one parental and one daughter strand) was distributed in a concerted manner between the parental and daughter duplex products without signiﬁcant generation of other intermediates. This strongly suggests that WRN-E84A, in a reaction mimicking fork regression, catalyzes direct and coordinated conversion of our short fork substrate with the 11 nt gap on the leading arm to parental and daughter duplexes. In all likelihood, this mechanism also applies to WRN-mediated (and possibly BLM-mediated) action on other, less favorable, fork substrates. The most straightforward analysis of these results is that WRN-E84A produces daughter (and parental) duplex from fork substrates by coordinately unwinding the parental–daughter arms and annealing the leading and lagging daughter strands by an intramolecular strand exchange reaction. However, we wanted to determine whether daughter duplex formation might occur by strand exchange between independent DNA molecules. To this end, we examined the action of WRN-E84A on two diﬀerent three-stranded forks, one containing only the lagging daughter strand and the other only the leading daughter strand. When WRN-E84A was incubated with only one of these three-stranded forks, no formation of daughter duplex was possible and, indeed, only other DNA species (leading daughter strand, parental duplex and parental–daughter duplexes) are produced (Supplemental Figure 1, lanes 1–6). As expected, produc- tion of daughter duplex (indicated by asterisk) is observed when WRN-E84A is incubated with four-stranded fork containing both leading and lagging daughter strands (Supplemental Figure 1, lanes 13–15). If WRN-E84A is incubated with both three-stranded forks simultaneously, a daughter duplex might conceivably occur via inter- molecular strand exchange between the diﬀerent forks. When this experiment was performed (with concentrations of both three-stranded fork substrates either half or equal to that of the four-stranded fork in the positive control), no daughter duplex was produced; instead, we observed only DNA species corresponding to those formed by unwinding of individual three-stranded forks (Supplemental Figure 1, lanes 7–12). This result indicates that WRN-mediated intermolecular strand exchange does not detectably occur between these three-stranded replica- tion forks under the same conditions in which daughter duplex is produced from a four-stranded replication fork substrate. Thus, daughter duplex formation from a four- stranded fork by WRN-E84A likely occurs by an intramolecular strand exchange mechanism, a concept even more strongly supported by our experiments showing that WRN can regress our longer fork substrates to form Holliday junctions (Figure 6). Although our kinetic experiments showed little or no production of free leading daughter strands at the near equimolar WRN-E84A concentrations that mediated daughter duplex formation, we wanted to more thor- oughly examine whether daughter duplex formation was a concerted process or the result of possibly independent unwinding and annealing steps. Thus, annealing reactions were performed both with complementary daughter and parental oligomers. Although WRN-mediated annealing of 80-mers can be achieved in reactions with or without Nucleic Acids Research, 2007, Vol. 35, No. 17 5735 ATP (51), these reactions were performed without ATP to minimize potential unwinding of duplex products. In a concentration-dependent manner, WRN-E84A annealed the parental oligomers to generate the 70 bp parental duplex (Supplemental Figure 2, lanes 27–31), conﬁrming its previously reported annealing capability (23). However, at WRN-E84A concentrations equal to and signiﬁcantly higher than needed for fork regression, enzyme-mediated annealing of lagging daughter oligomer (30lag) to any of the leading daughter oligomers was not detected (Supplemental Figure 2, lanes 1–25). These experiments and others (A. Machwe, unpublished results) indicate that WRN does not facilitate annealing of free oligomers when both are relatively short (<30 nt). A similar eﬀect of oligomer length on BLM-mediated annealing has been previously reported (24). More importantly, the inability of WRN-E84A (and BLM) to anneal these oligomers when free in solution demonstrates that formation of daughter duplex from our fork substrates does not occur by independent enzyme- mediated unwinding and annealing steps. Instead, daugh- ter strands appear to be paired while still associated with the fork, a process likely facilitated by some structural property inherent in WRN and BLM. These results indicate that daughter duplex formation results from an intimate linkage between unwinding of the parental– daughter arms and pairing of the daughter strands. Together with our analysis of the production of daughter and parental duplexes from 21lead fork (Figure 1F), WRN-mediated conversion of our fork substrates to daughter (and parental) duplexes reﬂects a bona ﬁde fork regression process. With regard to protein speciﬁcity, it is worthwhile to determine whether this fork regression function is limited to WRN (and BLM) or perhaps common to other enzymes with helicase activity. Since the UvrD, Rep and PriA proteins have been implicated in the response to replication fork blockage in E. coli (56,58), we examined the action of these helicases on our replication fork substrates. When increasing concentrations of these proteins were incubated with the 21lead, 27lead and 32lead fork substrates containing leading arm gaps of 11, 5 and 0 nt, respectively, no signiﬁcant production of daughter duplex (reﬂective of fork regression) was observed (Supplemental Figure 3), in contrast to our results with WRN-E84A. Both UvrD and Rep produced almost exclusively parental duplex and free leading daughter strand. This pattern suggests that UvrD and Rep displace both daughter strands, allowing the already linked parental strands to completely re-anneal but (unlike WRN) without pairing of the displaced daughter strands. The behavior of PriA varied depending on fork structure. On forks with 11 and 5 nt leading arm gaps, PriA not only produced parental duplexes and free leading daughter strand but also generated a signiﬁcant amount of parental–daughter partial duplexes, the latter due to forward unwinding of the fork. However, on 32lead fork without a leading arm gap, PriA generates predominantly a three-stranded fork (without concomitant release of labeled leading daughter strand). This indicates that PriA preferentially unwinds the lagging daughter strand on