Kinetic analyses of DNA unwinding of the 47-bp forked duplex DNA substrate by FANCJ-WT or FANCJ polymorphic variants in the presence of RPA or ESSB. (A) FANCJ-WT (4.8 nM) or its associated variants (FANCJ-M299I or FANCJ-P47A) was incubated with the 47-bp forked duplex in the absence or presence of 24 nM RPA heterotrimer under standard helicase reaction conditions. Incubation was at 30°C for the indicated times. Quantitation of results from helicase assays with SD are indicated by error bars. FANCJ-WT (△); FANCJ-WT + RPA (▲); FANCJ-M299I (○); FANCJ-M299I + RPA (●); FANCJ-P47A (□); FANCJ-P47A + RPA (■).(B) Coimmunoprecipitation of FANCJ (FANCJ) sequence variants with RPA. FA-J cells were transiently transfected with plasmid DNA encoding Myc-tagged FANCJ-WT, FANCJ-M299I, or FANCJ-P47A, and WCEs were used for coimmunoprecipitation experiments using FANCJ antibody. The blot was probed with rabbit anti-FANCJ (top) and mouse anti-RPA (bottom) antibodies. Input represents 15% of WCE input for coimmunoprecipitation experiments. (C) RPA directly binds to FANCJ variants. RPA (96 nM heterotrimer) was coated onto the ELISA plate. After blocking with 3% BSA, the wells were incubated with increasing concentrations of purified recombinant FANCJ-M299I or FANCJ-P47A protein (0-150 nM) for 60 minutes at 37°C. Wells were aspirated and washed 3 times, and bound FANCJ protein was detected by ELISA using a rabbit polyclonal antibody against FANCJ.