The effect of dnaA protein and n' sites on the replication of plasmid ColE1

The role of the dna A protein in the replication of plasmid ColE1 and its derivatives was examined. Wild-type and mutant ColE1 plasmids were compared as to their ability to replicate in an in vitro replication system supplemented with ammonium sulfate fractionated extracts from a dnaA-overproducing strain. Synthesis on plasmid templates containing the wild-type origin of replication was stimulated 1.3-fold by addition of the dnaA-overproducing extract. A larger effect was observed after deletion of the primosome assembly site, the n' site, on the leading strand. On the latter template, synthesis was only about one-half that observed with the wild-type templates, but synthesis could be restored to normal levels by addition of the dnaA-overproducing fractions. When the n' site on the lagging strand of pBR322 was deleted, synthesis in the in vitro replication system was reduced to less than 10% of levels seen with intact templates. dnaA-overproducing extract did not restore activity since the dnaA site was also deleted on these plasmids. To verify that the observed stimulation of wild-type and leading strand n' site mutants was due to the dnaA protein, dnaA protein was purified to greater than 50% homogeneity, and antiserum was prepared. The purified protein stimulated synthesis on the plasmid templates to the same extent as the overproducing extracts, and dnaA antiserum blocked stimulation both by extracts and by the purified protein. Thus, dnaA protein, and, by inference, the dnaA recognition site at the ColE1 origin of replication seem to be important for ColE1 replication. The effect of dnaA protein is enhanced when the n'site is defective, suggesting that the dnaA protein plays a role similar to that of the proteins i, n, n', and n'' in directing primosome assembly, as proposed by Seufert, W., and Messer, W. ((1987) Cell 48, 73-78).