Temperature effects on the fidelity of a thermostable HIV-1 reverse transcriptase.

ABSTRACT

Transcriptomics and gene expression analysis are largely dependent of the availability of efficient thermostable reverse transcriptases (RTs). However, the intrinsic fidelity of DNA synthesis catalyzed by retroviral RTs is low. Reported error rates are in the range 1.2 × 10(-5)-6.7 × 10(-4), with oncoretroviral RTs being the most faithful enzymes. Wild-type HIV-1 group O (HIV-1O) RT is a thermostable polymerase that is able to synthesize cDNA at temperatures as high as 70 °C. At 37 °C, its error rate has been estimated at 5.8 × 10(-5) in M13mp2 lacZ-based forward mutation assays. However, at higher temperatures (e.g. 50 and 55 °C), the accuracy of HIV-1O RT is increased by approximately two- to five-fold. At 55 °C, the HIV-1O RT error rate (1.3 × 10(-5)) was similar to that shown by the AffinityScript (Agilent Technologies Inc., La Jolla, CA, USA) RT, a commercially available thermostable murine leukaemia virus RT. At higher temperatures, the increased accuracy of the HIV-1 enzyme results from a lower base substitution error rate, although it shows a higher tendency to introduce frameshifts. Kinetic studies carried out with model template-primers suggest minor differences in nucleotide discrimination, although, at higher temperatures, HIV-1O RT showed a reduced ability to extend mispaired template-primers.