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Updated: Jun 23, 2026

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach
Published on: September 11, 2017
DnaAcos hyperinitiates by circumventing regulatory pathways that control the frequency of initiation in Escherichia
Magdalena M Felczak1, Jon M Kaguni
1Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
Abstract:
Mutants of dnaAcos are inviable at 30 degrees C because DnaAcos hyperinitiates, leading to new replication forks that apparently collide from behind with stalled forks, thereby generating lethal double-strand breaks. By comparison, an elevated level of DnaA also induces extra initiations, but lethality occurs only in strains defective in repairing double-strand breaks. To explore the model that the chromosomal level of DnaAcos, or the increased abundance of DnaA, increases initiation frequency by, escaping or overcoming pathways that control initiation, respectively, we developed a genetic selection and identified seqA, datA, dnaN and hda, which function in pathways that either act at oriC or modulate DnaA activity. To assess each pathway's relative effectiveness, we used genetically inactivated strains, and quantified initiation frequency after elevating the level of DnaA. The results indicate that the hda-dependent pathway has a stronger effect on initiation than pathways involving seqA and datA. Testing the model that DnaAcos overinitiates because it fails to respond to one or more regulatory mechanisms, we show that dnaAcos is unresponsive to hda and dnaN, which encodes the beta clamp, and also datA, a locus proposed to titer excess DnaA. These results explain how DnaAcos hyperinitiates to interfere with viability.
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