inhibits λ plasmid replication as efficiently as λcI 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’.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01568.x
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  • 8
    Electronic Resource
    Electronic Resource
    A comprehensive set of DnaA-box mutations in the replication origin, oriC, of Escherichia coli (1996)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 21 (1996), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We probed the complex between the replication origin, oriC, and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1–R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non-box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes. For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype. This demonstrates that all DnaA boxes in oriC have a function in the initiation process. Most mutants with point mutations retained some origin activity, and the in vitro DnaA-binding capacity of these origins correlated well with their replication proficiency. Inversion or scrambling of DnaA boxes R1 or M inactivated oriC-dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites. In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild-type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations. We conclude that these origins require more DnaA per origin for initiation than does wild-type oriC. Mutants in DnaA box R3 behaved essentially like wild-type oriC, except for those in which the low-affinity box R3 was replaced by the high-affinity box R1. Apparently, initiation is possible without DnaA binding to box R3, but high-affinity DnaA binding to DnaA box R3 upsets the regulation. Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build-up of the initiation complex and replication initiation in vivo
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1996.6481362.x
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  • 9
    Electronic Resource
    Electronic Resource
    High-affinity binding sites for the initiator protein DnaA on the chromosome of Escherichia coli (1998)
    Oxford BSL : Blackwell Science Ltd, UK
    Molecular microbiology 28 (1998), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The initiator protein DnaA of Escherichia coli binds with unusually high affinity to five regions on the chromosome, in addition to the replication origin, oriC. Using a solid-phase DNA binding assay, in which the DNA binding C-terminal domain of DnaA is bound via a biotin tag to magnetic beads, we could fish only fragments with these six regions from different chromosomal digests. Except for oriC, these fragments contain only one or two consensus DnaA binding sites, DnaA boxes. The distribution of these high-affinity DnaA boxes on the chromosome is random.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00813.x
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  • 10
    Electronic Resource
    Electronic Resource
    The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication (2002)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology reviews 26 (2002), S. 0 
    Details
    ISSN: 1574-6976
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The initiation of replication is the central event in the bacterial cell cycle. Cells control the rate of DNA synthesis by modulating the frequency with which new chains are initiated, like all macromolecular synthesis. The end of the replication cycle provides a checkpoint that must be executed for cell division to occur. This review summarizes recent insight into the biochemistry, genetics and control of the initiation of replication in bacteria, and the central role of the initiator protein DnaA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6976.2002.tb00620.x
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