Figure 4. The WRN N-terminal KBM promotes WRN exonuclease activity.

(a) Left, cartoon illustrating the GFP-tagged truncated recombinant WRN proteins employed in these experiments. The WRN N-terminal (‘nA') and C-terminal (‘cA') KBMs are indicated by red boxes and XLF-like motif (‘X') by a blue box. The exonuclease domain is indicated by a black box, and the position of the KBM mutation (W18G) by an asterisk and dotted line. Middle, U2-OS cells transiently expressing the indicated recombinant GFP-tagged WRN protein were imaged for GFP before and after UVA microirradiation, as in Fig. 2. Right, Ku80^−/− MEFs transiently co-expressing GFP-tagged WRN-Exo, RFP-Ku70, and either RFP (vector), RFP-Ku80 or RFP-Ku80^L68R as indicated were micro-irradiated as in Fig. 1. Data are the mean GFP fluorescence (±s.e.m.) in the laser track relative to the mean GFP fluorescence before irradiation (set at 100%) from >20 cells per experiment. (b) The indicated GFP-tagged WRN proteins were recovered from transiently transfected HEK293T cell lysates pre-treated or not as indicated with Benzonase and RNAse in pull-down assays using GFP-TRAP beads. Aliquots of the bead eluate were fractionated by SDS-PAGE and silver stained to detect GFP-WRN, GFP-WRNW18G, Ku80, and DNA-PKcs (‘CS'). (c) Cy3-labeled 30 bp duplex oligonucleotide (20 nM) with a 5′ overhang was incubated with 500, 100, 20 or 5 nM HIs-tagged WRN-Exo or WRN-Exo^W18G in the absence or presence of 100 nM Ku heterodimer (Ku70/Ku80, ‘Ku') and 5 mM MgCl₂. Exonuclease products were resolved on a 16% TBE-Urea gel. (d) Exonuclease assays were conducted as above in the presence of 5 mM MgCl₂ using 10 nM His-tagged WRN-Exo and 100, 20, 4 or 0.8 nM of either Ku heterodimer (Ku70/Ku80; ‘Ku'), KuΔC heterodimer (Ku70/Ku80ΔC; ‘KuΔC'), or mutant KuΔC heterodimer harbouring the Ku80 mutation, L68R (KuΔC^L68R). (e) Exonuclease assays were conducted as above using 100, 20 and 4 nM of the indicated His-tagged WRN protein and 10 nM wild-type Ku heterodimer (Ku70/Ku80; ‘Ku') in 5 mM Mg²⁺.