Putative Cooperative ATP-DnaA Binding to Double-Stranded DnaA Box and Single-Stranded DnaA-Trio Motif upon Replication Initiation Complex Assembly.

is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of s is universal; however, the structure of and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized of , which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA-ssDNA oligomer formation-stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in , constitute a new class of ATP-DnaA boxes. Indeed, in vitro ATP-DnaA unwinds more efficiently than ADP-DnaA. Our results expand the understanding of orisome formation, indicating another regulatory pathway of orisome assembly.