Limited unwinding reaction catalyzed by FANCJ is stimulated by RPA. (A) FANCJ helicase catalyzes a limited unwinding reaction. Helicase assays were as described,14 using the indicated concentrations of FANCJ-WT or FANCJ-M299I and forked duplex DNA substrates of 22 bp and 47 bp. Incubation was at 30°C for 15 minutes. Reaction products were analyzed by nondenaturing gel electrophoresis. Quantitation of results from helicase assays with standard deviations indicated by error bars. FANCJ-WT, 22 bp, ▲; FANCJ-M299I, 22 bp, △; FANCJ-WT, 47 bp, ■; FANCJ-M299I, 47 bp, □. Percentage of displacement is expressed as a function of FANCJ-WT or FANCJ-M299I protein concentration. (B-D) Stimulation of FANCJ-WT helicase activity by RPA. FANCJ-WT protein (4.8 nM) was incubated with the 47-bp forked duplex in the presence of the indicated concentrations of RPA heterotrimer (B) or ESSB homotetramer (C) under standard helicase reaction conditions. Incubation was at 30°C for 60 minutes. (D) Quantitation of results from helicase assays with SD indicated by error bars. Percentage of displacement is expressed as a function of single-stranded DNA-binding protein concentration (RPA heterotrimer or ESSB homotetramer). (Inset) Quantitation of results with percentage of displacement expressed as a function of the ratio (SSB binding unit)/(DNA-binding unit).
