Exonuclease activity of wild-type WRN and the N333 fragment. 6×his-tagged proteins were purified from baculovirus-infected insect cells using either nuclear (WRN, D82A, E84A, K577M, mock control) or cytosolic (N333, mock control) extracts. WRN, N-333, or mock proteins (10 ng, a,b; 5 ng, c,d) were incubated with 10,000 cpm of either a partial DNA duplex containing a 21-nt 5′ 32P-labelled fragment annealed to an unlabelled 43-nt fragment (a,c) or a 36-bp DNA fragment 32P-labelled at the 3′ end (b,d) in nuclease assay buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5 mM DTT, 0.1 mg/ml BSA) at 37 °C for the indicated intervals. Products were analysed on 20% polyacrylamide-8 M urea denaturing gels and visualized by autoradiography. M, mock; W, no protein addition. e, N333 or mock proteins (20 ng) were incubated with a 374-bp DNA fragment labelled with 32P at the 3′ end and 3H at internal thymidine residues for the indicated intervals. The products were precipitated with 70% ethanol, and ethanol-soluble radioactivity was determined by scintillation counting. The activity is presented as percentage of input label (10,000 cpm 32P; 6,000 cpm 3H) that was soluble in 70% ethanol. f, Equal amounts of 6×his-affinity purified N333 and mock proteins were resolved on a 8% non-denaturing polyacrylamide gel. The region containing N333 and a comparable region in the mock lane were excised, and the proteins eluted into nuclease buffer. The eluted N333 (20 ng) and mock proteins were incubated with 10,000 cpm of DNA substrates labelled to similar specific activities with 32P at either the 5′ or 3′ end (a,b, respectively) for 1 h, and the activity was determined as in (e).