5744 Nucleic Acids Research, 2007, Vol. 35, No. 17 First, WRN does not anneal the free daughter oligomers (due to their short length) that comprise daughter duplexes (Supplemental Figure 2), indicating that anneal- ing does not independently follow unwinding of the parental-daughter arms. Second, at limiting concentra- tions of WRN-E84A (Figure 1), there is almost exclusive production of daughter and parental duplexes with- out signiﬁcant generation of three-stranded forks or free leading daughter strands. Furthermore, free leading daughter strands are not signiﬁcantly produced at any time during our kinetic experiments (Figures 1D–F and 4A), strongly suggesting that daughter duplexes are formed from the fork substrates without release of daughter strands. Most convincingly, a quantitative analysis of WRN-E84A-mediated regression of 21lead fork substrate over time demonstrates that the parental and daughter duplexes are produced directly and essen- tially simultaneously from intact four-stranded fork (Figure 1F). Lastly, pairing of daughter strands during regression is a process mediated by WRN (or BLM) but not all unwinding enzymes, as other helicases such as UvrD and Rep unwind both daughter strands of our short fork substrates but do not produce daughter duplexes. These results indicate that daughter duplex formation does not simply occur spontaneously during daughter strand unwinding. Taken together, our results suggest that, during regression, the partially unwound daughter strands are juxtaposed by WRN in a manner that promotes their pairing to form a Holliday junction that, for our short fork substrates, is subsequently converted to daughter and parental duplexes. This is further supported by the WRN-dependent conversion of our long fork substrate to Holliday junctions (Figure 6) in which the daughter strands are paired while still associated with the parental strands. It seems very likely that WRN-mediated fork regression occurs through coordination between its unwinding function and its previously reported strand annealing activity (23). The results with our short fork substrates indicated that the leading arm structure had a major inﬂuence on the eﬃciency of fork regression. Speciﬁcally, daughter duplex formation by exonuclease-deﬁcient WRN-E84A was greatly enhanced when the single-stranded gap on the leading arm increased to 11 nt from 8 nt (Figures 1 and 2). Although WRN-wt was similarly eﬀective as WRN- E84A in daughter duplex formation from substrate with an 11 nt gap, it was much more eﬃcient on fork substrates with shorter leading strand gaps (Figure 2), suggesting that WRN’s 30 to 50 exonuclease activity might be digesting the leading daughter strand to increase the gap size. In addition, daughter duplexes formed by WRN-wt migrated slightly faster by native PAGE than those formed by WRN-E84A, in agreement with putative 30 to 50 processing of the leading daughter strand. Analysis by denaturing PAGE conﬁrmed that WRN-wt was speciﬁ- cally degrading the leading daughter strand during these regression reactions (Figures 3–5). However, the exonu- clease activity of WRN-wt could not detectably digest daughter duplexes (or displaced leading daughter strands) once formed (Figure 3), indicating that digestion of the leading daughter strand was occurring prior to or concomitant with formation of daughter duplex. Further analysis indicated that, during regression, exonucleolytic digestion of the leading daughter strand by WRN-wt was essentially limited to a deﬁned range, regardless of the original length of this strand in the fork substrate. The observed preference for digestion to a length of 20 or 19 nt corresponds to generation of a 12 or 13 nt single- stranded gap on the leading arm, respectively (Figure 5B and C). Taken together, these results indicate that WRN exonuclease activity promotes regression on substrates with gaps shorter than 11 nt by digesting the leading daughter strand to increase the leading arm gap size. The exonuclease-deﬁcient WRN-E84A protein is unable to alter the gap size and thus acts eﬃciently only on our short fork substrate with an 11 nt gap. By this reasoning, the optimum structure for regression by WRN, without assistance from its exonuclease function, is a fork with a leading arm gap of at least 11–13 nt. This conclusion is further supported by the highly eﬃcient regression and Holliday junction formation by WRN- E84A on the longer fork substrate containing a leading arm gap of 12 nt (Figure 6B and C). These experiments suggest WRN exonuclease activity may participate in processing the leading daughter strand during regression of blocked replication forks, but its degree of involve- ment may depend on the precise structure at the fork junction and perhaps the spatial relationship between leading and lagging daughter strands. The eﬃciency of WRN-mediated regression on forks with 11 nt leading arm gaps suggests that WRN may preferentially act on forks in which leading strand synthesis is blocked while lagging strand synthesis con- tinues, leaving a single-stranded gap on the leading arm. During fork regression, WRN exonuclease activity may further digest the leading daughter strand, a processing step that may simply generate the optimum structure for enzyme-mediated pairing of the daughter strands. However, there may be other advantages to this arrange- ment. On this series of substrates, WRN-mediated regression only occurs eﬃciently when the leading daughter is at least 10–12 nt shorter than the lagging daughter strand. If this structural relationship was maintained (or perhaps further exaggerated) in vivo, fork regression would always yield a Holliday junction contain- ing a single free end with a 50 overhang. Since formation of Rad51-mediated ﬁlaments occurs on 30 overhangs (61), this structure might inhibit recombination, perhaps in favor of alternate, less error-prone pathways such as (1) repair of the blocking lesion and reverse branch migration to re-establish a viable replication fork or (2) strand switching synthesis and reverse branch migration with concomitant bypass of the obstacle (on the parental leading strand) that originally impeded fork progression. Recent studies in E. coli suggest that lesion repair might be the ﬁrst alternative attempted following fork blockage caused by DNA damage (62). It is tempting to speculate that enzymatic regression processes have evolved to at least initially favor less error-prone pathways such as repair or strand switching. Cells from WS patients have a higher rate of spontaneous RAD51 foci formation than normal cells, supporting the idea that recombination