Reinitiation kinetics in eight <i>dnaA</i>(Ts) mutants of <i>Escherichia coli</i>: rifampicin‐resistant Initiation of chromosome replication

Hansen, Flemming

doi:10.1111/j.1365-2958.1995.tb02227.x

Cited by 15 publications

(16 citation statements)

References 29 publications

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“…The increase in oriC per mass ratio during rifampicin treatment was high for about 60 min and then virtually stopped. DnaA‐dependent rifampicin‐resistant initiations have been observed previously in different dnaA mutants, dnaA5 (Hanna and Carl, 1975), dnaA601 and dnaA606 (Hansen, 1995), but not in the dnaA46 mutant (Messer et al ., 1975; Hansen and Rasmussen, 1977). It is even more striking that the origin concentration in the ihf mutant after rifampicin treatment is very similar to that of the wild‐type control.…”

Section: Discussionmentioning

confidence: 67%

Rifampicin‐resistant initiation of chromosome replication from oriC in ihf mutants

Freiesleben

Rasmussen

Atlung

et al. 2000

Molecular Microbiology

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IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run‐out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from oriC in a rifampicin‐resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration. This shows that the initiation mass was substantially higher in the ihf mutants. The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild‐type strain. This indicates that cells lacking IHF replicate their chromosome(s) very fast.

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Section: Discussionmentioning

confidence: 67%

Rifampicin‐resistant initiation of chromosome replication from oriC in ihf mutants

Freiesleben

Rasmussen

Atlung

et al. 2000

Molecular Microbiology

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“…Many of these RNA polymerase mutants are also rifampin-resistant, suggesting that the sites of DnaA and rifampin interaction may be near each other. Rifampin-resistant initiation has also been found in some dnaA(Ts) mutants (dnaA606, dnaA601, and dnaA5) but not others (dnaA167 and dnaA46), upon shift-down to permissive temperature after prolonged incubation at nonpermissive temperature (17). It is possible that the former mutants retain the capacity of DnaA to "protect" RNA polymerase from rifampin, while the latter ones do not.…”

Section: Discussionmentioning

confidence: 92%

Deletion of the datA Site Does Not Affect Once-per-Cell-Cycle Timing but Induces Rifampin-Resistant Replication

2005

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In Escherichia coli, three mechanisms have been proposed to maintain proper regulation of replication so that initiation occurs once, and only once, per cell cycle. First, newly formed origins are inactivated by sequestration; second, the initiator, DnaA, is inactivated by the Hda protein at active replication forks; and third, the level of free DnaA protein is reduced by replication of the datA site. The datA site titrates unusually large amounts of DnaA and it has been reported that reinitiation, and thus asynchrony of replication, occurs in cells lacking this site. Here, we show that reinitiation in ⌬datA cells does not occur during exponential growth and that an apparent asynchrony phenotype results from the occurrence of rifampin-resistant initiations. This shows that the datA site is not required to prevent reinitiation and limit initiation of replication to once per generation. The datA site may, however, play a role in timing of initiation relative to cell growth. Inactivation of active ATP-DnaA by the Hda protein and the sliding clamp of the polymerase was found to be required to prevent reinitiation and asynchrony of replication.Chromosome replication of Escherichia coli is initiated at a single origin, oriC, by the initiator protein, DnaA (28). In rapidly growing cells, rounds of replication overlap and two, four, or even eight origins coexist and are initiated simultaneously only once per cell cycle (45). The new origins are immediately inactivated by sequestration. Sequestration relies on binding of the SeqA protein to the newly replicated, hemimethylated origins. Subsequent methylation by Dam methylase ends the sequestration (12,35,50). Sequestration is one of the processes necessary to limit initiation of replication to once and only once per cell cycle. In addition to the sequestration, a reduction of the initiation potential (i.e., sufficient amounts of active DnaA) must be achieved during the sequestration period, such that when origins are released from sequestration they cannot be reinitiated (46), and initiation cannot occur again until one generation later, when enough initiation potential again has accumulated. In this way the E. coli cell does not need to keep track of how many chromosomal origins have been initiated every generation; it is sufficient to first initiate all and then sequester all origins. This explains why E. coli can harbor large numbers of minichromosomes (oriC plasmids) without problems of incompatibility. The minichromosomal origins are initiated in synchrony with the chromosomal origins (30, 31) and sequestered in the same way.The reduction in initiation potential during sequestration can be achieved in two ways, (i) by reducing the availability of DnaA and (ii) by reducing the activity of DnaA. The availability of DnaA at oriC is affected by binding sites (DnaA boxes) distributed around the E. coli chromosome (19). One site in particular, datA, with five DnaA boxes, titrates unusually large amounts of DnaA protein in vivo (24) and is the main contributor to the DnaA ...

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“…Thus, in accordance with earlier reports, we find that, in rich medium, DNA replication in the ΔseqA strain is somewhat aberrant (von Freiesleben et al ., 1994; Lu et al ., 1994), whereas it is fairly normal in the dnaA204 strain (Skarstad et al ., 1988; Hansen, 1995; Hansen and Atlung, 1995).…”

Section: Resultsmentioning

confidence: 99%

The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein

Torheim¹,

Boye²,

Løbner-Olesen³

et al. 2000

Molecular Microbiology

100

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In wild‐type Escherichia coli cells, initiation of DNA replication is tightly coupled to cell growth. In slowly growing dnaA204 (Ts) mutant cells, the cell mass at initiation and its variability is increased two‐ to threefold relative to wild type. Here, we show that the DnaA protein concentration was two‐ to threefold lower in the dnaA204 mutant compared with the wild‐type strain. The reason for the DnaA protein deficiency was found to be a rapid degradation of the mutant protein. Absence of SeqA protein stabilized the DnaA204 protein, increased the DnaA protein concentration and normalized the initiation mass in the dnaA204 mutant cells. During rapid growth, the dnaA204 mutant displayed cell cycle parameters similar to wild‐type cells as well as a normal DnaA protein concentration, even though the DnaA204 protein was highly unstable. Apparently, the increased DnaA protein synthesis compensated for the protein degradation under these growth conditions, in which the doubling time was of the same order of magnitude as the half‐life of the protein. Our results suggest that the DnaA204 protein has essentially wild‐type activity at permissive temperature but, as a result of instability, the protein is present at lower concentration under certain growth conditions. The basis for the stabilization in the absence of SeqA is not known. We suggest that the formation of stable DnaA–DNA complexes is enhanced in the absence of SeqA, thereby protecting the DnaA protein from degradation.

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Reinitiation kinetics in eight dnaA(Ts) mutants of Escherichia coli: rifampicin‐resistant Initiation of chromosome replication

Cited by 15 publications

References 29 publications

Rifampicin‐resistant initiation of chromosome replication from oriC in ihf mutants

Rifampicin‐resistant initiation of chromosome replication from oriC in ihf mutants

Deletion of the datA Site Does Not Affect Once-per-Cell-Cycle Timing but Induces Rifampin-Resistant Replication

The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein

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