Figure 3

The processivity of WRN exonuclease is enhanced by ATP binding and hydrolysis. A) Experimental design to examine, under various conditions, processivity of WRN exonuclease activity on single-stranded substrates by addition of excess competitor single-stranded DNA during the reaction. In duplicate samples, labeled, single-stranded 80-mer is preincubated with WRN at 4°C for 5 min, then enzymatic reactions are initiated at 37°C. After 2.5 min, an excess of competitor DNA (unlabeled 80-mer) was added to one reaction while the other remained unchanged. For each sample, aliquots were removed for analysis just prior to the 37°C incubation (0 min), at the time of addition of the competitor (2.5 min), and at 5, 10, and 15 min into the 37°C incubation. B) As described above, reactions containing WRN (1.2 nM) and G80 (0.25 nM) without ATP (left panel), with 1 mM ATP (middle panel), or with 1 mM ATPγS (right panel) conditions were preincubated at 4°C then incubated at 37°C and, where indicated, an eightfold excess of unlabeled G80 (2 nM) was added 2.5 min into the 37°C incubation. Aliquots were removed after 0, 2.5, 5, 10, and 15 min of 37°C incubation for analysis of exonuclease activity as described in Methods. Vertical arrows indicate the timing of addition of excess competitor in the relevant reactions; asterisks indicate relatively short fragments appearing in reactions containing competitor as well as either ATP or ATPγS, but not in comparable reactions minus ATP. Horizontal arrows indicate the position of the reference point used in partitioning of long and short fragments for quantitation (presented in C) of the relative amounts of inward degradation under different conditions. C) For the reactions in B in which the unlabeled G80 competitor was added at 2.5 min, the percentage of radioactivity present below the reference point (defined by horizontal arrows in B) with respect to the total radioactivity in the reaction was quantitated. The bar graph directly compares the percentage of radioactivity below the reference point for the different reaction conditions (-ATP, +ATP, +ATPγS) at the individual time points; the exact percentage appears above its respective bar.