Reactivity of reverse transcriptase toward (s4U,U)n copolymers and spin-labeled nucleic acid lattices.

Abstract

Spin-labeled copolymers of 4-thiouridine and uridine [(ts4UU)] that contain various amounts of spin label (e) were synthesized by either (i) chemical alkylation of the 4-thiouridine-uridine copolymers (s4U,U). prepared by copolymerizing 4-thiouridine 5'-diphosphate (s4UDP) and UDP or (ii) copolymerization of spin-labeled S4UDP with UDP using polynucleotide phosphorylase. The effect of (s4U,U). and (tS4UU). on avian myeloblastosis virus (AMV) RNA-dependent DNA polymerase (RNA-dependent DNA nucleotidyltransferase, EC 2.7.7.7; reverse transcriptase) was studied to determine whether the presence of potentially reactive thiol groups or spin labels enhances the inhibitory properties of the copolymers as compared to (U)3. Inhibition by (s4U,U) gradually increases as the percentage of thiolation increases. Enhanced inhibition by (s4U,U). appears to be due to the interaction of the thiol groups of (s4U,U). with the thiol group(s) of the polymerase, because inhibition by (s4U,U). (8% thiolated) in the presence of dithiotreitol resembles that by (U)L. In contrast, inhibition by (ts4UU). containing 3% spin label resembles that by (U)L; however, increasing the spin label to 6% or 12% results in enhanced inhibition by (4s4U,U). as compared to that by (U)3, and dithiothreitol has no effect on enhanced inhibition by (t UU)3.These results suggest that the mechanism of inhibition observed with (ts4U,U). with a es4U:U ratio > 1:33 differs from the mechanism for (s4U,U). and involves complex formation between the spin label and the essential Zn2+ of RNA-dependent DNA polymerase.

Local base dynamics and local structural features in RNA and DNA duplexes

“…The chemical structures of the 5-and 4-substituted nitroxide-labeled ribonucleosides are given in Figure 1. (ls4U,U)" was prepared according to a published procedure from this laboratory (Warwick et al, 1980). All other spin-labeled RNAs were prepared under conditions similar to those described for (ls4U,U)" by copolymerizing uridine 5'-diphosphate and the appropriate nitroxide-containing nucleoside 5'-diphosphate analogues with polynucleotide phosphorylase.…”

Internal motions in ribonucleic acid duplexes as determined by electron spin resonance with site-specifically spin-labeled uridines

“…Synthesis of pplsAU. The position 4 spin-labeled uridine 5'-diphosphate analogue was synthesized according to a previously described procedure (Warwick et al, 1980). The nonexchangeable hydrogen resonances of the reduced 5'monophosphate of ls4U are the following: C5-H, 6.5 (d); C6-H, 8.1 (d); CH2 (exocyclic) and CH (piperidine), 3.7-4.4 (m); CH2 (piperidine), 1.8 (d); CH3 (piperidine), 1.2 (s) and 1.3 (s); Cl'-H, 5.8 (m); C2'-H to C5'-H, 3.7-4.4 (m).…”

“…Enzymatic Synthesis of Spin-Labeled RNA. (1 s4U,U)" was prepared according to a published procedure (Warwick et al, 1980). All other spin-labeled RNAs were prepared under conditions similar to those described for (ls4U,U)" by co-polymerizing uridine 5'-diphosphate with the appropriate nitroxide-containing nucleoside 5'-diphosphate analogue.…”