Figure 3. DNA binding specificity of Mei5-Sae3 and Mei5.

(A and B) Mei5-Sae3 (panel I; 0.1 μM, lane 2; 0.3 μM, lane 3; 0.6 μM, lane 4; 1.25 μM, lane 5; 2.0 μM, lane 6 and 7) and MBP-Mei5 (panel I; 0.07 μM, lane 9; 0.2 μM, lane 10; 0.4 μM, lane 11; 0.83 μM, lane 12; 1.38 μM, lane 13 and 14) were incubated with 0.05 pmol of radiolabeled fork, dsDNA and ssDNA (A, panel I) or radiolabeled fork, dsDNA, and OH (B, panel I) for 10 min at 37°C and subjected to non-denaturing-PAGE. The gels were dried and visualized using a Typhoon phosphorimager. Reactions with Mei5-Sae3 (A and B, panels II) and MBP-Mei5 (A and B, panel III) were quantified using ImageQuant (GE Healthcare) software. (C and D) Mei5-Sae3 and MBP-Mei5, respectively (panel I and II; 0.08 μM, lane 2 and 8; 0.26 μM, lane 3 and 9; 0.52 μM, lane 4 and 10; 1.3 μM, lane 5 and 11; 1.3 μM, lane 6 and 12) were incubated with 0.05 pmol of radiolabeled poly dT 10-mer (panel I, lanes 1–6), poly dT 20-mer (panel I, lanes 7–12), poly dT 40-mer (panel II, lanes 1–6), and poly dT 60-mer (panel II, lanes 7–12) for 10 min at 37°C and treated as described above. Where indicated, the reaction was treated with SDS (0.5% final) and Proteinase K (0.5 mg/mL) at 37°C for 10 min prior to analysis. S/P, SDS/Proteinase K; NP, no protein control; ss, ssDNA; ds, dsDNA.