Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for <i>Escherichia coli</i> DnaA protein

Kitagawa, Risa; Ozaki, T.; Moriya, Shigeki; Ogawa, Tohru

doi:10.1101/gad.12.19.3032

Cited by 167 publications

(295 citation statements)

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“…DnaA binding to datA is thought to reduce the number of DnaA molecules accessible to oriC, thereby inhibiting untimely initiations (22)(23)(24)(25). Originally, datA was speculated to bind ∼370 DnaA molecules (22); however, this figure is deduced from indirect measurements not yet supported by direct evidence.…”

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confidence: 61%

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DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation

Kasho

Katayama

2012

Proc. Natl. Acad. Sci. U.S.A.

102

231

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The initiation of chromosomal DNA replication is rigidly regulated to ensure that it occurs in a cell cycle-coordinated manner. To ensure this in Escherichia coli, multiple systems regulate the activity of the replication initiator ATP-DnaA. The level of ATP-DnaA increases before initiation after which it drops via DnaA-ATP hydrolysis, yielding initiation-inactive ADP-DnaA. DnaA-ATP hydrolysis is crucial to regulation of initiation and mainly occurs by a replication-coupled feedback mechanism named RIDA (regulatory inactivation of DnaA). Here, we report a second DnaA-ATP hydrolysis system that occurs at the chromosomal site datA. This locus has been annotated as a reservoir for DnaA that binds many DnaA molecules in a manner dependent upon the nucleoid-associated factor IHF (integration host factor), resulting in repression of untimely initiations; however, there is no direct evidence for the binding of many DnaA molecules at this locus. We reveal that a complex consisting of datA and IHF promotes DnaA-ATP hydrolysis in a manner dependent on specific inter-DnaA interactions. Deletion of datA or the ihf gene increased ATP-DnaA levels to the maximal attainable levels in RIDA-defective cells. Cell-cycle analysis suggested that IHF binds to datA just after replication initiation at a time when RIDA is activated. We propose a model in which cell cycle-coordinated ATP-DnaA inactivation is regulated in a concerted manner by RIDA and datA.AAA+ | bacteria | initiation regulation | in vitro reconstitution | nucleoprotein complex

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confidence: 61%

“…1A), is crucial for repressing untimely initiation events (22)(23)(24)(25). datA-deleted cells, like seqA-deleted cells, perform untimely initiations at a level that does not inhibit cell growth (23).…”

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confidence: 99%

DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation

Kasho

Katayama

2012

Proc. Natl. Acad. Sci. U.S.A.

102

231

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“…The growth rates were determined by measuring the optical density of the cultures at 450 nm. The thr : : Tn10dnaC2 (Withers & Bernander, 1998), DdatA : : Kan (Kitagawa et al, 1998) and recA938 : : Cam (Zyskind et al, 1992) alleles were transferred by P1 transduction from MG1655dnaC2, RSD448 and ALS972, respectively, to the recipient strains listed in Table 1. W3110dnaC2 and MOR165 (W3110DdatAdnaC2) were transformed with MiniR1 or MiniR1-datA (Morigen et al, 2001) by electroporation.…”

