Oligonucleotides as primers for polynucleotide phosphorylase.

Ribonucleoside diphosphates react to form polyribonucleotides and inorganic phosphate. Studies of the nature of nucleotide incorporation into nucleic acid in Escherichia coli led to the recognition of a similar reaction by Littauer and Kornberg (3). Beers (4,5) and Olmsted (6,7) have investigated the phosphorylase in Micrococcus lysodeikticus. Heppel et al. (8-10) have elucidated the structure of the polyribonucleotides formed in the polynucleotide phosphorylase catalyzed reaction. The polymers polyadenylic acid, polyuridylic acid, the mixed polymers of adenylic and uridylic acids, and of adenylic, uridylic, guanylic, and cytidylic acids conform to the structure of a normal polynucleotide, and poly AGUCY is chemically indistinguishable from natural ribonucleic acid. With relatively crude preparations of polynucleotide phosphorylase, only nucleoside diphosphate and Mg++ ion are required to demonstrate polymer formation (2-4). Nevertheless Ochoa and Heppel (8), as well as Littauer and Kornberg (3), discussed the possibility that, in analogy with the carbohydrate phosphorylase (11) , polymerization requires a polynucleotide primer. They suggested that polynucleotide material contaminating the enzyme preparations served this function. With highly purified enzyme preparations from A. agile, Mii and Ochoa (12) found that polymerization of nucleoside diphosphates occurs only after a lag period and that the lag can be overcome by RNA and the biosynthetic polymers. The data presented in this report show that oligonucleotides such as pApA, and ApU, and pApApApA also overcome the lag period and, in sufficient concentration, stimulate the reaction to the same extent as the polymers. Furthermore, they are incorporated into polymers formed in their presence. The polymer chains are built by successive additions of mononucleotide units to the unesterified C-3’ hydroxyl of the terminal nucleoside residue in the preformed oligonucleotide chain.