non-complementarity on each arm (again to prevent spontaneous branch migration) as well as the optimal size gap (12 nt) on the leading arm for regression by WRN that obviated the need for its exonuclease activity. Most importantly, both parental–daughter arms of this sub- strate were signiﬁcantly longer and contained homologous sequences proximal to the fork junction (denoted in green) but non-homologous sequences (30 bp on each arm) at the distal ends (denoted in red). Theoretically, this design would allow initiation of fork regression with branch migration and daughter strand pairing up to the point of heterology, at which further regression might be inhibited. Nevertheless, if regression was initiated, the fork should be converted, at least transiently, to a Holliday junction with limited mobility (Figure 6A, lower pathway). The high eﬃciency of WRN-mediated regression of fork substrates with similar-sized leading arm gaps (Figures 1 and 2) made us hopeful that we could detect transient formation of such Holliday junction structures by using known resolvases. Thus, consensus sequences (50-#CC-30) for cleavage by RusA, a Holliday junction-speciﬁc resolvase, were included on each strand within the homologous but not the heterologous regions of the parental–daughter arms. Notably, 50-CC-30 sequences were also present in the parental duplex region, serving as an internal control for the Holliday junction speciﬁcity of RusA. Unique Rsa I and Xmn I sites were included precisely at the junctions between homologous and heterologous regions on the leading and lagging arms, respectively. For this substrate, radioactive labels were placed at the 50 ends of the lagging daughter strand (82lag) and the leading parental strand (122lead). Notably for this series of experiments, the speciﬁc activity of the former was approximately 18 times that of the latter, inﬂuencing the relative intensities of individual DNA products. Moreover, this particular placement of radiolabels deter- mines the nature of DNA products that can be observed following treatment of both intact fork substrate with WRN-E84A plus RusA (Figure 6A, bottom right) and restricted fork with only WRN-E84A (Figure 6A, top right). We initially wanted to determine whether this longer fork substrate could be acted on by WRN to yield a daughter duplex product in a manner identical to the substrate above. To this end, this substrate was restricted with Rsa I and Xmn I (Figure 6A, top center) to release the heterologous (and unlabeled) regions of both arms; the resulting fork with entirely homologous leading and lagging arms was puriﬁed. When this substrate was treated with WRN-E84A, two new DNA products (Figure 6B, lanes 2–5, indicated by arrows) appeared with migration consistent with that of the potential daughter duplex (a 42 bp plus 10 nt 50 overhang) and the parental duplex (92 bp); note that the daughter duplex appears more intense due to the higher speciﬁc activity of the lagging daughter strand. As above, production of both species required ATP hydrolysis and occurred at extre- mely low WRN concentrations without detectable amounts of single-stranded products (data not shown). These experiments conﬁrm that this restricted fork could be regressed by WRN-E84A in the same manner as the Nucleic Acids Research, 2007, Vol. 35, No. 17 5741 shorter fork substrate (21lead fork) that contained a similar structure at the fork junction. These ﬁndings also suggest that the length of the homologous arms in our substrates does not signiﬁcantly aﬀect WRN’s regression function, as the homologous arms of the restricted fork are approximately twice as long as those in our short fork substrates. Next, the unrestricted long fork substrate was treated with WRN-E84A in the presence or absence of RusA. Over the same range of concentration used above, WRN-E84A alone did not generate a product resulting from pairing of the daughter strands (Figure 6C, lanes 3–6), in contrast to our results on forks with completely homologous arms. This suggests that the heterology on the distal ends of each arm inhibits WRN-mediated generation of ‘daughter duplex’ in a manner that cannot be overcome by unwinding activity at these limiting concentrations of WRN-E84A. Thus, if fork regression intermediates are formed by WRN-E84A from this substrate, they revert (by enzyme-mediated or sponta- neous reversal of branch migration) back to the fork structure during the reaction and/or electrophoresis. Small amounts of parental–daughter duplexes were detected, consistent with weak forward unwinding of this fork. In order to detect whether Holliday junction intermediates are transiently generated, RusA was added one min following the start of a 378C incubation of substrate with WRN-E84A. Addition of RusA to WRN-containing reactions yielded new DNA products (indicated by asterisks) when analyzed by native PAGE (Figure 6C, lanes 7–10). The more prominent product migrated a little slower than a 77 bp marker, while the other migrated just slightly above a 122/82 partial duplex marker, consistent with RusA resolution of a Holliday junction formed from this fork substrate—i.e. a 72 bp duplex with a 10 nt 50 overhang (more prominent because it contains the labeled strand with higher speciﬁc activity) and a 122 bp duplex, each putatively containing a nick (see Figure 6A, lower right). In an important control, RusA without WRN-E84A does not detectably act on the fork substrate (Figure 6C, lane 2). These results are consistent with the previously characterized speciﬁcity of RusA for Holliday junctions and also indicate that WRN-E84A is generating the Holliday junction structure for RusA cleavage. Importantly, RusA cleavage required not only WRN- E84A but also ATP hydrolysis, as cleavage was not detected in the absence of ATP or in the presence of the poorly hydrolyzable analog ATPgS (Figure 6D), demon- strating that WRN ATPase and helicase activities were necessary to convert the fork substrate to a Holliday junction intermediate. Similar reactions were analyzed to determine whether these new DNA products indeed corresponded with RusA-speciﬁc cleavage events on Holliday junctions generated by WRN-dependent fork regression. If a Holliday junction were generated by fork regression, positioning of RusA consensus (50-#CC-30) sequences in the labeled lagging daughter (82lag) and leading parental (122lead) strands within the homologous regions on the arms of this substrate would cause RusA to cut 30 and 70 nt from the labeled 50 ends of the respective strands