Reversibility of DnaA protein activity in the‘irreversible’<i>dnaA204</i> mutant of <i>Escherichia coli</i>

Hansen, Flemming; Atlung, Tove

doi:10.1111/j.1365-2958.1995.tb02228.x

Cited by 5 publications

(8 citation statements)

References 16 publications

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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%

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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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Section: Resultsmentioning

confidence: 99%

“…The present experiments do not address whether the DnaA204 protein is active and rapidly degraded at the non‐permissive temperature. It is certainly a possibility that rapid degradation accounts for the cessation of initiation of replication at high temperature (Hansen and Atlung, 1995).…”

Section: Discussionmentioning

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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“…whereas the mutational alterations in the other reversible dnaA mutants {dna.A46 an6 dnaA167) led to a decreased binding affinity ot DnaA protein to RNA polymerase, with the result that these two mutants exhibit virtually no rifampicinresistant initiation capacity, although they exhibit a significant chloramphenicol-resistanf initiation capacity. It should also be emphasized thaf when wild-type DnaA protein is overexpressed in wild-type or dnaA204 mutant ceils this results in considerable amounts ot rifampicin-resistant initiation capacity (Atlung and Hansen, 1993;Hansen and Atlung, 1995). Finally, if shouid also be kept in mind that it is possible to isolate allele-specitic dnaA{Ts) suppressor mutants in the rpoB gene, which, in many (most) cases, makes the mutated RNA polymerase rifampicin-resistant (Atlung, 1984).…”

Section: Discussionmentioning

confidence: 99%

“…The dnaA46, dnaAS, dna167, dnaA601, and dnaA604 mutants can reinitiate in the absence of protein synthesis and were classed as reversible mutants, whereas the dnaA508, dnaA204, and dnaA205 mutants cannot reinitiate in the absence of protein synthesis and, therefore. Received 10 May, 1994;revised 1 September, 1994;accepted 6 September, 1994. tPresent were called irreversible (see the accompanying paper by Hansen (1995) for references). Most dnaA mutants, both the reversible and the irreversibie ones, beoome temperature resistant if they contain pBR322-derived plasmids carrying the dnaA gene with the respective dnaA mutant alieie {Hansen etal., 1992).…”

Section: Introductionmentioning

confidence: 99%

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

Hansen

1995

Molecular Microbiology

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The kinetics of reinitiation of chromosome replication of eight dnaA(Ts) mutants was investigated in an isogenic set of strains. Five mutants (167, 46, 601, 606 and 5) are classified as reversible, since they can reinitiate at 30 degrees C without protein synthesis, whereas the other three (508, 205, 204) require protein synthesis. In the presence of protein synthesis, reversible mutants initiate one round of replication rapidly after a shift to 30 degrees C, indicating that they contain active or renaturable DnaA protein. The dnaA508 and dnaA204 mutants also reinitiate chromosome replication rapidly, whereas reinitiation is delayed 15-20 min in dnaA205. The dnaA508 and dnaA204 mutants might contain active DnaA protein just below the threshold level at 42 degrees C and only require synthesis of small amounts of new DnaA protein before initiation at 30 degrees C, whereas dnaA205 accumulates DnaA protein for some time at 30 degrees C before reaching the initiation threshold. Three of the reversible mutants (5, 601, and 606) exhibited, in addition to the protein synthesis-independent initiation capacity, an RNA synthesis-independent initiation capacity. The thermal stability of these initiation capacities is the same as for mutant DnaA protein, strongly suggesting that mutant DnaA protein is responsible for both.

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“…This may be due to contribution of the dnaA204 allele, which maintains residual activity at 42ЊC (29).…”

Section: Resultsmentioning

confidence: 99%

Novel alleles of the Escherichia coli dnaA gene are defective in replication of pSC101 but not of oriC

Sutton

Kaguni

1995

J Bacteriol

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Five novel alleles of the Escherichia coli dnaA gene that were temperature sensitive in maintenance of pSC101, a plasmid that is dependent on this gene for replication, were isolated. Nucleotide sequence analysis revealed that four of the five alleles arose from single base substitutions, whereas the fifth contained three base substitutions, two of which were silent. Whereas all five alleles were temperature sensitive in vivo for pSC101 maintenance, genetic and biochemical characterization indicated that only two were defective in replication from the chromosomal origin, oriC. As previously characterized mutations are defective in replication for both pSC101 and oriC, the dnaA mutations specifically defective in pSC101 maintenance represent a novel class. We speculate that one or more of these pSC101-specific mutants are defective in interaction with pSC101 RepA protein, which is also required for initiation of plasmid DNA replication.

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Reversibility of DnaA protein activity in the‘irreversible’dnaA204 mutant of Escherichia coli

Cited by 5 publications

References 16 publications

The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein

The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein

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

Novel alleles of the Escherichia coli dnaA gene are defective in replication of pSC101 but not of oriC

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