Figure 3.

MutSβ-stimulated DNA synthesis by Polβ requires a (CAG)_n or (CTG)_n hairpin. Unless mentioned otherwise, hairpin retention/removal assays were performed in a 40-μL purified system containing 0.15 pmol (CAG)₅ or (CTG)₅ DNA hairpin substrate, 4 pmol MutSβ, 110 fmol RFC, 2 pmol PCNA and 260 fmol Polβ. DNA synthesis products were analyzed by Southern blot analysis as described in Figure 1 legends. (A) Comparison of hairpin retention activity of Polβ in reactions with or without MutSβ. DNA substrate in non-hairpin reactions (reactions 1 and 2) used a ssM13mp18 derivative containing 15 CAG repeats to match the size of hairpin-retained products. (B) Hairpin retention activity in reaction with heat-inactivated MutSβ. (C) Dependence of hairpin retention activity on the incubation order of MutSβ and Polβ. Relative hairpin retention activity in each group was calculated by using the hairpin retention activity conducted by Polβ alone as a reference, i.e., dividing hairpin retention activity of each reaction with that catalyzed by Polβ alone (see *). The data were from three independent experiments and the error bar represents SD.