Abstract
DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein’s domain III, rather than confined to a particular sequence. We propose a new binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication.
Highlights
DnaA is a ubiquitous protein which initiates chromosome replication in bacteria and its activity in initiation is restricted to once per cell division cycle
The presence of DnaA in steady-state growing E. coli BL21 cells and in different cell fractions was detected by immunoblotting with further quantification using a range of amounts of purified His-tagged DnaA as a standard (Figure 1)
Concluding Remarks and Hypothesis Our results obtained both with native and with fluorescently tagged protein demonstrate that about 10% of cellular DnaA is located on the membrane in steady-state growing E. coli
Summary
DnaA is a ubiquitous protein which initiates chromosome replication in bacteria and its activity in initiation is restricted to once per cell division cycle. DnaA binds to distinct high-affinity sites on the origin of replication, oriC, called DnaA-boxes, and to other sites on the chromosome with lower affinity. In E. coli, deletion of domain II (amino acids 87–134) showed it was not essential for cell viability [5] it was recently shown to participate in regulation of replication initiation [11]. This expendability of domain II and its being virtually unfolded has been exploited by [12,13] and in this work to insert a gene encoding a fluorescent protein into the DnaA sequence so as to obtain an active fluorescent hybrid. Domain IV is responsible for DnaA-DNA interaction, binding to the major groove of DNA via a helix-turn-helix motif [7]
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