C-Terminal Extension of the Yeast Mitochondrial DNA Polymerase Determines the Balance between Synthesis and Degradation
Figure 5
DNA binding affinity of Mip1 and C-terminal deletion mutants.
DNA binding was measured with an electrophoretic mobility shift assay using an oligomeric substrate of 45 nt template strand and a radiolabeled 25 nt primer. 0.2–12.5 nM polymerase was incubated for 2 min at 0°C with 1 nM substrate. A. Reaction products were resolved on a native Tris-glycine 8% polyacrylamide gel. Positions of the free and bound substrate are indicated accordingly with empty and filled triangles. B. The dissociation constant KD (nM) was calculated from the logarithmic binding curve as the concentration of the polymerase when 50% of the substrate was bound. Data from three independent experiments was used to establish the average KD and standard deviation values.