Role of ATP in DNA synthesis in Escherichia coli

Abstract

Escherichia coli, rendered permeable by toluene treatment, is known to catalyze a limited amount of semi-conservative DNA synthesis; in addition to the four deoxynucleoside triphosphates, the reaction specifically requires ATP (or ADP). We show that this requirement was also evident in the conversion of Okazaki fragments to high molecular weight DNA in strains of E. coli carrying the pol A1 mutation. In strains containing DNA polymerase I, ATP was not required for this conversion. In addition, strains containing DNA polymerase I convert Okazaki fragments to a high molecular weight form more rapidly than strains deficient in this enzyme. The influence of nalidixic acid on this conversion process has been studied. In the presence of nalidixic acid, the 6 to 10 s Okazaki fragments are converted into a collection of molecules sedimenting at 30 to 40 s; there is essentially no breakdown of the fragments to an acid-soluble form under these conditions. When synthesis of DNA is inhibited by incubating temperaturesensitive mutants of the dnaB locus at the non-permissive temperature, however, there is a preferential degradation of the Okazaki fragments into an acidsoluble form. Prior incubation at the permissive temperature will prevent this degradation.