On the association of reverse transcriptase with polynucleotide templates during catalysis.
The association of avian myeloblastosis virus (AMV) DNA polymerase with polynucleotide templates during catalysis has been studied. During the course of polymerization, different template-primer complexes were added and the ability of the enzyme to switch from one polynucleotide template to another was determined. At 37 degrees C as well as at 4 degrees C, the polymerase is able to switch from certain template-primer complexes to others. For example, the addition of poly(A)-oligo(dT) during the course of synthesis with poly(C)-oligo(dG) results in the immediate cessation of dGMP polymerization and the start of dTMP polymerization without any lag. Early during the course of polymerization, the size of the product, as determined by alkaline sucrose gradient centrifugation, is, in part, a function of the ratio of the template-primer complex to the enzyme. These cumulative experiments indicate that catalysis on polynucleotide templates with avian myeloblastosis virus DNA polymerase under the conditions tested is not processive in a classical sense. Similar to cellular DNA polymerases the enzyme can shift from one template-primer to another. Using autoradiography after gel electrophoresis to estimate the product size, it can be calculated that the enzyme switches from one template to another within 0.25 min at 37 degrees C which corresponds to the incorporation of greater than 25 nucleotides. At 4 degrees C, switching can be calculated to occur in less than three nucleotide addition steps. Thus, with certain homopolymers, conditions can be found by which AMV DNA polymerase can switch from one template-primer complex to another, perhaps after each nucleotide addition step.
D001354:Avian Leukosis Virus / Q000201:enzymology*; D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001665:Binding Sites; D001667:Binding, Competitive; D007700:Kinetics; D008970:Molecular Weight; D011089:Polydeoxyribonucleotides; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D011089:Polydeoxyribonucleotides; D012194:RNA-Directed DNA Polymerase