Early steps of Bacillus subtilis primosome assembly

Primosomes are nucleoprotein assemblies designed for the activation of DNA replication forks. Their primary role is to recruit the replicative helicas e onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. Pri A is conserved in bacteria, but its primosomal partners are not. In Bacillu s subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB, and Dn aD proteins unravels their role in primosome assembly. They are both multim eric and bind individually to DNA. Furthermore, DnaD stimulates DnaB bindin g activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucle oprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferre d DNA substrate mimics an arrested DNA replication fork with unreplicated l agging strand, structurally identical to a product of recombinational repai r of a stalled replication fork.