The DNA replication initiation protein DnaD is recruited to a specific strand of the Bacillus subtilis chromosome origin

Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. The molecular mechanisms for helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC (DnaD Recognition Element, "DRE"). Considered with cryo-electron microscopy (cryo-EM) imaging of full length DnaD, we propose that the location of the DRE within the oriC orchestrates strand-specific recruitment of helicase to achieve bidirectional DNA replication. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.