Figure 3

Effects of hRPA on unwinding and annealing by WRN-E84A and BLM. A) Helicase reactions were performed for 7.5 min with the 80 bp, 15 nt 3' tail (labeled C80/G95) substrate (0.1 nM) with WRN-E84A (9 nM), BLM (4 nM), and/or hRPA (0.1–50 nM) and analyzed as described in Methods. B) Quantitation of data from the experiment depicted in A, the effect of hRPA on WRN-E84A and BLM unwinding. C) Annealing reactions were performed in the absence of ATP for 7.5 min using labeled C80 (0.1 nM) and unlabeled G95 (0.1 nM) and WRN-E84A (9 nM), BLM (4 nM), and/or hRPA (0.1–50 nM) and DNA products were analyzed as described in Methods. D) Quantitation of data from the experiment depicted in C, the inhibition of WRN-E84A- (WRN) and BLM-mediated annealing by hRPA. E) Annealing assays were performed as in C, except using eSSB (0.025–1.25 nM) instead of hRPA. F) Quantitation of data from experiments as described in E, the inhibition of WRN-E84A (WRN) and BLM-mediated annealing by eSSB. Each data point is the mean of two experiments. G) Annealing assays were performed with labeled C80 and unlabeled G95 (0.025 nM each) minus or plus WRN-E84A (4.5 nM) in reaction buffer without or with ATP (1 mM) and either normal (4 mM) or low (1 mM) MgCl₂ concentration as indicated. H) Helicase assays were performed with the 80 bp, 15 nt 3' tail substrate (0.025 nM) in reaction buffer containing 1 mM MgCl₂ with WRN-E84A (4.5 nM) minus or plus hRPA (0.1–50 nM). I) Quantitation of data depicted in H, the stimulation of WRN-E84A (WRN) unwinding by hRPA in low (1 mM) MgCl₂. J) Helicase reactions were performed using 1 mM MgCl₂ as in H with or without WRN-E84A (4.5 nM) and the indicated concentrations of hRPA or eSSB.