(Figure 4A, bottom panels). However, digestion of the leading daughter strand is much more pronounced with WRN-wt in the presence of ATP in comparison to each of the other conditions, as indicated both by the direct digestion patterns (Figure 4A, bottom panels) and by the quantitation of relative changes in leading daughter strand length ranging from 27 nt (undigested) to 17 nt after 5 min (Figure 4B). While the other conditions have remarkably similar digestion proﬁles, reactions containing WRN-wt in the presence of ATP show, by 5 min, a much higher level of exonucleolytic activity on the leading daughter strand by two criteria. They have 1) a much more dramatic (approximately 2.5-fold greater) loss of signal associated with undigested leading daughter strand (27 nt) and 2) a concomitantly increased generation of shorter leading daughter strand products with lengths between 26 and 19 nt (Figure 4B, compare black bars to others). Also notable in this digestion pattern are elevated amounts of 19- and 20-mers compared to several longer leading daughter strand products (see next paragraph for further clariﬁcation). Since only reactions including WRN-wt with ATP showed both formation of daughter duplex and enhanced exonuclease activity on the leading daughter strand, these results suggest that optimal WRN exonu- clease activity may be connected to partial unwinding of the leading daughter strand and perhaps regression of the fork substrate. These experiments not only conﬁrm that regression activity speciﬁcally requires the ATPase and helicase activities of WRN, but also indicate that the exonuclease activity of WRN on the leading daughter strand of replication fork substrates is positively corre- lated to its ability to perform regression. Finally, we tested how the 30 to 50 exonuclease activity of WRN processed the leading daughter strands of fork substrates with diﬀerent leading arm gaps in relation to the speciﬁc formation of daughter duplexes. For these experiments, fork substrates with leading arm gaps of 2–11 nt were incubated with WRN-wt and total DNA products were analyzed in parallel by native and denatur- ing PAGE. In addition, daughter duplex products from native PAGE were excised, extracted, and then analyzed by denaturing PAGE alongside the total DNA products from the regression reaction. As before, native PAGE (Figure 5A) showed that WRN-wt generated daughter and parental duplexes along with some short single- stranded products from these substrates. Signiﬁcantly, the migration of daughter duplexes generated from fork substrates with gap sizes of 2–8 nt was slightly faster than their respective (undigested) daughter duplex mar- kers (Figure 5A, compare lanes 5, 8 and 11 with lanes 6, 9 and 12, respectively). Analysis of total DNA products by denaturing PAGE shows that WRN-wt degrades the labeled leading daughter strand of each substrate, although the number of nucleotides removed decreases as the gap size increases (Figure 5B, lanes 2, 5, 8, 11 and 14). Conspicuously, for each fork substrate, there is substantial degradation until the leading daughter strand reaches 19–20 nt, but digestion beyond that point decreases precipitously. In this analysis of total products, the leading daughter strand could be associated with daughter duplex, displaced leading daughter strand and/or four-stranded Nucleic Acids Research, 2007, Vol. 35, No. 17 5739 fork, but not to any signiﬁcant extent with other DNA structures. Parallel analysis of the extracted daughter duplexes (denoted a-d in Figure 5A and B) shows that the leading daughter strand component of these duplexes is strictly degraded in a range extending down to 19 nt (Figure 5B, lanes 3, 6, 9, 12 and 15). Thus, the lengths of leading daughter strand speciﬁcally associated with the daughter duplex product reﬂects a deﬁned subset of the WRN exonuclease activity occurring during the entire reaction. Keeping in mind that WRN exonuclease does not act on the daughter duplex once formed (Figure 3), this result indicates that the exonuclease activity of WRN- wt digests the leading daughter strand of each fork substrate, decreasing its length and thereby increasing the leading arm gap size to as much as 13 nt, prior to or concomitant with unwinding and pairing of daughter strands to yield the (faster-migrating) daughter duplexes. To scrutinize this further, the relative amounts of radioactivity associated with each band present in lanes corresponding to the extracted daughter duplexes pro- duced from each fork substrate were determined and directly compared (Figure 5C). This analysis shows that, for each substrate, a minor portion of the daughter duplexes contained undegraded leading daughter strands—i.e. 15.1, 9.5, 19.1 and 25% (Figure 5C, see striped shapes) for the daughter duplexes from the fork substrates with 2, 5, 8 and 11 nt gaps, respectively. Thus, digestion of the leading daughter strand (prior to or concomitant with daughter duplex formation) occurs during the vast majority of fork regression events catalyzed by WRN-wt. The most favored length of the leading daughter strand within the daughter duplex product was, by far, 20 nt followed closely by 19 nt, regardless of the leading strand gap size in the original fork substrate. Although the length of the leading daughter strand in these daughter duplexes is certainly not uniform, there is a deﬁnite preference for a leading daughter strand length of 19 or 20 nt corresponding to a gap size of 13 or 12 nt on the original fork substrate. When considered in combination with our comparison of WRN-E84A and WRN-wt on fork substrates with diﬀerent leading arm gaps (Figure 1), these ﬁndings convincingly demonstrate that the exonuclease activity inherent in wild type WRN enhances the regression of forks with small (2–8 nt) leading strand gaps, apparently by generating a better structure for the regression process. Furthermore, our data thus far indicate that the favored structure for regression by the coordinated helicase and annealing activities of WRN is a fork with a leading strand gap of at least 11–13 nt. WRN regresses replication forks to form Holliday junction intermediates The experiments above demonstrate that WRN (and BLM) can readily produce a daughter duplex from model replication forks with homologous parental–daughter arms of limited length. However, in vivo the parental- daughter arms are essentially continuous, extending back to the origin of replication. Thus, during the process of fork regression, the daughter strands would remain