might be utilized more often when WRN is non-functional (43). Importantly, the notion that WRN regresses replication forks to speciﬁcally generate intermediates that suppress instead of promote recombination would be consistent with the hyperrecombination phenotypes of cells that have lost WRN function. Fork structures with heterologous arms have previously been shown to be excellent substrates for WRN helicase (63). Here, we demonstrate that WRN speciﬁcally regresses forks with homologous arms. With regard to its action on fork structures, WRN binds in the vicinity of the fork junction as judged by DNase I footprinting (Machwe et al., unpublished results). It is quite relevant that WRN-wt and WRN-E84A regression activities are highly eﬃcient when the structure of the fork substrate is favorable. As judged by daughter duplex formation, regression of the fork substrate with an 11 nt gap on the leading arm is readily detectable after 5 min using sub- and near-equimolar levels of enzyme compared to substrate (Figure 1B, lanes 19–23). Furthermore, at low concentra- tions, WRN is more eﬀective at catalyzing regression of this fork substrate than unwinding a 27 bp partial duplex (Figure 2). WRN also eﬃciently regresses our longer fork substrate containing a 12 nt gap at near-equimolar concentrations (Figure 6B and C). Although these experiments cannot determine the stoichiometry of WRN with respect to the DNA substrate, the requirement for unwinding of both parental-daughter arms during regression might imply at least a dimeric structure. Irregardless, the eﬃciency by which WRN catalyzes this multi-faceted regression reaction suggests that it is particularly suited to this task. It is noteworthy that BLM, another human RecQ helicase that possesses unwinding and strand pairing activities and DNA substrate speciﬁcity similar to WRN, also performs fork regression (49,64). Like WRN, BLM produces Holliday junctions from our long unrestricted fork substrate that are recognized and cleaved by RusA (data not shown). However, BLM appears to be consistently less eﬃcient in fork regression of each of our short fork substrates than WRN-wt (Figure 2A and B). Although RecG can also regress model forks as previously reported (59), other helicases (including UvrD, Rep and PriA implicated in resolution of fork blockage in E. coli) tested thus far could not perform this function, suggesting that fork regression capability appears relatively limited to a small group of helicases that includes WRN and BLM. Thus, some RecQ helicases are structurally designed to catalyze fork regression by combining their helicase and strand pairing activities. Notably, two other human RecQ family members, RecQ1 and RecQ5b, have also been shown to possess both DNA unwinding and annealing activities (25,26). It may be relevant in a physiological context that BLM and other human RecQ helicases do not possess exonuclease activity and alone cannot modify the struc- ture at the fork junction in the way that WRN-wt can. We speculate that WRN is preferentially involved in regres- sion of blocked forks, while BLM may be more likely to participate (in combination with topoisomerase IIIa and BLAP75) in other DNA transactions, such as double Nucleic Acids Research, 2007, Vol. 35, No. 17 5745 Holliday junction resolution to prevent crossing over during homologous recombination (65). RecQ helicases have previously been postulated to play roles in resolution of replication fork blockage (38,39,66). The ﬁnding that both WRN and BLM can readily regress model replication forks greatly strengthens this hypothesis. However, formation of Holliday junctions resulting from replication fork regression in vivo is somewhat speculative and further proof is needed of speciﬁc genome maintenance pathways utilizing fork regression and under what circumstances they are implemented. It is also noteworthy that WRN has been hypothesized to participate in several other pathways that preserve genome stability (such as telomere main- tenance). Despite these caveats, potential involvement of WRN in replication fork regression is consistent with previous ﬁndings regarding WRN and certain properties of WRN-deﬁcient cells. WRN-deﬁcient cells are slower than their normal counterparts in completing S phase and show asymmetric replication fork progression, consistent with an inability to properly resolve fork blockage (41,42). They are also hypersensitive to agents that severely inhibit DNA replication including (1) hydroxyurea, which depletes deoxynucleotide pools, (2) topoisomerase inhibitors that induce strand breaks and DNA-protein crosslinks and (3) interstrand crosslinking agents (such as mitomycin C and cisplatin) that prevent unwinding of the parental strands (8,11,43,44). Furthermore, in normal cells, WRN migrates rapidly to sites of DNA synthesis following treatment with these and other DNA damaging agents (21,45–47), ﬁndings that suggest that WRN is recruited to sites where replication is blocked. Our results suggest the reason for this relocalization—i.e., WRN is brought to blocked replication forks to catalyze their regression as part of a pathway that maintains genome stability. It is possible that, although they lack exonuclease activity, other human RecQ helicases such as BLM can partially compensate for loss of WRN function in fork regression. However, if such redundancy exists, it is likely imperfect and there may be situations in which WRN-deﬁcient cells are still compromised in dealing with blocked replication forks. As a result, cells lacking WRN are hyperrecombi- nant and accumulate chromosomal abnormalities that are almost assuredly responsible for the increased cancer incidence of WS patients. Alternatively, in response to these DNA metabolic problems some cell types may trigger apoptosis or permanent cell cycle exit (senes- cence). With time, the cumulative loss of either cells by apoptosis or reduction in proliferative capacity within a tissue may cause the premature aging phenotypes of WS. Although more research is needed to conﬁrm these hypotheses, a speciﬁc function in fork regression is highly consistent with existing knowledge regarding WRN and WS. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online.