PubMed · 7679340
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C Fehlmann, R Krapf, M Solioz. 1993. Reverse transcriptase can block polymerase chain reaction.. https://pubmed.ncbi.nlm.nih.gov/7679340/
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Pyrimidine analogues, N4-hydroxycytosine (C(oh)), N4-methoxycytosine (C(mo)) and 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P) can form base pairs with both adenine and guanine. We examined the mutagenic properties of these ribonucleotide analogues in RNA in reverse transcription with HIV and AMV reverse transcriptases. Both reverse transcriptases incorporated dATP and dGTP opposite these analogues in RNA. The incorporation ratio of dGTP to dATP opposite each analogue was measured to estimate the potential for inducing U-to-C mutations. The potentials may be rC(oh) > rC(mo) > rP for both reverse transcriptases. It might be possible to induce mutations in the retroviral genomes and to develop a new antiviral therapy, if these analogues are incorporated by a human RNA polymerase.
We compared the thermal stabilities of wild-type recombinant avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) with those of mutants of the recombinant enzymes lacking RNase H activity. They differed in resistance to thermal inactivation at elevated temperatures in the presence of an RNA/DNA template-primer. RNase H-minus RTs retained the ability to efficiently synthesize cDNA at much higher temperatures. We show that the structure of the template-primer has a critical bearing on protection of RT from thermal inactivation. RT RNase H activity rapidly alters the structure of the template-primer to forms less tightly bound by RT and thus less able to protect the enzyme at elevated temperatures. We also found that when comparing wild-type or mutant AMV RT with the respective M-MLV RT, the avian enzymes retained more DNA synthetic activity at elevated temperatures than murine RTs. Enzyme, template-primer interaction again played the most significant role in producing these differences. AMV RT binds much tighter to template- primer and has a much greater tendency to remain bound during cDNA synthesis than M-MLV RT and therefore is better protected from heat inactivation.
R2 is a retrotransposable element that specifically inserts into the 28 S rRNA genes of arthropods. The element encodes a single protein with endonuclease activity that cleaves the 28 S gene target site and reverse transcriptase (RT) activity that uses the cleaved DNA to prime reverse transcription. Here we compare various properties of the R2 RT activity with those of the well characterized retroviral RT, avian myeloblastosis virus (AMV). In processivity assays using heterogeneous RNA templates, R2 RT can synthesize cDNA over twice the length of that synthesized by AMV RT and can synthesize cDNA over 4 times longer than AMV RT in assays with poly(rA) templates. The higher processivity of R2 RT compared with retroviral RTs is a result of the slower rate of dissociation of the enzyme from RNA templates. The elongation rates of the two enzymes are similar. Finally, a highly distinct property of the R2 RT, compared with retroviral enzymes, is its ability to displace RNA strands annealed to RNA templates during cDNA synthesis. We suggest that both the higher processivity and displacement properties of R2 RT compared with retroviral RT result from the greater affinity of the R2 protein for the RNA template upstream of its active site.