Multiple homeostatic mechanisms regulate initiation of chromosome replication in Escherichia coli: Characterization of the Hda protein

Leise Riber, Anders Løbner-Olesen (Supervisor)

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In Escherichia coli, initiation of chromosome replication is highly regulated in such a way that multiple origins are initiated virtually simultaneously, and only once per cell cycle. Several mechanisms seem to act in the regulation of replication initiation of which most are described in previous publications. However, a higher understanding of these control mechanisms remains to be elucidated. Sequestration of the dnaA gene promoter is thought to prevent de novo synthesis of DnaA protein during origin sequestration. Cells in which the dnaA gene and oriC were not sequestered simultaneously were found to display initiation asynchrony, and reinitiation occasionally occurred at some origins within the same cell cycle, probably because newly synthesized DnaA protein, mainly ATP-bound, would accumulate during origin sequestration. This suggests that coordinated sequestration of dnaA and oriC is important and involved in maintaining controlled once-per-cell cycle initiation in E. coli. Initiation of DNA replication in E. coli requires the ATP-bound form of the DnaA protein. The conversion of DnaA-ATP to DnaA-ADP by hydrolysis seems to be facilitated by a complex of DnaA, Hda (Homologous to DnaA), and DNA-loaded β-clamp proteins in a process termed RIDA (Regulatory Inactivation of DnaA). Hda deficiency was found to result in severe growth impairment so suppressors were readily generated. Several types of suppressors of the growth impairment of Hda deficient cells were obtained of which some were found to reduce the number of initiations from oriC (DnaA box R4 mutations), and some seemed related to the processing of collapsed replication forks (multicopy plasmids carrying polA, dnaK, or the initiator-helicase loader gene pairs of λ and Rac). An infrequent type of hda suppressor mutation (hsm-2) was found to map in the dnaA gene (dnaAF349V), which probably resulted in reduced binding of nucleotides to this mutant DnaA protein. Finally, multicopy plasmids carrying the nrdAB genes, encoding ribonucleotide reductase, were found to suppress the Δhda mutant growth defect. The elevated level of DnaA-ATP in Δhda cells may repress transcription of the nrdAB operon, suggesting that the growth defect in Δhda cells is caused by a limited synthesis of nucleotides. As the various types of Δhda suppressors may directly or indirectly compensate for a lack of dNTP’s, the finding of these lends some support to this proposal. In Δhda mutant cells most DnaA protein is ATP-bound, the form active for initiation of replication. However, Hda deficiency led only to moderate effects on cell cycle control, partly because they were offset by altered dnaA gene expression. The increased fraction of DnaA-ATP in Δhda cells was found to repress dnaA gene expression, whereas Hda overproduction led to an increased level of DnaA-ADP, and consequently derepression of dnaA transcription. This suggested that Hda-mediated conversion of DnaA-ATP to DnaA-ADP would fine-tune dnaA gene expression so that supply of the initiator to the oriC region was adjusted. RIDA and dnaA gene autoregulation may therefore act in concert as homeostatic mechanisms to ensure once-per-cell cycle initiation. These regulatory mechanisms, probably including titration of DnaA to non-origin DnaA binding sites, may serve to respond to either an increase or decrease in initiator or oriC copy number by decreasing or increasing the initiation rate, respectively. They seem to act as back up for each other, as only moderate effects on cell cycle control are obtained when either of these are inactivated. However, the inactivation of several mechanisms severely affects timely initiation, suggesting that all regulatory mechanisms contribute and are necessary for maintaining once-per-cell cycle initiation in wild type cells.
Original languageEnglish
Number of pages59
Publication statusPublished - Feb 2007
Externally publishedYes


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