Robust helicase loading mechanism underlies diverse architectures of bacterial replication origins.

The Escherichia coli chromosomal origin, oriC, contains a duplex-unwinding element (DUE) flanked by two clusters of the initiator DnaA-binding sites (DnaA boxes). ATP-DnaA oligomerizes to construct Left- and Right-DnaA subcomplexes on these clusters. These complexes coordinately promote DUE unwinding and DnaB helicase loading onto the unwound region for replication initiation. However, despite the strong conservation of DnaA and DnaB across bacterial species, oriC architectures vary largely. Remarkably, bipartite and bilateral configurations contain the two DnaA box-clusters separated by an insertion of the dnaA gene or positioned on both sides of the DUE, respectively. Here, we demonstrate that E. coli oriC variants mimicking these configurations remain fundamentally functional. Notably, DnaB loading activity of these variants showed increased dependence on single-stranded DUE binding by the relocated DnaA subcomplex and on the AT cluster upstream of the DUE, reflecting reduced stability of DUE unwinding. Consistent with these in vitro results, these variants supported cellular replication initiation with only slow cell growth rates. Together, these findings reveal remarkable architectural flexibility, mechanistic robustness, and a conserved mechanism in oriC. Also, those highlight unwinding stability as a key determinant of oriC adaptation to rapid cell growth, providing insight into the evolution of oriC architecture.