Reconstitution of in vitro inactivation and reactivation systems of DnaA protein for the control of chromosomal replication initiation in Escherichia coli

2006 ◽  
Author(s):  
Masayuki Su'etsugu ◽  
Kazuyuki Fujimitsu ◽  
Tsutomu Katayama
Keyword(s):  
Escherichia Coli ◽  
Replication Initiation ◽  
Chromosomal Replication ◽  
Dnaa Protein

scholarly journals DnaA, the Initiator of Escherichia coliChromosomal Replication, Is Located at the Cell Membrane

2000 ◽  
Vol 182 (9) ◽  
pp. 2604-2610 ◽  
Author(s):  
Gillian Newman ◽  
Elliott Crooke
Keyword(s):  
Cell Membrane ◽  
Spatial Organization ◽  
Active Form ◽  
Chromosomal Replication ◽  
E Coli ◽  
Dnaa Protein ◽  
Section Electron ◽  
Acidic Phospholipids

ABSTRACT 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.

scholarly journals Involvement of the Escherichia coli folate-binding protein YgfZ in RNA modification and regulation of chromosomal replication initiation

2006 ◽  
Vol 59 (1) ◽  
pp. 265-275 ◽  
Author(s):  
Tomotake Ote ◽  
Masayuki Hashimoto ◽  
Yoshiho Ikeuchi ◽  
Masayuki Su'etsugu ◽  
Tsutomu Suzuki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Protein ◽  
Replication Initiation ◽  
Rna Modification ◽  
Chromosomal Replication ◽  
Folate Binding Protein

scholarly journals Cnu, a Novel oriC-Binding Protein of Escherichia coli

2005 ◽  
Vol 187 (20) ◽  
pp. 6998-7008 ◽  
Author(s):  
Myung Suk Kim ◽  
Sung-Hun Bae ◽  
Sang Hoon Yun ◽  
Hee Jung Lee ◽  
Sang Chun Ji ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Replication Initiation ◽  
In Vitro Assays ◽  
Wild Type ◽  
Genetic Method ◽  
Dna Replication Initiation ◽  
Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

scholarly journals The Escherichia coli datA site promotes proper regulation of cell division

Microbiology ◽  
2014 ◽  
Vol 160 (4) ◽  
pp. 703-710 ◽  
Author(s):  
Morigen Morigen ◽  
Ingvild Flåtten ◽  
Kirsten Skarstad
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Binding Sites ◽  
Replication Initiation ◽  
Permissive Temperature ◽  
Initiator Protein ◽  
Dnaa Protein ◽  
Daughter Cells ◽  
Initiation Of Replication ◽  
Replication Initiator

In Escherichia coli inhibition of replication leads to a block of cell division. This checkpoint mechanism ensures that no cell divides without having two complete copies of the genome to pass on to the two daughter cells. The chromosomal datA site is a 1 kb region that contains binding sites for the DnaA replication initiator protein, and which contributes to the inactivation of DnaA. An excess of datA sites provided on plasmids has been found to lead to both a delay in initiation of replication and in cell division during exponential growth. Here we have investigated the effect of datA on the cell division block that occurs upon inhibition of replication initiation in a dnaC2 mutant. We found that this checkpoint mechanism was aided by the presence of datA. In cells where datA was deleted or an excess of DnaA was provided, cell division occurred in the absence of replication and anucleate cells were formed. This finding indicates that loss of datA and/or excess of DnaA protein promote cell division. This conclusion was supported by the finding that the lethality of the division-compromised mutants ftsZ84 and ftsI23 was suppressed by deletion of datA, at the lowest non-permissive temperature. We propose that the cell division block that occurs upon inhibition of DNA replication is, at least in part, due to a drop in the concentration of the ATP–DnaA protein.

scholarly journals Feedback Control of DnaA-Mediated Replication Initiation by Replisome-Associated HdaA Protein in Caulobacter

2009 ◽  
Vol 191 (18) ◽  
pp. 5706-5716 ◽  
Author(s):  
Justine Collier ◽  
Lucy Shapiro
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Regulatory Mechanism ◽  
Caulobacter Crescentus ◽  
Replication Initiation ◽  
Dnaa Protein ◽  
Additional Layer ◽  
Initiation Of Dna Replication ◽  
The Right ◽  
Feedback Regulatory Mechanism

ABSTRACT Chromosome replication in Caulobacter crescentus is tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. The timing of replication initiation is controlled by both CtrA and DnaA. CtrA binds to and silences the origin. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. Here, we identify an additional layer of replication initiation control that is mediated by the HdaA protein. In Escherichia coli, the Hda protein inactivates DnaA after replication initiation. We show that the Caulobacter HdaA homologue is necessary to restrict the initiation of DNA replication to only once per cell cycle and that it dynamically colocalizes with the replisome throughout the cell cycle. Moreover, the transcription of hdaA is directly activated by DnaA, providing a robust feedback regulatory mechanism that adjusts the levels of HdaA to inactivate DnaA.

Involvement of the Escherichia coli folate-binding protein YgfZ in RNA modification and regulation of chromosomal replication initiation

2006 ◽  
Vol 60 (1) ◽  
pp. 252-252 ◽  
Author(s):  
Tomotake Ote ◽  
Masayuki Hashimoto ◽  
Yoshiho Ikeuchi ◽  
Masayuki Su'etsugu ◽  
Tsutomu Suzuki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Protein ◽  
Replication Initiation ◽  
Rna Modification ◽  
Chromosomal Replication ◽  
Folate Binding Protein

scholarly journals DiaA, a Novel DnaA-binding Protein, Ensures the Timely Initiation ofEscherichia coliChromosome Replication

2004 ◽  
Vol 279 (44) ◽  
pp. 45546-45555 ◽  
Author(s):  
Takuma Ishida ◽  
Nobuyoshi Akimitsu ◽  
Tamami Kashioka ◽  
Masakazu Hatano ◽  
Toshio Kubota ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Replication Initiation ◽  
Temperature Sensitive ◽  
Minichromosome Maintenance ◽  
Chromosomal Replication ◽  
Timely Initiation ◽  
Initiation Of Replication ◽  
Direct Binding ◽  
Cold Sensitive

The DnaA protein is the initiator ofEscherichia colichromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitivednaAmutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. AdiaA::Tn5mutation suppresses the cold-sensitive growth of an overinitiation typednaAmutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in thediaAmutant cells as well as wild-type cells with pBR322 expressing thediaAgene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in anin vitrosystem especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication.

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