DnaC Inactivation in Escherichia coli K-12 Induces the SOS Response and Expression of Nucleotide Biosynthesis Genes

Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark.
PLoS ONE (Impact Factor: 3.23). 02/2008; 3(8):e2984. DOI: 10.1371/journal.pone.0002984
Source: PubMed


Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations.
DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38 degrees C and 42 degrees C. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation.
We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart.

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Available from: Flemming G. Hansen
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    • "dnaA46 is a temperature-sensitive allele of the oriC-specific replication initiator that can sustain oriC-dependent replication initiation and therefore cell division at 30°C but not at 42°C. However, even at 30°C this allele is not fully functional[43,44]. We tested dnaB overexpression in a dnaA46 strain at the permissive temperature and found that, although colonies could form, they displayed significant growth defects as compared with dnaA + cells (Figure 6A, compare i and ii). "
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    ABSTRACT: Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The E. coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6-DnaC6 complex that is required for loading DnaB onto ssDNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6-DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased re-initiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6-DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6-DnaC6 complexes with excess DnaC [1, 2]. Thus there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication.
    Full-text · Article · Jan 2016 · Journal of Molecular Biology
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    • "We may also exclude the possibility that a ΔsfiA mutation – which we introduced in priA2 cells to prevent the priA2-induced SOS response to inhibit cell division – modulate the stability of RF or the rate at which they are inactivated since we established that the rate, at which inactivated RF accumulate, was identical in dnaC2 sfiA+ and in dnaC2 sfiA− cells (data not shown). An additional point may be made with regards to the SOS response: is it possible that the SOS response, which is induced in dnaC2 cells at non-permissive temperature [23], modifies the fate of ongoing rounds of replication? In this respect, an over-stabilization of active RF in dnaC2 cells in response to the induction of the SOS response may be excluded since this response is induced in priA2 cells as well. "
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    ABSTRACT: Replicative helicases unwind double-stranded DNA in front of the polymerase and ensure the processivity of DNA synthesis. In Escherichia coli, the helicase loader DnaC as well as factors involved in the formation of the open complex during the initiation of replication and primosomal proteins during the reactivation of arrested replication forks are required to recruit and deposit the replicative helicase onto single-stranded DNA prior to the formation of the replisome. dnaC2 is a thermosensitive allele of the gene specifying the helicase loader; at non-permissive temperature replication cannot initiate, but most ongoing rounds of replication continues through to completion (18% of dnaC2 cells fail to complete replication at non-permissive temperature). An assumption, which may be drawn from this observation, is that only a few replication forks are arrested under normal growth conditions. This assumption, however, is at odds with the severe and deleterious phenotypes associated with a null mutant of priA, the gene encoding a helicase implicated in the reactivation of arrested replication forks. We developed an assay that involves an abrupt inactivation of rounds of synchronized replication in a large population of cells, in order to evaluate the ability of dnaC2 cells to reactivate arrested replication forks at non-permissive temperature. We compared the rate at which arrested replication forks accumulated in dnaC2 priA(+) and dnaC2 priA2 cells and observed that this rate was lower in dnaC2 priA(+) cells. We conclude that while replication cannot initiate in a dnaC2 mutant at non-permissive temperature, a class of arrested replication forks (PriA-dependent and DnaC-independent) are reactivated within these cells.
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