5736 Nucleic Acids Research, 2007, Vol. 35, No. 17 forks without a leading arm gap, in agreement with previous biochemical analyses (56). Thus, fork regression is not a common property of all helicases, but limited to a subset that includes WRN and BLM. Notably, E. coli RecG can also regress our model replication fork substrates (data not shown), as would be expected from previous studies (59). A role for WRN exonuclease activity in fork regression Our data above indicate that fork substrates containing larger single-stranded gaps on the leading arm are superior structures for regression by exonuclease-deﬁcient WRN- E84A. Since an increase in gap size within these forked structures could be accomplished by degradation from the 30 end of the leading daughter strand, we hypothesized that the 30 to 50 exonuclease activity of WRN-wt might also participate in the fork regression process. To initially test the merit of this theory, we compared the action of exonuclease-proﬁcient WRN-wt with that of WRN-E84A on our fork substrates with diﬀerent gap sizes on the leading arm. Wild-type BLM, which does not contain exonuclease activity, was also examined to directly compare its action with that of both WRN proteins. First, we measured unwinding by WRN-wt, WRN-E84A, and BLM on a 27 bp partial duplex substrate (70lag/27lag, with both 30 ends modiﬁed to block WRN exonuclease) containing a 30 overhang of 43 nt (Figure 2A) to determine the amounts of each enzyme needed for comparable activity within the linear range of unwinding. Notably, the same molar concentrations of WRN-E84A and WRN-wt catalyzed nearly equal levels of unwinding; BLM- mediated unwinding was also of similar strength on this 30 overhang substrate. Then, the action of these enzymes (using concentrations with the same relative unwinding strength) was directly compared on our fork substrates containing leading strand gaps of 11, 8, 5, 2 and 0 nt. These experiments conﬁrm that each of our replication fork substrates can be acted upon by both WRN-E84A and WRN-wt to primarily yield parental duplex and daughter duplex products as well as minor amounts of leading daughter strand (Figure 2B). As judged by formation of daughter duplex, the regression activities of WRN-wt and WRN-E84A on fork substrate with an 11 nt gap are comparable. However, as the gap size on the leading arm decreases, daughter duplex formation becomes much more eﬃcient with WRN-wt than with WRN-E84A (Figure 2C). This diﬀerence is particularly notable on the 27lead fork with a 5 nt leading arm gap (Figure 2B, lanes 13 versus 14, and C). A titration of both proteins on this fork substrate also demonstrated that WRN-wt is consistently much more eﬃcient than WRN- E84A in forming the daughter duplex reﬂective of the fork regression process (Figure 2D). BLM is less eﬃcient at fork regression than WRN-wt on all forks except the substrate without a gap on the leading arm (for which regression activity is very weak for each protein), as evidenced by consistently lower daughter duplex forma- tion and higher production of free leading daughter strand (Figure 2B and C). In assessing the action of WRN-wt on these fork substrates, two other diﬀerences from WRN-E84A (and BLM) were observed. In reactions containing WRN-wt, there was a faint smear extending down from the leading daughter strand band, reﬂecting minor generation of shorter leading daughter strands. More importantly, for the fork substrates with gaps of less than 11 nt, daughter duplexes produced by WRN-wt migrated slightly faster than those formed by WRN-E84A or BLM, indicating that one or both strands that compose this DNA species had been altered by WRN-wt. As the other strands in the fork substrates are blocked at their 30 ends, these eﬀects appear to be due to the 30 to 50 exonuclease activity inherent in WRN-wt speciﬁcally on the leading daughter strand during the course of the reaction. Importantly, this data could imply that the exonuclease activity of WRN-wt may digest the leading daughter strand prior to or during formation of daughter duplexes, particularly on replication fork substrates with gaps on the leading arm shorter than 11 nt. The above analysis of WRN-wt action on our replica- tion fork substrates suggests that its 30 to 50 exonuclease activity clearly participates in production of faster- migrating daughter duplexes. Since the 30 ends of other strands in our fork substrates are blocked, WRN- mediated degradation is likely targeted speciﬁcally to the leading daughter strand. However, the timing of this exonucleolytic digestion with respect to other enzymatic events (i.e. regression) was unclear. One way to approach this question was to determine whether (and how) WRN exonuclease activity might digest possible reaction pro- ducts that contain the leading daughter strand. Thus, the action of WRN-wt on both isolated lead daughter strand oligomers (21lead, 24lead, 27lead and 30lead) and daughter duplex substrates (21lead/30lag, 24lead/30lag, 27lead/30lag and 30lead/30lag) as well as our replication fork substrate (27lead fork) with a 5 nt gap was examined by denaturing PAGE. The 27lead fork substrate was utilized here and subsequently due to the obvious beneﬁt that the exonuclease activity of WRN-wt provides for the regression eﬃciency on this substrate (Figure 2B–D). In this experiment (as well as later ones), the labeled lagging parental strand (70 nt) of the fork substrate that is modiﬁed at its 30 end is not detectably digested by WRN exonuclease (Figure 3, lanes 2–3). Importantly, at enzyme concentrations comparable to those used in regression assays above, the exonuclease activity of WRN-wt could not detectably digest either pre-formed daughter duplexes (Figure 3, lanes 4–15) or isolated leading daughter strand oligomers (Figure 3, lanes 16–27). In dramatic contrast, the leading daughter strand was extensively digested in the context of the intact 27lead fork substrate (Figure 3, lanes 1–3). This result on single-stranded oligomers is in agreement with earlier studies showing that oligomers of <30 nt are very poor substrates for WRN exonuclease (60). These experiments on isolated daughter duplexes and daughter strand oligomers are highly relevant to the timing of WRN exonuclease activity in our reactions containing short fork substrates. Speciﬁcally, once daugh- ter duplexes are formed or leading daughter strands are displaced, they are not subject to WRN exonuclease activity. Therefore, digestion of the leading daughter strand of the fork substrate by WRN-wt must have