Section: Methodsmentioning

confidence: 99%

The Escherichia coli datA site promotes proper regulation of cell division

Flåtten

Skarstad

2014

Microbiology

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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. INTRODUCTIONIn Escherichia coli cell division involves the formation of a septum that separates the cell into two daughter cells. It is crucial that the two daughter nucleoids are segregated into each half of the cell, so that each daughter cell receives a copy of the genetic material and that none of the nucleoids are trapped in the septum. To ensure this, cell division and chromosome segregation are highly regulated processes involving a number of different proteins (see Egan & Vollmer, 2013;Harry et al., 2006;and Weiss, 2004, for reviews), the most studied being FtsZ. This protein polymerizes to form a ring-like structure at the site of division and serves as a scaffold for recruitment of other division proteins (Bi & Lutkenhaus, 1991). How the timing of the formation of the FtsZ ring is regulated is not clear.It is known that the chromosome replication cycle is independent of the cell division cycle, but that cell division is dependent on chromosome replication (Boye & Nordström, 2003; Nordström et al., 1991). That is, a blockage of chromosome replication stops cell division (Helmstetter & Pierucci, 1968;Walker & Pardee, 1968). Thus, a checkpoint mechanism must exist. The mechanism ensures that cell division does not occur in the absence of replication and thus prevents the formation of DNA-free (anucleate) daughter cells (Boye & Nordström, 2003; Nordström et al., 1991). Whether this mechanism is related to other regulatory circuits necessary for coupling the division cycle to cell physiology and to the growth rate of the cells, is not clear (see Haeusser & Levin, 2008, for a review).The DnaA protein is one of the main players in initiation of replica...

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“…In E. coli, there are Ϸ300 DnaA-binding sites scattered all over the chromosome (11,12). Titration of the initiator to these sites is believed to reduce availability of the protein after replication initiation, because new sites are created during replication elongation.…”

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confidence: 99%

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Venkova-Canova¹,

Srivastava²,

Chattoraj³

2006

Proc. Natl. Acad. Sci. U.S.A.

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The oriCII region that includes the initiator gene, rctB, can function as a plasmid in E. coli. Here we show that RctB suffices for the oriCII-based plasmid replication, and rctA in cis or trans reduces the plasmid copy number, thereby serving as a negative regulator. The inhibitory activity could be overcome by increasing the concentration of RctB, suggesting that rctA titrates the initiator. Purified RctB bound to a DNA fragment carrying rctA, confirming that the two can interact. Although rctA apparently works as a titrating site, it is nonetheless transcribed. We find that the transcription attenuates the inhibitory activity of the gene, presumably by interfering with RctB binding. RctB, in turn, repressed the rctA promoter and, thereby, could control its own titration by modulating the transcription of rctA. This control circuit appears to be a putative novel mechanism for homeostasis of initiator availability.O ur knowledge of DNA replication control in bacteria comes largely from studying the chromosome and plasmids of Escherichia coli (1, 2). In general, controlling proteins that initiate replication, initiator proteins, seems to be the central mechanism to regulate replication. A variety of mechanisms have been found to control the initiator.Transcriptional autoregulation of the initiator gene is common in plasmids with iterated initiator-binding sites (iterons), where the initiator serves as the repressor of its own synthesis (3). The E. coli initiator, DnaA, also is autoregulated (4). In addition, the dnaA promoter is inactivated by sequestration to the cell membrane for approximately one-third of the cell generation (5). More traditional modes of transcriptional control using repressors and activators also have been found in phage and plasmid RK2 (6, 7).Initiator translation is regulated by antisense RNAs in a variety of plasmids, the classic examples being plasmids R1 and pT181 (8, 9). The antisense RNA either blocks the ribosomebinding site of the initiator gene directly or of a regulatory peptide for the initiator gene (10).Most other mechanisms operate posttranslationally either by titrating or inactivating the initiator protein. In E. coli, there are Ϸ300 DnaA-binding sites scattered all over the chromosome (11,12). Titration of the initiator to these sites is believed to reduce availability of the protein after replication initiation, because new sites are created during replication elongation.Initiator inactivation is the most widely used mechanism to prevent premature replication reinitiation. The pT181 initiator is inactivated by covalent modification at the end of one round of replication (13). E. coli DnaA is converted from an active ATP bound form to an inactive ADP form after replication (14-16). A similar mechanism also operates in Bacillus subtilis (17). The initiators of iteron-carrying plasmids are inactivated by dimerization, the active form being monomers (18). Dimerization not only reduces monomer concentration, the dimers also participate in inactivating origins (19)(20)(21). Euk...

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Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein

Cited by 167 publications

References 48 publications

DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation

DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation

The Escherichia coli datA site promotes proper regulation of cell division

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

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