Question

In the Harris paper, they used the Minus dNTP Assay to measure the fidelity of the mutant RTs and compared their fidelity with that of the WT RT. They took a select group of mutants and tested this select groups fidelity on the U5-PBS DNA primer-template in the presence of Mn²⁺. (i) Why didn’t the researchers choose these mutants to test with Mn²⁺; (ii) How does the Mn²⁺ affect the fidelity of the select group?

Answer 1

i) The researchers did not choose these mutants to test with Mn2+ because this divalent cation rescues polymerase activity even during sub-optimal conditions where Mg2+ fails to do so. Thus, Mn2+ exerts infidelity to the polymerase by aiding its processivity even when conditions are not otimal (such as right concentrations of dNTPs). Therefore, Mn2+ is not a wise option to opt for when two groups, WT RTs and mutant RTs are tested for their fidelity.

ii) It has been seen that the fidelity of polymerase activiy is significantly compromised when Mg.dNTP was replaced with Mn.dNTP. In the presence of Mn2+, high fidelity mutants are able to catalyze misinsertion which is not witnessed in presence of Mg2+.