The role of template-primer in protection of reverse transcriptase from thermal inactivation

Abstract

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.

Insight into the Mechanism of the Stabilization of Moloney Murine Leukaemia Virus Reverse Transcriptase by Eliminating RNase H Activity

“…Gerard et al 8) and we 9) have found that T/P-mediated stabilization of AMV RT was more potent than that of MMLV RT, suggesting that AMV RT has higher affinity toward the T/P than MMLV RT. Recently, Arezi et al discovered five novel mutations (Glu69 !…”

“…7) Gerard et al have found that elimination of the RNase H activity of RT markedly enhanced the thermal stability of reverse transcription activity. 8) We have found that the reduction of reverse transcription activity of RT enhanced the thermal stability of RNase H activity. 6) Because RNase H activity is not required for in vitro cDNA synthesis, genetically engineered MMLV RT lacking RNase H activity is now widely used as a tool to synthesize cDNA.…”

Effects of Organic Solvents on the Reverse Transcription Reaction Catalyzed by Reverse Transcriptases from Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus

“…Both Gerard et al 22) and our own laboratory group 13) have found that T/P-mediated stabilization of AMV RT was more potent than that of MMLV RT, suggesting that AMV RT has higher affinity toward the T/P than does MMLV RT. Arezi et al discovered five novel mutations (Glu69!…”

“…Formamide at 10% inhibited PCR amplification by non-engineered Taq DNA polymerase, but increased the activity of the genetically engineered thermostable variant AmpliTaq DNA polymerase. 19,21) This suggests that genetically engineered thermostable MMLV RT variants [22][23][24] might also be highly resistant to DMSO and formamide effects.…”

Stabilization of Moloney murine leukemia virus reverse transcriptase by site-directed mutagenesis of surface residue Val433

“…The thermostability of MMLV RT was first improved by eliminating RNase H activity. [3][4][5] Recently, Arezi and Hogrefé increased thermostability by random mutagenesis in combination with activity screening at high temperature and generated a highly stable multiple variant, E69K/E302R/W313F/L435G/N454K 6) (In this report, mutation of a residue, e.g. Glu69 to Lys, is designated Glu69→Lys, and the MMLV RT variant, e.g.…”