Abstract
The requirement of DnaA protein binding for plasmid RK2 replication initiation the Escherichia coli was investigated by constructing mutations in the plasmid replication origin that scrambled or deleted each of the four upstream DnaA boxes. Altered origins were analyzed for replication activity in vivo and in vitro and for binding to the E. coli DnaA protein using a gel mobility shift assay and DNase I footprinting. Most strikingly, a mutation in one of the boxes, box 4, abolished replication activity and eliminated stable DnaA protein binding to all four boxes. Unlike DnaA binding to the E. coli origin, oriC, DnaA binding to two of the boxes (boxes 4 and 3) in the RK2 origin, oriV, is cooperative with box 4 acting as the "organizer" for the formation of the DnaA-oriV nucleoprotein complex. Interestingly, the inversion of box 4 also abolished replication activity, but did not result in a loss of binding to the other boxes. However, DnaA binding to this mutant origin was no longer cooperative. These results demonstrate that the sequence, position, and orientation of box 4 are crucial for cooperative DnaA binding and the formation of a nucleoprotein structure that is functional for the initiation of replication.
Highlights
Whereas the interactions of DnaA at oriC have been studied in considerable detail, much less is known about the formation of DnaA-nucleoprotein structures at the replication origins of plasmid replicons
DnaA binding to this mutant origin was no longer cooperative. These results demonstrate that the sequence, position, and orientation of box 4 are crucial for cooperative DnaA binding and the formation of a nucleoprotein structure that is functional for the initiation of replication
It was demonstrated that there are two DnaA boxes in addition to two previously described boxes located in the leftward part of the RK2 origin, oriV, and that all four boxes bind the E. coli DnaA protein [11]
Summary
Plasmids, and Proteins—Plasmid pKD19L1, a dual RK2R6K mini-replicon, was used for constructing all mutations using the QuickChange polymerase chain reaction-based site-directed mutagenesis kit (Stratagene, Inc.) as described previously [30]. Binding of DnaA protein to origin DNA containing mutated DnaA box sequences was performed essentially as described previously [11], except that DnaA protein was incubated with DNA at 37 °C for 15 min and DNase I (New England Biolabs, Inc.), 0.67 units/reaction, at 37 °C for 30 s. After incubating the reaction containing labeled DNA template and DnaA protein for 15 min at 37 °C, DNase I was added (6.7 units/reaction) for 2 min at the same temperature. Gel Mobility Shift Assay—Complexes between oriV DNA and DnaA protein were formed and analyzed essentially as described previously [11] except that the reaction mixture, based on standard conditions used for RK2 in vitro replication [29], contained 40 mM Hepes/KOH, pH 8.0, 25 mM Tris-HCl, pH 7.4, 80 g/ml bovine serum albumin, 4% sucrose, 4 mM dithiothreitol, 11 mM magnesium acetate.
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