Nucleic Acids Research, 2007, Vol. 35, No. 17 5733 Table 1. Oligonucleotides used to construct model replication forksa Short fork substrate series 21lead GCTATCGTACATGATATCCTC 24lead GCTATCGTACATGATATCCTCACA 27lead GCTATCGTACATGATATCCTCACACTC 30lead GCTATCGTACATGATATCCTCACACTCACT 32lead GCTATCGTACATGATATCCTCACACTCACTTA 30lag TCAGAGTGTGAGGATATCATGTACGATAGC 70lead CGTGACTTGATGTTAACCCTAACCCTAAGAATTCGGCTTAAGTGAGTGTGAGGATATCATGTACGATAGC 70lag GCTATCGTACATGATATCCTCACACTCTGAATAGCCGAATTCTTAGGGTTAGGGTTAACATCAAGTCACG Long fork substrate 72lead GCAGCGTCGCTGCTAGCGTGCAGCGCTTGTACTTCAGCTGATAGACACGTGGCAATTGCCTACATGTAT-CCT 82lag TCAGAGTGTGAGGATACATGTAGGCAATTGCCACGTGTCTATCAGCTGAAGTTGTTCGCGACGTGCGAT-CGTCGCTGCGACG 122lead CGTGACTTGATGTTAACCCTAACCCTAAGATATCGCGTTAAGTGAGTGTGAGGATACATGTAGGCAATT-GCCACGTGTCTATCAGC TGAAGTACAAGCGCTGCACGCTAGCAGCGACGCTGC 122lag CGTCGCAGCGACGATCGCACGTCGCGAACAACTTCAGCTGATAGACACGTGGCAATTGCCTACATGTAT-CCTCACACTCTGAATAC GCGATATCTTAGGGTTAGGGTTAACATCAAGTCACG aAll sequences are depicted in 50 to 30 orientation. phosphoimager and ImageQuant software (GE Healthcare). In fork regression assays, radioactivity associated with individual DNA species was measured. For speciﬁc kinetic experiments (Figure 1F), the amounts (as a percentage of total radioactivity) of each DNA species were determined and directly compared. To calculate enzyme-mediated generation of DNA products, the percentage of each product with respect to the total (molar) amount of original fork substrate in that reaction was quantitated following subtraction of background levels of respective DNA species in reactions without enzyme. For analysis of WRN exonuclease activity during regression reactions, the amounts of radioactivity associated with intact and digested products of the leading daughter strand were determined and the percentage of each length product with respect to the total radioactivity derived from the leading daughter strand in that lane was quantitated. This data for each product derived from the leading daughter strand from individual lanes is comparatively presented for daughter duplexes extracted from native PAGE (Figure 5C). Alternatively, the percentages of each product formed after 5 min of digestion are compared with the amount of the respective product in the undigested substrate (0 min time point), and the percent change over time for each product is plotted (Figure 4B). RESULTS Fork regression by exonuclease-deficient WRN and the influence of leading arm structure Our earlier experiments (49) indicated that WRN and BLM could act on a model fork structure with homo- logous arms to generate both parental and daughter duplexes, consistent with the possibility that these enzymes might regress replication forks in vivo. We next wanted to determine whether and how the precise structure at the fork junction might inﬂuence these novel regression activities. To this end, a series of model four- stranded replication fork substrates was constructed from individual oligomers (for details, see ‘Materials and methods’ section and Table 1 for nucleotide sequences). These short fork substrates (Figure 1A) contained a 38 bp parental duplex region, a lagging parental–daughter arm of 30 bp plus a 2 nt single-stranded gap at the fork junction, and a leading parental–daughter arm with a parental strand region of 32 nt but, on individual fork substrates, the leading daughter strand varied in length from 32 to 21 nt resulting in single-stranded gaps of 0–11 nt at the fork junction. Individual short fork substrates are identiﬁed below by their leading daughter strand (32lead fork) and/or the size of the single-stranded gap on the leading arm. Importantly, lagging and leading parental–daughter arms of these substrates were entirely homologous (except for 5 non-complementary nt on each