Overinitiation of chromosome replication in the Escherichia coli dnaAcos mutant depends on activation of oriC function by the dam gene product

Katayama, Tsutomu; Akimitsu, Nobuyoshi; Mizushima, Tohru; Miki, Takeyoshi; Sekimizu, Kazuhisa

doi:10.1046/j.1365-2958.1997.5001872.x

Cited by 21 publications

(32 citation statements)

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“…By carrying out transposon mutagenesis of the dnaAcos mutant, which exhibits overinitiation of replication and inhibition of growth at 30 ° C (Katayama & Kornberg 1994;Katayama 2001), we previously confirmed that the dam gene, which encodes the Dam methyltransferase, is a stimulatory factor for initiation ( Katayama et al . 1997).…”

Section: Introductionmentioning

confidence: 83%

“…To test this idea, we first quantified cellular levels of DnaA protein in wild-type and dnaA508 strains with or without hspQ::mini-kan by immunoblot analysis as previously described (Katayama & Kornberg 1994;Katayama et al 1997). The introduction of hspQ::mini-kan did not affect the level of DnaA in the wild-type dnaA strain (Table 2).…”

Section: Figurementioning

confidence: 99%

“…Immunoblot analysis of DnaA was performed as previously described (Katayama & Kornberg 1994;Katayama et al 1997). Briefly, cells were grown exponentially at the indicated temperature in LB medium.…”

Section: Immunoblot Analysismentioning

confidence: 99%

“…1988;Chiaramello & Zyskind 1989;Hansen et al . 1991;Katayama & Kornberg 1994;Katayama et al . 1997).…”

Section: Introductionunclassified

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Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli

et al. 2004

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Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle. In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature-sensitive dnaA508 and dnaA167 mutants at 40 °°°° C. The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ , a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor σ σ σ σ 32. Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 °°°° C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ . Cellular DnaA508 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ -independent manner. Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins.

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Section: Introductionmentioning

confidence: 83%

Section: Figurementioning

confidence: 99%

Section: Immunoblot Analysismentioning

confidence: 99%

“…1988;Chiaramello & Zyskind 1989;Hansen et al . 1991;Katayama & Kornberg 1994;Katayama et al . 1997).…”

Section: Introductionunclassified

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Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli

et al. 2004

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“…pTOA5 (2.7 kb) and pTOA24 (6.1 kb) minichromosomes bearing oriC-mioC and gidA-oriC-mioC regions, respectively, in addition to the ␤-lactamase gene, were kindly provided by Dr. Tohru Ogawa (32). The media used are described in a previous report (24,26,33). M9 medium was supplemented with 0.2% glucose, 2 g/ml thiamine, 20 g/ml tyrosine, and 40 g/ml each of isoleucine, valine, threonine, methionine, and tryptophan.…”

Section: Methodsmentioning

confidence: 99%

DiaA, a Novel DnaA-binding Protein, Ensures the Timely Initiation of Escherichia coli Chromosome Replication

Ishida¹,

Akimitsu²,

Kashioka³

et al. 2004

Journal of Biological Chemistry

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111

191

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The DnaA protein is the initiator of Escherichia coli chromosomal 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-sensitive dnaA mutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. A diaA::Tn5 mutation suppresses the cold-sensitive growth of an overinitiation type dnaA mutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in the diaA mutant cells as well as wild-type cells with pBR322 expressing the diaA gene. 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 an in vitro system 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 DnaAassociating factor that is crucial to ensure the timely initiation of chromosomal replication.

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Countermeasures to survive excessive chromosome replication in Escherichia coli

2017

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In Escherichia coli, like all organisms, DNA replication is coordinated with cell cycle progression to ensure duplication of the genome prior to cell division. Chromosome replication is initiated from the replication origin, oriC, by the DnaA protein associated with ATP. Initiations take place once per cell cycle and in synchrony at all cellular origins. DnaA also binds ADP with similar affinity as ATP and in wild-type cells the majority of DnaA molecules are ADP bound. In cells where the DnaA/DnaA ratio increases or in cells where DnaA has increased access to oriC, premature initiations take place, often referred to as overinitiation. Overinitiating cells are generally characterized by their slow growth and in the most severe cases lethal accumulation of DNA strand breaks. Here, we review the different strategies adopted by E. coli to survive overinitiation. We propose a unifying model where all mutations that suppress overinitiation keep replication forks separated in time and, thereby, reduce the formation of strand breaks. One group of mutations does so by lowering the activity of oriC and/or DnaA to reduce the frequency of initiations to an acceptable level. In the other group of mutations, replication forks are kept apart by preventing formation of damages that would otherwise cause replication blocks, by allowing bypass of replication blocks and/or by slowing down replication forks. This group of suppressors restores viability despite excessive chromosome replication and provides new insights into mechanisms that safeguard DNA integrity.

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Overinitiation of chromosome replication in the Escherichia coli dnaAcos mutant depends on activation of oriC function by the dam gene product

Abstract: SummaryThe activity of DnaA protein, the initiator of chromosome replication in Escherichia coli, is regulated by adenine nucleotide binding; the ATP-bound form, not the ADP-bound form, is active. DnaAcos is a mutant protein that is insensitive to negative regulation by

Cited by 21 publications

References 60 publications

Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli

Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli

DiaA, a Novel DnaA-binding Protein, Ensures the Timely Initiation of Escherichia coli Chromosome Replication

Countermeasures to survive excessive chromosome replication in Escherichia coli

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