Kinetic analysis of reverse transcriptase activity of bacterial family A DNA polymerases

Abstract: Some bacterial thermostable, wild-type or genetically engineered family A DNA polymerases have reverse transcriptase activity. However, difference in reverse transcriptase activities of family A DNA polymerases and retroviral reverse transcriptases (RTs) is unclear. In this study, comparative kinetic analysis was performed for the reverse transcriptase activities of the wild-type enzyme of family A DNA polymerase (M1polWT) from Thermus thermophilus M1 and the variant enzyme of family A DNA polymerase (K4polL329A), in which the mutation of Leu329→Ala is undertaken, from Thermotoga petrophila K4. In the incorporation of dTTP into poly(rA)-p(dT)45, the reaction rates of K4polL329A and M1polWT exhibited a saturated profile of the Michaelis–Menten kinetics for dTTP concentrations but a substrate inhibition profile for poly(rA)-p(dT)45 concentrations. In contrast, the reaction rates of Moloney murine leukemia virus (MMLV) RT exhibited saturated profiles for both dTTP and poly(rA)-p(dT)45 concentrations. This suggests that high concentrations of DNA-primed RNA template decrease the efficiency of cDNA synthesis with bacterial family A DNA polymerases. [Copyright &y& Elsevier]