RNA dependent DNA polymerase of avian sarcoma virus B77. II. Comparison of the catalytic properties of the α, β₂, and αβ enzyme forms

The α, β₂, and αβ forms of the RNA dependent DNA polymerase of avian sarcoma virus B77 grown in duck embryo fibroblasts have been compared with respect to several kinetic properties. The following results were obtained. The K(m) values for dTTP and dGTP for enzyme forms α, β₂, and αβ were 77, 39, and 74, and 6.8, 3.1, and 6.1 μM, respectively. The affinity of 70 S Rous sarcoma virus RNA for enzyme form αβ was about twice that for the other two forms. The relative specific activities of the three enzyme forms on synthetic primer templates such as poly(rA).poly(dT) were almost the same. The viral 70 S RNA dependent specific activities were 2 to 3 orders of magnitude lower and in the ratio of 1:3:5 for enzyme forms α:β₂:αβ. Addition of exogenous oligo(dT) stimulated the 70 S viral RNA dependent activity of enzyme forms αβ and β₂ by a factor of 3, and that of enzyme form α by a factor of 30, so that it then became the most active transcriptase of viral 70 S RNA. The largest transcripts formed by the three enzyme forms with 70 S viral RNA as primer template were about 4,500 nucleotides long. About one third of the total amount of polynucleotides polymerized by the αβ enzyme was in the form of such transcripts. This proportion was far higher than for the other two enzyme forms. All three enzyme forms were capable of transcribing single stranded into double stranded DNA. The 3 propylcyclohexyl piperidyl derivative of rifamycin SV, at a concentration of 100 μg/ml, inhibited enzyme forms β₂ and αβ by over 99.5 and 96%, respectively, but enzyme form α by only about 60%. The β₂ and αβ forms of the enzyme were processive DNA polymerases, the α form a nonprocessive polymerase. In general, these results indicate that in most respects the properties of the dimeric enzyme forms resemble each other much more closely than those of the α form. In some very important respects, such as affinity for viral RNA and the size of transcripts formed from it, the αβ enzyme form performs significantly better than either of the other two enzyme forms.