Structural analyses of the bacterial primosomal protein DnaB reveal that it is a tetramer and forms a complex with a primosomal re-initiation protein.

The DnaB primosomal protein from Gram-positive bacteria plays a key role in DNA replication and restart as a loader protein for the recruitment of replisome cascade proteins. Previous investigations have established that DnaB is composed of an N-terminal domain, a middle domain, and a C-terminal domain. However, structural evidence for how DnaB functions at the atomic level is lacking. Here, we report the crystal structure of DnaB, encompassing the N-terminal and middle domains (residues 1-300), from (DnaB) at 2.8 Å resolution. Our structure revealed that DnaB forms a tetramer with two basket-like architectures, a finding consistent with those from solution studies using analytical ultracentrifugation. Furthermore, our results from both GST pulldown assays and analytical ultracentrifugation show that DnaB is sufficient to form a complex with PriA, the primosomal reinitiation protein. Moreover, with the aid of small angle X-ray scattering experiments, we also determined the structural envelope of full-length DnaB (DnaB) in solution. These small angle X-ray scattering studies indicated that DnaB has an elongated conformation and that the protruding density envelopes originating from DnaB could completely accommodate the DnaB C-terminal domain (residues 301-461). Taken together with biochemical assays, our results suggest that DnaB uses different domains to distinguish the PriA interaction and single-stranded DNA binding. These findings can further extend our understanding of primosomal assembly in replication restart.