5740 Nucleic Acids Research, 2007, Vol. 35, No. 17 Figure 5. The exonuclease activity of WRN-wt performs limited digestion of the leading daughter strand of fork substrates to create an optimal structure for fork regression. Reactions containing fork substrates (50 pM) with leading strand gaps of 11, 8, 5 and 2 nt were incubated for 5 min at 378C without ( ) or with (+) WRN-wt (300 pM). (A) Aliquots of these reactions were analyzed by native PAGE along with markers (M) for undigested daughter duplexes and leading daughter strands speciﬁc for each fork substrate (lanes 3, 6, 9 and 12). Positions of migration of parental duplex (70lag/70lead), daughter duplex and leading daughter strand products are indicated at left. The letters (a–d) associated with daughter duplexes (generated from each fork substrate) identify the speciﬁc products that were excised and analyzed by denaturing PAGE. (B) In parallel, aliquots of the same reactions were subject to denaturing PAGE with the total DNA products from reactions containing WRN-wt (lanes 2, 5, 8 and 11) run alongside the corresponding daughter duplex products (a–d) individually extracted from native PAGE (lanes 3, 6, 9 and 12). The positions of migration and sizes (in nt) of both undigested leading daughter strands for each fork substrate and the primary 19 and 20 nt digestion products (denoted with arrowheads) are indicated at right. (C) The percentage of the total radioactivity associated with each band in the daughter duplex digestion proﬁles (B, lanes 3, 6, 9 and 12) was quantitated and then plotted with respect to the product (leading daughter strand) lengths derived from each original fork substrate [back to front, 11 nt gap (purple blocks), 8 nt gap (blue cylinders), 5 nt gap (orange pyramids) and 2 nt gap (green cones) substrates, respectively]. Striped shapes represent the amount of undigested leading daughter strand for each substrate, also signifying the maximum length possible for each corresponding substrate. associated with the parental strands resulting in a Holliday junction or ‘chicken foot’ structure. Such a structure is believed to be the key intermediate in pathways that cope with blocked forks and restart replication (33–37). To determine whether WRN could generate Holliday junction intermediates, a longer, more complex replication fork substrate was designed. Precisely at the fork junction, this substrate (Figure 6A, top left) contained 5 nt of