Processive nature of reverse transcription by avian myeloblastosis virus deoxyribonucleic acid polymerase

Abstract

The RNA-dependent DNA polymerase from avian myeloblastosis virus synthesized poly(dt) transcripts by a processive mechanism using poly(rA)1100.cntdot.oligo(dT)12-18 as template and primer. Template challenge experiments demonstrate that at low temperature and ionic strength, the polymerase remains bound to the completed template-daughter strand complex after completion of daughter strand elongation. Higher temperaturs and ionic strength increase the dissociation of the enzyme from the complex, thus reducing transcript length. Analysis of product size and quantity indicates that the degree of processivity is also influenced by the types and concentrations of metal ions present and indicates that the metal ions affect the activity of the poly(rA) as a template more than they affect processivity or enzyme activity. The results also lead to the conclusion that initiation is the rate-limiting step under all of the experimental conditions. The arguments for a processive as opposed to a distributive mechanism are based on an analysis of enzyme-template interactions, product size and amount of product made under specific reaction conditions.