Template Specificity of Eucaryotic DNA‐Dependent RNA Polymerases: Effect of DNA Structure and Integrity

The routine procedures used to prepare DNA from eucaryotic cells and nuclei give rise to material which is considerably degraded. This degradation takes the form of haplotomic (single‐stranded) breaks arising from deoxyribonuclease action action and diplotomic cleavage (double‐stranded scission) of the DNA duplex due to mechanical shearing during the isolation procedure. Attempts to study the template specificities of the eucaryotic DNA‐dependent RNA polymerases must take into account how these modifications to the structure of the DNA affect its template properties for these enzymes. Techniques are described which permit an accurate assessment of the state of integrity of the DNA. Treatment of the DNA with pancreatic deoxyribonuclease or sonication is shown to give rise to single‐stranded interruptions in the duplex which are competent sites for the initiation of RNA synthesis by both forms AI and B rat liver RNA polymerases. However both these treatments also give rise to diplotomic cleavage of the DNA. This results in an inhibition of RNA synthesis which may be correlated with the formation of non‐productive complexes by the RNA polymerases at DNA ends. Therefore the degree of template activation by pancreatic deoxyribonuclease and sonication for RNA synthesis as seen in the assay in vitro is a delicate balance between two opposing effects and is highly dependent upon the polymerase : DNA ratio. This data serves to emphasise that experiments designed to study the template specificity of eucaryotic RNA polymerases must make use of new, sophisticated techniques for the preparation of a template which is both intact and of high molecular weight.