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Figure 2 | BMC Molecular Biology

Figure 2

From: Competition between the DNA unwinding and strand pairing activities of the Werner and Bloom syndrome proteins

Figure 2

Addition of trap strand stimulates unwinding of long duplexes by WRN-E84A and BLM. A) The 80 bp with 15 nt 3’ tail (labeled C80/G95) substrate (0.1 nM) and WRN-E84A (9 nM) without or with the addition of unlabeled C80 oligomer (0.05-2.5 nM, 0.5-25´ with respect to C80/G95 substrate concentration) were incubated at 37oC for 7.5 min and DNA products were analyzed as specified in Methods for the helicase assay that measures conversion of duplex reactants to single-stranded products (indicated here and subsequently by a downward arrow between DNA structures at left). B) Helicase assays were performed as in A, except using BLM (4 nM) without or with unlabeled C80 (0.1-10 nM, 1-100´). C) Quantitation of the stimulation of WRN-E84A (WRN) and BLM unwinding by unlabeled C80 (trap strand) from experiments as those described in A and B. The WRN and BLM data points are the means of three and four independent experiments, respectively. D) Helicase assays were performed as in A, except using variable concentrations of WRN (1.5- 12 nM) and a fixed concentration of unlabeled C80 oligomer (0.5 nM, 5´), respectively. E) Helicase assays were performed as in A, except using a non-complementary unlabeled NC77 oligomer (0.05-2.5 nM, 0.5-25´). F) Helicase assays performed without the addition of unlabeled C80, using four-fold lower C80/G95 substrate (25 pM) and WRN-E84A (3-12 nM), with quantitation of WRN-dependent unwinding presented above relevant lanes.

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