A DNAA BOX CAN FUNCTIONALLY SUBSTITUTE FOR THE PRIMING SIGNALS IN THEORIV OF THE BROAD-HOST-RANGE PLASMID RSF1010

The initiation of replication from oriV(RSF1010), the replication orig in of the broad host-range plasmid RSF1010, depends on RepA (helicase) , RepB' (primase), and RepC (intiator protein), encoded by RSF1010 its elf, while this initiation event in E. coli is independent of dnaA, dn aB, dnaC, and dnaG [Scherzinger et al, (1984) Proc. Nat], Acad. Sci. U SA 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(RS F1010) and a DnaA protein binding site, the dnaA box, inserted near or iV(RSF1010) (oriV(RSF1010)-dnaA box) could function without RepB' prim ase, but required RepA and RepC, This oriV(RSF1010)-dnaA box could not replicate in a dnaA46 strain in which only RepA and RepC were supplie d, even at a permissive temperature, These results indicate that an in serted dnaA box can functionally substitute for the RSF1010-specific s si signals, the RepB' dependent priming signals in OriV(RSF1010), and can direct a priming pathway different from the RSF1010-specific one, but related to DnaA protein.