FIG. 3.
“Manganese suppressor” mutants display an increased polymerization rate in the presence of elevated manganese levels. (A) Time course assay with the WT and representative mutants in the absence of manganese. (B) Time course assay with the WT and representative mutants in the presence of a 0.1 Mn2+/Mg2+ ratio. Assays of recombinant WT and mutant Ty1 RT proteins were performed in 30-μl reaction mixtures [50 mM HEPES-KOH (pH 7.8), 3 mM DTT, 0.2 μM dGTP, 0.5 μCi of [α-32P]dGTP, 1 μg/ml oligonucleotide (dG)12-18, 10 μg/ml poly (rC)n]. Reactions were set up on ice and carried out at the indicated times (3, 6, 10, 22, and 32 min) at 22°C, and products were kept on ice while being spotted onto DE81 anion-exchange paper. The RT activity of manganese suppressor mutant proteins was normalized to WT RT activity.