Structures of DnaA domain I reveal a dimer conserved across Actinomycetes

DNA replication is a fundamental process in biology, and initiation marks a key regulatory step. In bacteria, DNA replication is initiated by the DnaA protein. DnaA exhibits multidomain architecture, consisting of an N-terminal domain I, linker region, AAA+ family ATPase cassette, and C-terminal DNA-binding motif. Taxon-specific regulatory functions are primarily coordinated by the DnaA domain I (DnaADI), which exhibits substantial sequence variation across bacteria. Notably, although DnaADI has been shown to be essential, its contributions to initiation are not completely understood. Previous studies suggested a role for DnaADI in the assembly of the initiation complex at the origin. However, the molecular mechanisms behind DnaADI functions have not been resolved. Here, we report the DnaADI structures from 10 species in the class Actinomycetes. Strikingly, all structures reveal the same, unique dimer, and our analyses show that key elements that support DnaADI self-interaction are broadly conserved across the class Actinomycetes. Further, a suite of biochemical oligomerization assays and HDX-MS (hydrogen-deuterium exchange mass spectrometry) studies support the formation of dimers with µM affinities. These findings suggest that weak DnaADI dimerization, which is a broadly conserved mechanism across the Actinomycetes, likely contributes to proper replication initiation in these bacteria.