5732 Nucleic Acids Research, 2007, Vol. 35, No. 17 Some exonucleolytic processing of either the leading or lagging daughter strand may also be involved in the fork regression process. A preliminary report from our laboratory has shown that BLM and an exonuclease- deﬁcient WRN mutant, WRN-E84A, can catalyze fork regression (49). In this study, a series of replication fork substrates have been used to determine the eﬀect of leading arm structure on the fork regression capabilities of WRN and BLM. Our results show a pronounced eﬀect of leading arm structure on the eﬃciency of regression mediated by WRN-E84A and BLM. Importantly, the 30 to 50 exonuclease activity of wild-type WRN enhances regression on a number of these structures through limited degradation of the leading daughter strand. Thus, our results indicate that the multiple enzymatic activities of WRN act together to mediate regression of replication forks. Furthermore, we demonstrate (on another model fork substrate) that WRN can mediate fork regression to form the Holliday junction or ‘chicken foot’ structure characteristic of a bona ﬁde fork regression process. A function of WRN to speciﬁcally regress blocked forks during replication fork repair would be highly consistent with the speciﬁc genomic instability phenotypes associated with WS. MATERIALS AND METHODS Enzymes Wild-type WRN (WRN-wt), WRN-E84A, and WRN- K577M were overexpressed in insect cells and puriﬁed essentially as described previously (50), except that 0.1% Nonidet P40 (NP40) was included in all liquid chromato- graphy buﬀers. WRN-E84A contains a point mutation in the conserved nuclease domain that inactivates its 30 to 50 exonuclease activity (29); this mutant retains DNA unwinding and annealing activities (23,51). WRN-K577M contains a point mutation inactivating its unwinding activity but still retains exonuclease and annealing activities (23,31,52). Recombinant human BLM, puriﬁed after overexpression in yeast as described previously (53), was provided by Joanna Groden (Ohio State University). The E. coli Holliday junction resolvase RusA was puriﬁed as previously described (54), except that RusA over- expression in E. coli was at 258C and the lysis step was performed in 1.5 M KCl. UvrD was provided by Steven Matson (University of North Carolina) while both PriA and Rep were from Ken Marians (Sloan-Kettering); these proteins were puriﬁed by previously described methods (55,56) Standards of known concentration were used to determine protein concentrations using the Bradford assay and/or SDS–PAGE. All proteins were stored at 808C prior to use. DNA substrate construction Nucleotide sequences of gel-puriﬁed oligonucleotides (Midland Certiﬁed Reagent Company, Midland, TX) are speciﬁed in Table 1. The 30 ends of the 70lag, 70lead and 30lag oligomers were modiﬁed with phosphate groups that block the 30 to 50 exonuclease activity of WRN-wt and WRN-K577M (57). For construction of short fork substrates with both the lagging parental and leading daughter strands labeled, the 70lag, 21lead, 24lead, 27lead, 30lead and 32lead oligomers were 50 end-labeled with 32P-g-ATP and T4 polynucleotide kinase, 30-phosphatase free (Roche Molecular Biologicals, Indianapolis, IN) and unincorporated nucleotides were removed using standard procedures. In an initial annealing step to form parental- daughter partial duplexes, labeled 70lag was heated to 908C and slow-cooled with excess unlabeled 30lag, while unlabeled 70lead was treated similarly in individual reactions with excess labeled 21lead, 24lead, 27lead, 30lead or 32lead. The resulting lagging and leading parental–daughter partial duplexes were then mixed together at 378C for 18 h. The long fork substrate was prepared similarly, except it contained radiolabels on both the lagging daughter (82lag) and leading parental (122lead) strands. Three-stranded forks were also prepared from sequential high and low-temperature annealing reactions, but without one of the leading or lagging daughter strands. Double-stranded substrates were prepared from single-step annealing reactions. Single-stranded oligonucleotides used for markers and in annealing reactions were simply labeled and gel-puriﬁed. After separation by native 8% polyacrylamide gel electrophoresis (PAGE), all DNA substrates were excised, extracted into TEN buﬀer (10 mM Tris, pH 8.0, 1 mM EDTA and 10 mM NaCl), and stored at 48C prior to use. Enzymatic assays All enzymatic assays were conducted in WRN reaction buﬀer (40 mM Tris–HCl, pH 7.0, 4 mM MgCl2, 0.1% NP40, 100 ug/ml bovine serum albumin and 5 mM dithiothreitol); fork regression, exonuclease and helicase assays also contained ATP (1 mM) unless otherwise indicated. For these assays, labeled fork (regression), partial duplex (helicase) or oligomeric (annealing) DNA substrate (50–200 pM) was pre-incubated for 5 min at 48C with enzyme (WRN-E84A, WRN-K577M, WRN- wt, BLM, UvrD, Rep or PriA) at the concentrations in ﬁgure legends, then transferred to 378C for the indicated times. In annealing reactions, complementary single-stranded oligomer (50 pM) was added just prior to incubation at 378C. For potential detection of Holliday junctions during regression assays with long fork substrate, RusA (10–40 nM) was added 1 min into the 378C incubation. Reactions (or aliquots thereof) were stopped by adding either one-sixth volume of helicase dyes (30% glycerol, 50 mM EDTA, 0.9% SDS, 0.25% bromphenol blue and 0.25% xylene cyanol) or an equal volume of formamide dye (95% formamide, 20 mM EDTA, 0.1% bromphenol blue and 0.1% xylene cyanol) for analysis by native 8% PAGE or denaturing 14% PAGE, respectively. Speciﬁc DNA species (daughter duplexes and RusA-generated products) identiﬁed on native PAGE were excised and extracted using a gel extraction kit (Qiagen) then re-analyzed by denaturing 14% PAGE. DNA products on native and denaturing gels were visualized and quantitated using a Storm 860