Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein

Abstract

Replication of the -y origin ofEscherichia coli plasmid R6K requires i protein, encoded by the R6Kpir gene, and many host factors, including DnaA protein. zr has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory function of w is counteracted by integration host factor and a specific sequence of the origin called the enhancer. This 106-bp DNA segment contains a binding site for DnaA protein (DnaA box 1). In this study, we mutated this site to determine if it was required for the enhancer's function. Using -y origin derivative plasmids with the DnaA box 1 altered or deleted, we show that this site is necessary to protect the origin against levels of wild-type wF protein that would otherwise inhibit replication. To show that the base substitutions in DnaA box 1 weakened the binding of DnaA, we developed a new application of the agarose gel retardation assay. This quick and easy assay has broad applicability, as shown in binding studies with DNA fragments carrying a different segment of the R6K origin, the chromosomal origin (oriC), or the pUC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays.Many replication origins have a bipartite organization, including an essential segment and an auxiliary segment which augments replication. Such enhancers have been discovered in organisms and systems as diverse as, for example, the bacteriophage fl (8, 23), the Staphylococcus aureus plasmid pT181 (21), and the yeast autonomously replicating sequence ARS121 (49). In each of these cases, the enhancer carries, or is believed to carry, a protein-binding site (20, 37a, 49, 50).The Escherichia coli plasmid R6K contains three origins of replication, a, y, and ,3 ( Fig. 1), which all require the initiator protein 7r, encoded by the R6K pir gene. All three origins require the essential 277-bp core segment, which contains repeated binding sites (iterons) for TT (14,19,33); each origin also needs a unique cis-acting DNA segment in addition to the core (28,29,36,41,42,44). When the flanking ot and P sequences are removed, the remaining y origin can replicate autonomously (18,43,46) if thepir gene is provided in cis or in trans (22,29). The y origin responds to the intracellular concentration of uz protein, and it contains the most critical information required for replication (12,28). Therefore, we use the y origin system (for the most recent review, see reference 10) as the simplest model derived from R6K to study the mechanisms regulating initiation of replication of an iteron-containing origin.The -y origin consists of two modules, the essential 277-bp core segment and the adjoining 106-bp enhancer (51a) (Fig. 1). The enhancer is not required for the a or ,B origin (36, 41, 42, 44). The isolated core is able to replicate if the level of wild-type (wt) wT is decreased (Sla), a situation which reduces the stringency of replication control (12). At the normal level of wt 7r (4,000 to 10,000 IT (9,11,25), the p...

Preponderance of Fis-binding sites in the R6K gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of Fis

“…These three ␥-origin plasmids have different replication, copy number, and stability properties (80,81). However, they all can replicate at a high copy number in wt cells producing copy-up variants (80). All three plasmids transformed the fis ϩ strains, but no transformants were obtained with the fis recipients producing copy-up variant 116 or 200 (Table 3, group A plasmids; also data not shown).…”

“…In pFW12, the enhancer had been deleted, but the core is also intact. These three ␥-origin plasmids have different replication, copy number, and stability properties (80,81). However, they all can replicate at a high copy number in wt cells producing copy-up variants (80).…”

“…In addition to causing an increase in plasmid copy number compared with the same concentration of wt (12, 20, 23, 40, 72a, 80), copy-up variants are known to allow replication of the ␥ origin under many conditions under which wt does not. These conditions include the following: the absence of host protein IHF (12) or mutation of the ihf1 site in the core (11), the deletion of the enhancer segment of the ␥ origin (79) or the mutation of a DnaA box in the enhancer (80), mutations in a promoter (P2) in the core (77), or the presence of extra -binding sites which cause incompatibility (59). In vitro replication studies have shown that copy-up variants are much more active in initiating replication than wt protein (11).…”

“…IHF binding to the AT-rich region and DnaA binding to the enhancer act synergistically to counteract the negative effect of high levels of wt on ␥-origin replication (11, 80). Copy-up variants also allow replication of the ␥ origin under many conditions under which wt cannot (11,12,59,77,79,80).…”

“…The ␥ origin, for example, includes the core and the adjacent enhancer (for a recent review, see reference 16). The enhancer contains DnaA box 1 (79,80) and a small segment, stb, involved in stable maintenance of ␥-origin-containing plasmids (81). We subdivided the core into three distinct regions (16): (i) the AT-rich region, bound by and IHF (11,13,15,17,19,45,52); (ii) seven 22-bp direct repeats (DRs) bound by (19,23,28,60); and (iii) a multiprotein-binding region interacting with DnaA, IHF, and RNA polymerase (11,13,15,32,77,79).…”