The initiation of replication from oriV R S F 1 0 1 0 , the replication origin of the broad host-range plasmid RSF1010, depends on RepA (helicase), RepB (primase), and RepC (intiator protein), encoded by RSF1010 itself, while this initiation event in E. coli is independent of dnaA, dnaB, dnaC, and dnaG [Scherzinger et al. (1984) Proc. Natl. Acad. Sci. USA 81, 654-658; Scholz et al. (1985) in: Plasmids in Bacteria, pp. 243-259, Plenum, New York; Haring and Scherzinger (1989) in: Promiscuous Plasmids of Gram-negative Bacteria, pp. 95-124, Academic Press, London; Scherzinger et al. (1991) Nucl. Acids Res. 19, 1203-1211]. We showed in this work that a newly constructed origin consisting of an oriV R S F 1 0 1 0 and a DnaA protein binding site, the dnaA box, inserted near oriV R S F 1 0 1 0 (oriV R S F 1 0 1 0 -dnaA box) could function without RepB primase, but required RepA and RepC. This oriV R S F 1 0 1 0 -dnaA box could not replicate in a dnaA46 strain in which only RepA and RepC were supplied, even at a permissive temperature. These results indicate that an inserted dnaA box can functionally substitute for the RSF1010-specific ssi signals, the RepB dependent priming signals in oriV R S F 1 0 1 0 , and can direct a priming pathway different from the RSF1010-specific one, but related to DnaA protein.