Figure 4.
BCCIPβ stimulates human RAD51-mediated D-loop formation in the presence of calcium. (A) Schematic of the D-loop assay. (B) RAD51 (1.5 μM) was incubated with 32P-OL90 (ssDNA; 4.5 μM) in the absence (lane 2) or presence of increasing concentrations of BCCIPβ (lanes 3–6; 0.75 μM, 1.5 μM, 3 μM, 4.5 μM, respectively). The supercoiled plasmid pBluescript (sc; 35 μM base pairs) was introduced to the reaction. After 6 min, the reactions were deproteinized with SDS and Proteinase K. Lane 1 was devoid of protein, lane 7 contained RAD51 and BCCIPβ (4.5 μM) in the absence of ATP, and lane 8 contained BCCIPβ (4.5 μM) alone. (C) The reactions in B were tested in the presence of (1.8 mM) calcium. (D) The D-loop assay was performed with ScRad51 (1.5 μM) in presence of BCCIPβ (lanes 4–6; 1.5 μM, 3 μM, 4.5 μM, respectively) or ScRad54 (0.2 μM; lane 3). All reaction products were subjected to 0.9% agarose gel electrophoresis and visualized using a phosphorimager. Lane 1 contained no protein, and lane 2 contained only ScRad51. S.e.m. (n = 3) are plotted as error bars; P-value *< 0.05, **< 0.01.