(see Figure 6A, lower right). When reactions containing WRN-E84A and/or RusA were analyzed by denaturing PAGE, neither WRN-E84A alone nor RusA alone detectably produced any change to the labeled (122lead and 82lag) strands of the fork substrate (Figure 6E, lanes 6 and 7). In contrast, reactions that contained WRN-E84A followed by RusA showed new bands that co-migrated with 30 and 70 nt markers (Figure 6E, lane 8). To further clarify the nature of the events catalyzed by WRN-E84A plus RusA, the new DNA products detected by native PAGE (Figure 6E, lane 4, denoted a and b) were individually excised, extracted, and subjected to denatur- ing PAGE. This analysis indicated that the lower product (a) contained both cleaved and uncleaved 82-mer (Figure 6E, lane 10). The upper product (b) contained both cleaved and uncleaved 122-mer with also some 82-mer (Figure 6E, lane 11), the latter apparently derived from lagging parental–daughter duplex that nearly co- migrates by native PAGE with this RusA cleavage product. Regarding this outcome, it is important to reiterate that CC sequences were also present on the unlabeled (122lag and 72lead) strands within the homo- logous regions. Thus, we conclude that after WRN-E84A mediates fork regression, RusA can cleave either both labeled or both unlabeled strands of the resulting Holliday junction (see Figure 6A, bottom left, solid and dashed arrows, respectively). While each set of cleavages generates diﬀerent locations of nicks, analysis by native PAGE shows only two new products, a 72 bp duplex with 10 nt 30 overhang plus a 122 bp duplex, regardless of whether the nicks are present in the labeled or unlabeled strands. When these products are extracted and analyzed by denaturing PAGE, concerted RusA cleavage of the labeled strands yields shorter single-stranded products (30- and 70-mers) while concerted cleavage of the unlabeled (undetectable) strands leaves the labeled strands intact (82- and 122-mers). These results clearly demon- strate that WRN-E84A regresses this fork substrate to generate a Holliday junction or ‘chicken foot’ structure that is cleaved by RusA at its speciﬁc recognition sequences. DISCUSSION Replication fork blockage is such a common event that cells have evolved specialized pathways to handle these situations. Regression of a blocked replication fork is theorized to be the initial step in dealing with these serious challenges to completion of DNA replication, genome stability and cell survival (33–37). Fork regression would involve pairing of nascent daughter strands and Nucleic Acids Research, 2007, Vol. 35, No. 17 5743 re-annealing of parental strands to form a Holliday junction or ‘chicken foot’ intermediate. Following fork regression, obstacles to replication might be addressed by alternative pathways including (1) repair of a blocking lesion and reverse branch migration to regenerate a forked structure, (2) a strand-switching replication step in which the lagging daughter strand serves as template followed again by reverse branch migration, bypassing the blocking lesion and re-establishing the fork or (3) resolution of the Holliday junction to generate a double-strand break that could initiate recombinational pathways to restore a functional replication fork. A preliminary report from our lab established that the human RecQ helicases WRN and BLM have the ability to regress a speciﬁc replication fork substrate (49). In this study, we have examined the ability of WRN to act on several model replication fork substrates, including a series of short fork substrates containing structural diﬀerences at the fork junction and another longer substrate that allowed Holliday junction formation and detection during a potential regression reaction. Importantly, fork regression by WRN on our long fork substrate directly forms a Holliday junction that is detected using the RusA resolvase (Figure 6). Fork regression requires the ATPase and helicase activities of WRN, as it does not occur in reactions (1) lacking ATP or including the poorly hydrolyzable analog ATP-g-S or (2) using the WRN-K577M mutant that lacks both ATPase and unwinding activity. Most notably, our experiments with shorter fork substrates indicate a speciﬁc role for the 30 to 50 exonuclease activity of WRN during fork regression—i.e. controlled digestion of the leading daugh- ter strand to generate a more favorable structure for regression and thereby increase the eﬃciency of this process. These ﬁndings suggest a novel role for the exonuclease activity of WRN during fork regression that operates in coordination with its unwinding and annealing activities. Thus, all of the DNA-dependent activities of WRN may cooperate to promote replication fork regression. Enzymatic reactions containing WRN-E84A or WRN-wt showed a concentration- and time-dependent conversion of our short fork substrates to both parental and daughter duplex products (see Figures 1 and 5A). Daughter duplex formation is speciﬁc for a regression event as it requires both unwinding and pairing of the physically unlinked daughter strands (Figure 1A). Although generation of free leading daughter strand product is detectable in reactions containing relatively high WRN concentrations, several lines of evidence demonstrate that daughter duplex formation occurs through an intimate linkage between unwinding of parental–daughter arms and pairing of daughter strands. WRN-E84A (100, 200, 400 or 600 pM) were initiated at 378C, supplemented with RusA (10 nM) after 1 min as indicated, and stopped after 5 min total. DNA products were analyzed as in B, along with a marker (lane 11) containing labeled 82lag/122lag, 122lead, 82lag and a 77 bp duplex. (D) Reactions containing unrestricted long fork (50 pM) and WRN-E84A (200 pM) and RusA (10 nM) where indicated were performed without ( ) or with (+) ATP (1 mM) or ATPgS (1 mM, denoted gS) and analyzed as in C, with the position of speciﬁc markers at right. (E) Reactions containing unrestricted long fork substrate (200 pM), WRN-E84A (800 pM) and ATP were incubated at 378C for 15 min total with RusA (40 nM) added at 1 min where indicated. Aliquots were analyzed in parallel by native (left panel, lanes 1–4) and denaturing PAGE (right panel, lanes 5–8). For native PAGE, the bands denoted a and b represent DNA species that were excised, extracted and analyzed subsequently by denaturing PAGE (right panel, lanes 10 and 11, respectively). Also run on this gel were labeled 30-mer (lane 9) and 70-mer (lane 12) as markers for RusA cleavage at its speciﬁc sites on the labeled 82lag and 122lead strands, respectively, of putative Holliday junction structures formed from unrestricted long fork substrate.