Template functions in the enzymic formation of polyribonucleotides, I. Integrity of the DNA template.

Abstract

Despite much excellent work, one of the most fundamental problems in biology, namely, the mechanism through which the cell produces and reproduces sequentially specific heteropolymers (proteins, nucleic acids, polysaccharides, etc.), still remains far from being well understood. The nearest we have come to an understanding is through the postulation of template mechanisms in which a preformed polynucleotide prescribes, either directly by base pairing or through an intricate coding procedure, the order in which the precursors of the newly forming polymer, simultaneously or subsequently linked by enzyme action, are aligned. It is, perhaps, not surprising that the conventional concepts of enzymology are not entirely adapted to dealing with enzyme complexes whose action depends on, or is directed by, high-molecular templates that surpass the enzyme protein itself in size and often in complexity and sensitivity. We may describe the dilemma as follows. An enzyme can be defined by its substrate or by its product. If we define RNA polymerase, the enzyme studied here, by its substrate, the situation is clear: the ribonucleoside triphosphates are the only substrate and the enzyme is a ribonucleotidyl transferase. But in trying to define the enzyme by its product, we encounter a predicament of ever-shifting variables: there are as many products, and consequently as many enzymes, as there are templates. The central function of DNA in specifying the enzymic formation of polyribonucleotides of specific composition and, presumably, sequence, prompted us to investigate the problem of a template in some detail--a study that will be continued in subsequent communications. RNA polymerase (EC 2.7.7.6) has formed the subject of several recent reviews.1-3 The abolition or the alteration of template activity has been observed in several instances, e.g., on storage,4 irradiation with ultraviolet light5' 6 or X rays,7 and also by chemical modification.8' In the present study, we have used the enzyme preparation from E. coli and the DNA of calf thymus; we have investigated the changes in template activity exhibited by the latter when subjected to ultrasonic oscillations, changes in pH, and the partial removal of the purine constituents. The effects of temperature will be discussed later, as will be the actioln as templates of such extreme degradation products of DNA as apurinic and apyrimidinic acids. As we were naturally interested in both the quantity and the quality of the products of the enzyme action, we also examined the composition of the polyribonucleotides synthesized in the presence of the treated templates.