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Published March 27, 2023 | Submitted
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Robust control of replication initiation in the absence of DnaA-ATP ⇋ DnaA-ADP regulatory elements in Escherichia coli

Abstract

Replication initiation, especially the widely conserved master initiator protein DnaA, is one of the most well-studied biological problems in bacteria. Specifically, conversion between the ATP and ADP form of DnaA during growth is critical for initiation control. In Escherichia coli, the regulatory elements for conversion have been discovered and extensively characterized over decades. However, they are not widely conserved in bacteria, raising questions on how to generalize the findings in E. coli to other organisms. In this work, we show that the intrinsic ATPase activity of DnaA itself is sufficient for robust and precise initiation control. We constructed and studied E. coli mutants lacking the extrinsic control of either DnaA-ATP → DnaA-ADP (by hda and datA) or DnaA-ADP → DnaA-ATP (by DARS1 and DARS2) at the single-cell and population levels. These cells showed distinct and opposing characteristics in initiation timing, the degree of initiation asynchrony, and cell-to-cell variability. Strikingly, when all four regulatory elements were deleted, E. coli exhibited a near wild-type phenotype, with only mildly increased intrinsic and extrinsic initiation noise. By further characterizing the DnaA variants with increased and decreased ATPase activity, we conclude that DnaA is the only requirement for robust initiation, shedding new evolutionary light on cell-cycle control in bacteria.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. The authors have declared no competing interest.

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Additional details

Created:
August 20, 2023
Modified:
October 18, 2023