5742 Nucleic Acids Research, 2007, Vol. 35, No. 17 Figure 6. WRN regresses long fork substrate to Holliday junction structures cleaved by RusA. (A) A model replication fork substrate (top left) was constructed using four oligomers (identiﬁed in italics) in a two-step annealing process (see ‘Materials and methods’ section). Lagging and leading arms were homologous (green) proximal and heterologous (red) distal to the fork junction; unique restriction sites for Xmn I and Rsa I speciﬁed blunt end cleavage at the boundary between homologous and heterologous regions on the lagging and leading arms, respectively (blue arrows). Parental strands contained non-complementarity (5 nt, in orange) precisely at the fork junction to prevent spontaneous branch migration. Digestion with Xmn I and Rsa I generated a replication fork with shorter, homologous arms that, upon fork regression (by WRN-E84A) would yield daughter and parental duplex products (top pathway). In contrast, potential WRN-E84A-mediated regression of the unrestricted fork would yield a Holliday junction structure (bottom left) in which branch migration would be limited by the heterologous regions. Since both the labeled and unlabeled strands within the homologous region of each arm contain consensus 50-CC-30 sequences for RusA cleavage (sequences indicated in yellow and white while cleavage sites denoted by solid and dotted arrows, respectively), putative Holliday junctions may be resolved by RusA to yield two nicked duplexes (bottom right). Although resolution by RusA may occur on the labeled or unlabeled strands of the Holliday junction, only products generated by cleavage of the labeled strands are depicted. (B) Rsa I- and Xmn I-restricted long fork substrate (50 pM) was incubated at 378C for the indicated times with WRN-E84A (100 pM). DNA products were analyzed by native PAGE with phosphorimaging, along with heat-denatured fork preparations either slow-cooled to produce various annealing products (Mkr 1) or rapidly cooled to maintain oligomers in single-stranded form (Mkr 2). Migration of relevant DNA structures is denoted at right. (C) Reactions containing unrestricted long fork substrate (50 pM) without or with