Enzymatic Synthesis of Deoxyribonucleic Acid I. PREPARATION OF SUBSTRATES AND PARTIAL PURIFICATION OF AN ENZYME FROM ESCHERICHIA COLZ

In considering how a complex polynucleotide such as DNA1 is assembled by a cell, the authors were guided by the known enzymatic mechanisms for the synthesis of the simplest of the nucleotide derivatives, the coenzymes. The latter, whether composed of an adenosine, uridine, guanosine, or cytidine nucleotide, are formed by a nucleotidyl transfer from a nucleoside triphosphate to the phosphate ester which provides the coenzymatically active portion of the molecule (1, 2). This condensation, which has been regarded as a nucleophilic attack (3) on the innermost or nucleotidyl phosphorus of the nucleoside triphosphate, results in the attachment of the nucleotidyl unit to the attacking group and in the elimination of inorganic pyrophosphate. By analogy, the development of a DNA chain might entail a similar condensation, in this case between a deoxynucleoside triphosphate with the hydroxyl group of the deoxyribose carbon-3 of another deoxynucleotide. Alternative possibilities involving other activated forms of the nucleotide (as, for example, nucleoside diphosphates which have proved reactive in the enzymatic synthesis of ribonucleic acid (4)) were not excluded. Earlier reports (1, 2, 5-7) briefly described an enzyme system in extracts of Escherichia coli which catalyzes the incorporation of deoxyribonucleotides into DNA. Purification of this enzyme led to the demonstration that all four of the naturally occurring deoxynucleotides, in the form of triphosphates, are required. In addition, polymerized DNA and Mg* were found to be indispensable for the reaction. Deoxynucleoside diphosphates are inert; and as a further indication of the specificity of the enzyme for the triphosphates, the synthesis of DNA is accompanied by a release of inorganic pyrophosphate, and reversal of the reaction is specific for inorganic pyrophosphate. These considerations have led to a provisional formulation of the reaction as follows: