5738 Nucleic Acids Research, 2007, Vol. 35, No. 17 Figure 3. Comparison of WRN exonuclease activity on substrates and products of fork regression reactions. WRN-wt (0, 120 or 480 pM) was incubated at 378C for 5 min with 50 pM of fork substrate containing a leading arm gap of 5 nt [ 27lead/70lead- 70lag/30lag (lanes 1–3)], pre- formed daughter duplex substrates [ 21lead/30lag (lanes 4–6)], 24lead/ 30lag (lanes 7–9), 27lead/30lag (lanes 10–12) and 30lead/30lag (lanes 13–15)] or isolated leading daughter strand oligomers [ 21lead (lanes 16–18), 24lead (lanes 19–21), 27lead (lanes 22–24) and 30lead (lanes 25–27)], with the asterisks above indicating the radiolabeled strands. The resulting DNA species were analyzed by denaturing PAGE and phosphorimaging, with the lengths (in nt) and positions of migration of the labeled, undigested oligomers within various substrates indicated at left. Importantly, WRN exonuclease activity is only detectable on the leading daughter strand in the context of the intact fork substrate. occurred before or concomitant with (but not after) the formation of daughter duplex. To further investigate a possible relationship between digestion of the leading daughter strand and regression, the eﬀect of ATP on WRN exonuclease activity was examined. Reactions with 27lead fork substrate and either WRN-wt or the ATPase- and helicase-deﬁcient WRN- K577M mutant were analyzed in parallel for regression and exonuclease activity in the presence or absence of ATP. For these experiments, concentrations of WRN-wt and WRN-K577M that yielded approximately equal levels of exonuclease activity on the leading daughter strand within this fork substrate in the absence of ATP were used. When DNA products of kinetic assays over 5 min with and without ATP were analyzed by native PAGE, formation of daughter (and parental) duplexes occurs readily with WRN-wt in the presence of ATP but not detectably in its absence; moreover, WRN-K577M does not catalyze formation of daughter duplex regardless of the presence or absence of ATP (Figure 4A, top panels). WRN-wt and WRN-E84A also do not catalyze formation of daughter duplex in the presence of the weakly hydrolyzable analog ATPgS (data not shown). These results demonstrate that formation of daughter duplex indicative of fork regression requires the ATPase and helicase activities of WRN. When these same reactions were analyzed by denaturing PAGE, some degradation of the leading daughter strand by WRN-wt and WRN- K577M is observed whether or not ATP is present Figure 4. Eﬀects of ATP on WRN regression and exonuclease activities on fork substrates. (A) Reactions containing 27lead fork substrate (50 pM) with a leading arm gap of 5 nt and either WRN-wt (150 pM, lanes 1–8) or WRN-K577M (150 pM, lanes 10–17) were incubated at 378C in the presence or absence of ATP as indicated. Duplicate aliquots were removed from each reaction at 0, 1, 2.5 and 5 min for analysis in parallel by native (top panels) and denaturing PAGE (bottom panels). The native PAGE analysis also contained daughter duplex (27lead/ 30lag) and leading daughter strand (27lead) markers (lane 9) and the migration of relevant DNA structures (top) and various lengths of single-stranded species (bottom) are indicated between panels. (B) For the 0 and 5 min time points in A analyzed by denaturing PAGE, the amount of radioactivity in each band of 27 nt was quantitated and the percentage of each band with respect to total signal derived from the leading daughter strand was determined. Then, the relative (loss or gain) change in the percentage of each DNA species ranging between 17 and 27 nt from the 0 to the 5 min time point is presented in bar graph form to directly compare exonuclease digestion proﬁles under the following conditions: WRN-K577M minus ATP (white), WRN-K577M plus ATP (light gray), WRN-wt minus ATP (dark gray), WRN-wt plus ATP (black). The table at bottom shows these numerical values for the relative change in each DNA species over 5 min. Negative values associated speciﬁcally with the 27-mer reﬂect the reduction in the amount of intact leading daughter strand as a result of WRN exonuclease activity.