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Fig. 4. WRN binds directly to RAD51 and RAD54B. (A) Reciprocal ELISAs showing direct binding between RAD51 and WRN. WRN, RAD52 (positive control), BSA (negative) and RAD51 were coated on microtiter plates. Binding of WRN, RAD52 and RAD51 to each other were measured as described in Materials and Methods. Values (OD_490nm) for 1:1 molar ratio between the proteins are given. (B) Relative binding of RAD54B, RAD54, RAD51 and RAD52 to WRN coat in ELISA. Both results (A and B) are representative of one out of at least three independent experiments. Absorbance (OD_490nm) is given as mean of duplicates adjusted for background binding to BSA in parallel duplicate wells. (C) Dot blot assay demonstrating a direct interaction between WRN and RAD51, and WRN and RAD54B. PVDF membranes with immobilized proteins were incubated in milk-solutions with or without WRN, and analyzed for WRN binding. (D) RAD51 and RAD54B do not modulate WRN helicase activity on a 12 bp bubble substrate. Native gels showing WRN (6.0 nM and 4.0 nM) mediated unwinding of a 12 bp 5'-labeled bubble substrate after adding increasing amounts of RAD51 (6, 12, 24 and 48 nM) and RAD54B (2, 4, 20 and 40 nM).

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