Figure 4.

Fidelity of PR-RTs. 20 nM of a 5′ ³²P endlabeled P₃₀/T₅₀dA DNA/DNA substrate was incubated with 1.25 mM of the correct (dTTP) or incorrect (dATP) nucleotide for polymerization. Reaction products were separated on a 10% sequencing gel. (A) Schematic representation of primer extension with the correct nucleotide and primer extension after one nucleotide mismatch. (B) Primer extensions in the presence of the next templated nucleotide (dTTP). Control, assay without enzyme. The diagram (top) depicts the mean values and standard deviations (black bars) of three independent experiments. The autoradiogram (bottom) shows the result of a typical extension experiment. The length of the extensions (P₃₁ to P₃₅) is indicated on the left. Since the bands above P₃₁ all comprise the first correct dTTP incorporation they were also included in quantification of the primer extension. For quantification of the extended products the total amount of labeled DNA per lane was set to 100%. (C) Mismatch primer extensions in the presence of a non-templated nucleotide (dATP). Evaluation of the results as in (B). P-values ≤ 0.05 represent statistically significant differences to the WT protein (* p-value ≤ 0.05; ** p-value ≤ 0.01;*** p-value ≤ 0.001).