5734 Nucleic Acids Research, 2007, Vol. 35, No. 17 parental strand precisely at the fork junction included to prevent spontaneous branch migration), permitting pairing between daughter strands and re-annealing of parental strands to form Holliday junction intermediates and eventually produce both parental and daughter duplexes (Figure 1A). With the exception of the leading daughter strand, the other 30 ends of these substrates were modiﬁed to block the 30 to 50 exonuclease activity of WRN. These short fork substrates were radiolabeled on the 50 ends of both the lagging parental strand (70lag) and the leading daughter strand (21lead, 24lead, 27lead, 30lead or 32lead) to facilitate identiﬁcation of multiple DNA products. In assays on this series of replication fork substrates, we initially used an exonuclease-deﬁcient protein, WRN- E84A, to avoid potential degradation of the leading daughter strand that might complicate interpretation of our results. To determine the inﬂuence of leading arm structure on regression activity, WRN-E84A was incu- bated with individual fork substrates with gaps of 0, 2, 5, 8 and 11 nt on the leading arm at the fork junction. Our fork substrates contain three duplex regions potentially subject to unwinding when treated with a DNA helicase such as WRN. Speciﬁcally, forward unwinding of the parental duplex region of the fork would yield two parental– daughter partial duplexes (PDs), while unwinding of either parental–daughter arm would yield a three-stranded fork and a displaced daughter strand. However, experi- ments performed with these substrates with low (sub- equimolar to a 3-fold molar excess) concentrations of WRN-E84A produced, within 5 min, primarily two species that co-migrated with markers for the parental (70 bp) and daughter duplexes (Figure 1B). In comparison, other DNA species (some present in low amounts in substrate preparations) including three-stranded forks, parental– daughter partial duplexes, and leading daughter strands were not produced in signiﬁcant amounts by WRN-E84A. The parental duplex could be the result of a fork regression event but also might be generated from spontaneous annealing of the partially hybridized parental strands following unwinding of both parental–daughter duplex regions. However, the daughter duplex could only arise from the unwinding of both parental–daughter arms of the fork combined with annealing of the daughter strands and thus speciﬁcally reﬂects fork regression. Importantly, the generation of daughter duplex (as well as parental duplex) products was observed for all fork substrates but was dramatically increased using the fork substrate (21lead fork) with an 11 nt gap as compared to fork substrates with smaller gaps (Figure 1B). Higher concentrations of WRN-E84A mediated increased con- version to daughter duplexes for each substrate, but the relative eﬃciencies of regression were preserved between substrates. Quantitation of data obtained over a wider range of WRN-E84A concentration (Figure 1C) demon- strated clearly that daughter duplex formation preferen- tially occurred when the fork substrate contained an 11 nt gap. Using a fork with a smaller gap of 8 nt reduced daughter duplex formation precipitously, while further shortening of the gap lowered the eﬃciency of this reaction further. This data was corroborated by kinetic experiments performed using a ﬁxed concentration of WRN-E84A on each substrate. In these assays (Figure 1D), the daughter duplexes formed were clearly detectable and increased linearly with time but were relatively modest for fork substrates with gaps of 0, 2, 5 and 8 nt. In contrast, daughter duplex formation from the substrate with an 11 nt gap was markedly higher at each time point (reaching about 60% conversion by 5 min) than for the substrates with smaller gaps. It is notable that generation of the leading daughter strand product is minimal over the same time frame for each substrate (Figure 1D, inset), again suggesting that the daughter duplex formation occurs through direct coordination between unwinding of both parental–daughter arms and pairing of the daughter strands. Taken together, our data indicates that daughter duplex formation (indicative of fork regression) by exonuclease-deﬁcient WRN-E84A occurs much more readily on replication fork substrate with a larger (11 nt) gap on the leading strand than on forks with smaller gaps. The eﬃciency of regression by WRN-E84A drops considerably when the gap is shortened to 8 nt and decreases further on substrates with even smaller gaps. Our results on these substrates conﬁrm our earlier observation (using a structurally diﬀerent fork substrate) that WRN catalyzes a reaction reminiscent of fork regression (49). Moreover, they suggest that, although WRN-E84A has a certain amount of structural ﬂexibility, the eﬃciency of this reaction is determined by structure of the leading arm at the fork junction. Mechanistic considerations Since daughter duplex formation catalyzed by WRN- E84A on 21lead fork substrate containing an 11 nt leading arm gap was so much more eﬃcient than on other substrates, a more in-depth analysis of WRN-E84A action on this substrate was performed. The amount of each detectable DNA species from a kinetic experiment on this substrate (Figure 1E) was determined at each time point. Then, the contribution (expressed as percentage) of each DNA species to the total radioactivity was plotted over time (Figure 1F). Before the beginning of the reaction, the fork substrate contained 91.6% of the total radio- activity with no other individual species contributing more than 2.5%. After initiation of the WRN-E84A-mediated reaction, only the amounts of four-stranded fork, daughter duplex and parental duplex changed signiﬁ- cantly; at any time point, not one of the other DNA species (lagging parental strand, parental-daughter partial duplexes, three-stranded fork or leading daughter strand) ever contributed more than 6.3% to the total radio- activity. Most notably, the amount of fork substrate decreased dramatically with time while the amounts of parental and daughter duplex increased (Figure 1E and F). Importantly, the combined increases in parental and daughter duplex products (positive values shown between bars, at bottom) almost exactly reﬂected the decreases (negative values between bars, at top) in fork substrate between individual time points (Figure 1F). Thus, it can be concluded that, during the course of this reaction, the radioactivity in the fork substrate (labeled on