DnaA protein binding to the plasmid origin region can substitute for primosome assembly during replication of pBR322 in vitro

Abstract

We analyzed the significance of DnaA protein binding to the origin region of pBR322. Replication of pBR322 in vitro was stimulated by DnaA protein. Moreover, the primosomal component protein i was no longer essential for replication after addition of DnaA protein, whereas, among others, proteins DnaB and DnaG were still required. Complete replication products were synthesized under these conditions. We constructed pBR322 deletion derivatives missing the primosome assembly sites. Efficient replication of these deletion plasmids was dependent on the presence of DnaA protein and its binding site, but independent of protein i activity. We conclude that DnaA protein binding to the pBR322 origin region substitutes for primosome assembly by directing DnaB, DnaC, and DnaG proteins to the origin. We term this process DnaA-directed pre-replisome formation.