Reverse transcriptases from HIV-1 and MuLV respectively prefer Mg 2+ and Mn2+ for their polymerase activity, with variable fidelity, on both RNA and DNA templates. The function of the RNase H domain with respect to these parameters is not yet understood. To evaluate this function, two chimeric enzymes were constructed by swapping the RNase H domains between HIV-1 RT and MuLV RT. Chimeric HIV-1 RT, having the RNase H domain of MuLV RT, inherited the divalent cation preference characteristic of MuLV RT on the DNA template with no significant change on the RNA template. Chimeric MuLV RT, likewise partially inherited the metal ion preference of HIV-1 RT. Unlike the wild-type MuLV RT, chimeric MuLV RT is able to use both Mn.dNTP and Mg.dNTP on the RNA template with similar efficiency, while a 30-fold higher preference for Mn.dNTP was seen on the DNA template. The metal preferences for the RNase H activity of chimeric HIV-1 RT and chimeric MuLV RT were, respectively, Mn 2+ and Mg2+, a property acquired through their swapped RNase H domains. Chimeric HIV-1 RT displayed higher fidelity and discrimination against rNTPs than against dNTPs substrates, a property inherited from MuLV RT. The overall fidelity of the chimeric MuLV RT was decreased in comparison to the parental MuLV RT, suggesting that the RNase H domain profoundly influences the function of the polymerase domain.
All Science Journal Classification (ASJC) codes
- Infectious Diseases