as a result of the inability to properly maintain their telomeres (14–16). Accumulation of senescent cells or the cumulative loss of cells by apoptosis is almost assuredly the cause of premature aging in WS; these mechanisms are also postulated to play a role in normal aging (17–19). Thus, the WS phenotypes may point to speciﬁc tissues in which these mechanisms may be at work during the development of aging phenotypes in normal individuals. All RecQ helicases including WRN are highly homo- logous within deﬁned amino acid sequence motifs that are also identiﬁable but less conserved in a larger group of enzymes from helicase superfamilies 1 and 2. These sequence motifs anchor a domain that, in RecQ helicases, uses the energy derived from ATP hydrolysis to unwind DNA with a 30 to 50 polarity deﬁned by the strand upon which the enzyme translocates. WRN and other RecQ helicases unwind short duplexes and a variety of unusual DNA structures including forks, D-loops, G-quartets and triplexes (20). Both WRN and BLM also readily branch migrate Holliday junctions (21,22). Recently, a number of RecQ helicases, including the human WRN, BLM, RECQ1, RECQL4 and RECQ5b proteins, have been shown to facilitate annealing of complementary DNA strands (23–27). Under certain circumstances, the unwind- ing and annealing activities of WRN, BLM or RecQ5b can function coordinately to achieve strand exchange (23,28). Taken together, biochemical studies suggest that some RecQ helicases are structurally designed to act on three- or four-stranded replication or recombination intermediates. Importantly, WRN is the only human RecQ homolog to have a nuclease domain in its N-terminal region (29) that confers a 30 to 50 exonuclease activity that is particularly robust on complex DNA structures and thus similar to the speciﬁcity of its unwinding activity (30–32). Although each of these DNA-dependent activities has been independently exam- ined in vitro, it remains unclear whether and how they might act together in a speciﬁc DNA metabolic pathway to help maintain genome stability. Although multiple DNA repair systems are present in all cells, encounters between replication forks and Nucleic Acids Research, 2007, Vol. 35, No. 17 5731 persistent DNA damage cannot be completely avoided. Recent investigations indicate that cells have evolved important pathways to respond to and overcome replica- tion fork blockage caused by lesions in the DNA template or other circumstances (33–37). It has been proposed that the initial step in dealing with a blocked replication fork involves its regression—a process by which the parental strands re-anneal and the daughter strands are paired to generate a Holliday junction or so-called ‘chicken foot’ structure (Figure 1A). Following Holliday junction for- mation, several alternative pathways might be employed for removing or circumventing the obstacle and restarting replication. With each pathway, eventual re-establishment of a functional replication fork is crucial for maintaining genomic stability and permitting cell survival. Because of the cellular phenotypes caused by RecQ deﬁciencies, they are often postulated to participate in pathways responding to fork blockage (38–40). More speciﬁcally, WRN-deﬁcient cells show an extension of S phase and speciﬁc replication abnormalities including asymmetry in the normal bidirectional progression of replication forks, suggesting diﬃculty in overcoming obstacles to replication (41,42). They are also hypersensitive to compounds such as hydroxyurea, topoisomerase inhibitors and interstrand crosslinking agents that inhibit replication fork progression (8,11,43,44). Moreover, immunoﬂuorescence studies in normal cells demonstrate that WRN is present in some replication foci and is actively recruited to these foci by treatment with certain genotoxic agents (21,42,45–48). If WRN or other RecQ helicases act in pathways that respond to replication fork blockage, loss of their function might cause sporadic replication fork collapse, leading to generation of double-strand breaks, elevated genomic instability and an increased likelihood of cell death. At the minimum, the regression of blocked replication forks to form Holliday junctions would entail unwinding of both parental–daughter duplex arms and pairing of the nascent daughter strands with concomitant re-annealing of the parental strands. Accurate completion of this complex process would be facilitated by an enzyme that possesses both unwinding and strand annealing capability such as WRN, or perhaps another RecQ helicase. Figure 1. Short replication fork substrates and the inﬂuence of leading arm gap size on regression eﬃciency of WRN-E84A. (A) A series of model replication fork substrates with homologous parental–daughter arms of the indicated lengths was generated by a two-step annealing process (see ‘Materials and methods’ section). The parental strands (gray) were entirely complementary except for 5 nt (indicated in dark gray) precisely at the fork junctions to prevent spontaneous branch migration, while the daughter strands (black) are completely complementary except where they overlap this 5 nt region. For diﬀerent short fork substrates, the length of the leading daughter strand ranged from 32 to 21 nt (denoted by dashed line) resulting in a leading arm gap of 0–11 nt at the fork junction. The putative WRN- or BLM-mediated conversion of these substrates through Holliday junction intermediates to parental and daughter duplex products is diagramed. (B) Reactions containing fork substrates (50 pM) with leading strand gaps of 0, 2, 5, 8 or 11 nt and WRN-E84A (25–150 pM) were incubated at 378C for 5 min and analyzed by native PAGE and visualized by phosphorimaging. The migration of speciﬁc DNA markers is indicated at left, with brackets encompassing the positions of diﬀerent daughter duplexes and leading daughter strands generated from diﬀerent fork substrates. (C) Quantitation (presented as% conversion, in molar terms, from fork substrate) of WRN-E84A-concentration dependent formation of daughter duplex products from fork substrates with leading strand gaps from 0 to 11 nt. (D) Reactions containing WRN-E84A (200 pM) and fork substrates (50 pM) with leading strand gaps from 0 to 11 nt were incubated at 378C for the indicated times and analyzed as in (B). Quantitation (as described earlier) of enzyme-dependent formation of daughter duplex and leading daughter strand (inset) products over time is graphed for each fork substrate. (E) Reactions containing WRN-E84A (200 pM) and fork substrate (50 pM) with an 11 nt leading arm gap (21lead fork) were incubated for the indicated times and analyzed as in (B). Lane 6 contains markers for the daughter duplex (21lead/30lag) and leading daughter strand (21lead). (F) Radioactivity associated with detectable DNA species in panel E (lanes 1–5) was quantitated and the percentage that each species contributed to the total radioactivity (100%) at each time point is plotted as a bar graph, with legend at right. The numbers between lanes correspond to the decreases in four-stranded fork substrate (top) and the sum of the increases in daughter and parental duplex species (bottom) from the previous to the subsequent time point. The near exact correspondence of these increases to the reductions in the four-stranded fork at each time point indicates that daughter and parental duplex are generated simultaneously and directly from the fork substrate.