Figure 5
ATP-dependent enhancement of WRN exonuclease on overhang substrates. A) The 5, 10, 15, 20, or 25 nt 3' overhang substrate (0.25 nM) was incubated for 10 min at 37°C without enzyme, with helicase-deficient WRN-K577M (hel-, 3 nM), or with wild type WRN (WT, 2.7 nM) in the presence or absence of ATP or ATPγS (1 mM) as indicated. The initial lengths of the labeled strands for each substrate are indicated at left. B) Reactions containing the 10 nt 3' overhang C55/G45 substrate (0.25 nM) and wild type WRN (2.7 nM) were incubated without or with ATP (1 mM) as indicated at 37°C. At 0, 2.5, 5, 7.5, and 10 min, two aliquots were removed from each reaction; one was analyzed for exonuclease activity by denaturing PAGE (top) while the other was run on non-denaturing PAGE (bottom) to determine whether WRN was unwinding the substrate. The single-stranded, labeled C55 oligomer was loaded as a marker on the non-denaturing gel (lane 11) and the positions of undegraded 3' overhang substrate and single-stranded 55-mer are indicated at left. The extensive degradation observed under +ATP conditions (top) is almost exclusively occurring on the overhang substrate while still in duplex form, as indicated by its slightly faster migration with time (bottom, lanes 7–10). The small amount of signal migrating faster than the single-stranded marker in both the – and +ATP reactions is primarily due to degradation of the small amount of single-stranded component present in the original substrate preparation (bottom, see lanes 1 and 6). C) Reactions containing the 5, 10, or 15 nt overhang substrate (0.05 nM) were incubated 37°C with WRN (3.5 nM) in the presence of ATP (1 mM). Aliquots were removed at the indicated times and analyzed for exonuclease activity. Nucleotide size markers are indicated at left. D) As described in Methods, EMSA was performed with reactions containing ATPγS, WRN-E84A (2.4, 4.8, or 7.2 nM), and 5, 10, 15, 20, or 25 nt overhang substrate (0.025 nM) and as indicated. E) From EMSA experiments as in D, quantitation and graphical representation of WRN binding to the 25 (■), 20 (●),15 (▲),10 (◇), and 5 (×) nt overhangs. Each data point is the mean of two independent experiments.