Figure 1

WRN exonuclease activity on single-stranded DNA is dependent on length and ATP hydrolysis. A) Standard exonuclease assays were performed on single-stranded, labeled G80 (0.1 nM) for 15, 30 or 60 mins at 37°C with WRN (10.8 nM) and ATP (1 mM). B) Similarly, exonuclease assays were also performed for 15 min at 37°C on labeled oligomers of different lengths (G80, G45, G40, G35, G30, G24, 0.1 nM each) with WRN (2 or 4 nM) either without (left panel) or with 1 mM ATP (right panel). C) Reactions containing WRN (10.8 nM), labeled G24 (0.1 nM) and without (lanes 1–4) or with (lanes 5–8) 1 mM ATP were incubated for 15, 30 or 60 min and assayed for exonuclease activity. D) Exonuclease-deficient WRN-E84A (22 nM) was incubated with 0.1 nM of labeled G40 (lanes 1–3) or G30 (lanes 4–6) and 1 mM ATP for 0, 15 or 30 min. E) As indicated, either WRN (2–4 nM) or ATPase/helicase-deficient WRN-K577M (2–10 nM) was incubated with G35 or C55 (0.1 nM each) for 15 min at 37°C in the presence of 1 mM ATP. F) WRN (3.4 nM) was incubated with G80 or G45 (0.1 nM each) without or with 1 mM ATP as indicated for 1, 2.5 or 5 min. For each experiment above, reactions were stopped and DNA products were analyzed as described for the exonuclease assay in Methods; the positions of migration and lengths of the original substrates (in nt) are indicated between or at left of the panels above.