When temperature-sensitive dnaA mutants are switched from nonpermissive to permissive temperatures, DNA synthesis begins at oriC. Subsequent bidirectional replication produces a gene dosage effect in which genes closest to oriC are present in twice the abundance of those further away. The OriC region of the E. coli chromosome shows oric and some of the genes that surround it at minute 84 of the E. coli genetic map.
The E. coli chromosome's large size and fragility precludes studying oriC structure and function in the intact chromosome in vitro. Fortunately, the desired goals can be achieved by constructing a minichromosome that depends on oriC for its replication. A restriction endonuclease cuts the bacterial chromosome into fragments and the plasmid DNA into a fragment containing a drug-resistance gene (ampicillin in this example) but lacking the plasmid origin of replication. After mixing, the bacterial and plasmid DNA fragments are joined by DNA ligase and the resulting recombinant plasmid DNA is used to transform bacteria. The cells are then spread onto growth medium that contains ampicillin. Only cells bearing recombinant plasmid DNA with a drug-resistance gene and oriC will form colonies under these growth conditions. Intracellular nucleases eventually destroy DNA molecules that cannot replicate autonomously.
The minimal sequence needed for oriC function, which was determined by trimming the ends of the cloned site, is 245 bp long. This sequence contains several elements that are essential for normal function. Five elements within the minimal sequence contain a 9 bp common sequence 5'_TTATNCACA (where N is any nucleotide). These 9-mers are called DnaA boxes because DnaA · ATp andDnaA · ADP complexes bind tightly to them. The individual DnaA boxes are designated R1-R5. DnaA also binds to three additional elements in oriC called I boxes, which also contain 9 bp. However, DnaA binding to I boxes differs from its binding to DnaA boxes in two important respects. First, only DnaA · ATP can bind to the 1 boxes, which explains why ATP is required for the initiation of replication. Second, DnaA · ATp complexes bind to I boxes only after the DnaA boxes are occupied. The binding of DnaA protein to the DnaA and I boxes causes three AT-rich regions on the left side oriC, each containing 13 bp (13-mer), to unwind. Additional DnaA · ATP binds to the single strands that are generated by this unwinding. Electron microscopy studies indicate that 20 to 30 DnaA monomers bind to the oriC region, forming a large aggregate that is probably stabilized by interactions involving the cause of their involvement in other processes, so their names do not provide any information about how the factors function at oriC. In fact, the two factors have opposite effects on initiation: IHF stimulates initiation and FIS inhibits it. Additional enzymes contribute to the initiation process. For instance, RNA polymerase helps to unwind DNA in the oriC region as it moves away from oriC, while transcribing genes in this region and RNase H helps to maintain specificity for the oriC region by hydrolyzing RNA molecules hybridized to DNA that might otherwise be able to serve as priming sites. The enzyme is called RNase H because it is specific for RNA in DNA-RNA hybrids.