Transcription of single base oligonucleotides by ribonucleic acid-directed deoxyribonucleic acid polymerase

Abstract

The synthesis of DNA products complementary to artificial templates by the enzyme RNA-directed DNA polymerase isolated from avian myeloblastosis virus has been studied. Of the single base polyribonucleotides, poly(rC), poly(rA), and poly(rI) were active while poly(rG) and poly(rU) were almost inactive. The minimum length showing activity for an oligo(rC) template was 9; the minimum primer length of oligo(dG) was 3 or 4. In order to examine the fidelity of transcription, single base oligoribonucleotides of defined length were studied. Using (rC)1 as template and (dG)8 as primer, the oligo(dG) product coelectrophoresed w th the template. However, using (rA)0 as template and (dT) as primer, a large (10-16S) product was formed.

Poliovirus RNA-dependent RNA Polymerase (3Dpol) Is Sufficient for Template Switchingin Vitro

“…The pulse-quench experiment showed that the only products formed were those consistent with utilization of the 15-nt oligo(dT) primer. Moreover, 30 24.6 Ϯ 2.8 9.1 Ϯ 0.5 dT 10 /rA 16 122 Ϯ 44 4.8 Ϯ 0.9 dT 15 /rA 16 38.2 Ϯ 8. pol and incubated at 37°C for 5 min. The reaction volume was 50 l. B, reactions were performed as described above with poly(rC), oligo(dG) n , and GTP.…”

“…Both the duration of linearity and the reaction end points differed substantially when the kinetics of nucleotide incorporation into dT 15 /rA 16 (Fig. 3A) was compared with that into dT 15 /rA 30 (Fig. 3B) or dG 15 /rC 30 (Fig.…”

“…Products Greater than Unit Length Form Using dT 15 /rA 16 , dT 15 /rA 30 , and dG 15 /rC 30 Primer/Templates-It has been reported by several investigators studying the oligo(dT)-primed, poly(rU)-polymerase activity of 3D pol that this enzyme extends, at best, 0.1-1% of primers in reactions which typically contain a concentration of primer in the 1 M range (28,29). Such a low fraction of primer utilization is not surprising, however, given a K m of 3D pol for dT 15 /rA 400 of 45 M. Therefore, it was comforting to note that the concentration of UMP incorporated was almost stoichiometric with respect to primer under saturating conditions of dT 15 /rA 16 (Fig.…”

“…While a distributive slippage mechanism has been proposed for DNA polymerase I to explain formation of long products from short primer/templates consisting of 3-nt repeats (33), a processive slippage mechanism has been suggested for T7 DNA polymerase on the same substrates (33). In the case of the reverse transcriptases from avian myeloblastosis virus (30,31) and human immunodeficiency virus (32), a template-switching mechanism has been proposed to explain products greater than unit length on a poly(rA) template.…”

“…(20 M), and heparin 6000 (0, 10, or 100 M). 3D pol was incubated with dT 15 /rA 30 at 37°C for 1 min, and the reaction was initiated by addition of UTP (q), UTP and 10 M heparin (f), or UTP and 100 M heparin (‚). In the control experiments, heparin was included during the initial incubation at either 10 M (Ⅺ) or 100 M (OE) and the reaction initiated by addition of UTP.…”