Bacillus subtilis DnaB forms multiple protein–protein interactions essential for DNA replication initiation

DNA replication is initiated at specific chromosomal loci termed origins. In bacteria, the master replication initiation protein DnaA unwinds the origin (oriC), allowing a pair of replicative helicases to be loaded around each strand of the DNA duplex. The molecular mechanisms for managing bacterial helicase loading at oriC are unclear. Here we have investigated the role of the essential accessory helicase loader DnaB in Bacillus subtilis. By identifying and characterizing DnaB residues that are critical for its role during DNA replication initiation, we have located three necessary protein-protein interactions that DnaB makes with initiation proteins DnaA, DnaD, and DnaI. Combining single particle cryo-electron microscopy, AlphaFold3 predictions, and two-hybrid interaction analyses, the data suggests that DnaB acts as an interaction hub to orchestrate dual helicase loading at the origin. We propose a model for DNA replication initiation in B. subtilis and related Firmicutes pathogens that employ DnaB-type helicase loaders.