Mutational Analysis of DnaA‐trio Motifs in E. coli OriC Reveals Multiple Modes for Bacterial Replication Origin Activation

Abstract

The chromosomal replication origin (oriC) in bacteria is a unique region that directs assembly of the orisome, a nucleoprotein complex that unwinds origin DNA and assists with the loading of the replicative helicase. All orisome functions are mediated by the initiator protein, DnaA, which binds to specific sites in oriC, and, after DNA unwinding, binds to and stabilizes the unwound region. Orisome assembly is well characterized in E. coli, but bacterial origins are not conserved with regard to the number and spacing of DnaA recognition sites, and so it is difficult to determine whether there are common sequence patterns that are essential for origin activity. However, one promising feature, the “DnaA‐trio” motif, can be found in the origins of most bacterial types, with multiple copies residing near the AT‐rich region where DNA strand separation takes place. DnaA‐trios are essential for B. subtilis chromosome replication initiation, where they facilitate delivery of DnaA‐ATP oligomeric filaments to the AT‐rich region. Three DnaA‐trio motifs also reside in E. coli oriC, but their role is unclear, since we recently reported that E. coli oriC could be activated without formation of DnaA‐ATP oligomers. Therefore, to test their essentiality in E. coli, we used site‐directed mutagenesis to inactivate the trios, and then used recombineering to replace wild type chromosomal oriC with the mutated versions. Eliminating trio sites had no effect on E. coli oriC activity or initiation timing. Based on these results, we hypothesized that E. coli might use an alternative mechanism to deliver DnaA to the AT‐rich region, one that does not depend on trios or DnaA‐ATP filaments. We hypothesized that the sharp DNA bend formed late in E. coli orisome assembly and stabilized by the DNA bending protein IHF, might eliminate the need for trio sites by positioning the AT‐rich region for cross‐strand DnaA donations provided by the DnaA bound to oriC's left half. To test this, we removed the bend region from oriC using site‐directed mutagenesis, and even though the mutated origin (oriCno bend) contained all three DnaA trios, it was unable to function as the sole chromosomal origin of replication. In vitro unwinding studies suggested that the defect could be caused by a failure to deliver DnaA to the AT‐rich region, so we tried to rescue oriCno bend function by activating a trio‐dependent mode of DnaA delivery. Successful rescue was achieved by inserting a DnaA binding site between R1 and the trio sites, and origin activity became completely dependent on the DnaA‐trio elements. However, rescued cells displayed perturbed initiation timing, suggesting that the bend mode of DnaA delivery plays both mechanical and regulatory roles in E. coli. We propose that there are at least two possible modes of bacterial origin activation; one that depends on DnaA‐trio elements, and one that uses DNA bending and cross‐strand DnaA binding to regulate late stage orisome assembly.Support or Funding InformationNational Institutes of Health (GM54042 to A.L.) and Florida Institute of Technology Holzer Lequear Fund for Molecular Genetics (to J.G.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.