Enzymatic Synthesis of Polynucleotides

Several publications from this laboratory have dealt in a preliminary fashion with the preparation and properties of highly purified polynucleotide phosphorylase from Azotobacter vinelan&i (2-4). A more detailed account of this work is presented in thii paper. Although polynucleotide phosphorylase preparations can catalyze the synthesis of a variety of homopolymers (polyadenylic acid, polyuridylic acid, etc.) and copolymers (of adenylic and uridylic acids, of adenylic, guanylic, uridylic, and cytidylic acids, etc.), the evidence so far available indicates that a single enzyme is involved. The best preparations represent a 500-fold purification of the enzyme from the initial extracts and are largely, although not totally, devoid of nuclease activity. These preparations contain about 3 y0 of a firmly attached, small polyribonucleotide. It has not been possible to remove this nucleotide without denaturing the enzyme, and it remains unsettled whether it is a prosthetic group or merely a tenacious contaminant. Although the oligonucleotide appears to act as a primer of polymer synthesis, the need for added oligonucleotide (5) or polynucleotide primers (2-4) can be shown readily under appropriate conditions.