FIGURE 9.

WRN-depleted cells exhibit increased mutagenesis. SV40-immortalized fibroblasts were infected with pBabe retrovirus expressing WRN-specific shRNA sequences. Seven days post-infection, short oligonucleotides bearing TaqI restriction enzyme cleavage sites and resembling primer-template (A) or D-loop (B) DNA (as described in supplemental Methods) were introduced in to WRN-depleted or control cells. The in vivo extended oligonucleotide probes were retrieved with streptavidin-coated magnetic beads from whole cell extracts (cyto) or nuclei, digested with TaqI restriction enzyme to eliminate wild-type sequences, and amplified by quantitative PCR. Mutagenesis was scored by the ability to amplify full-length PCR products due to absence of cleavage of the extended oligonucleotide by TaqI restriction enzyme. B in the box, 5′-biotin; 3′-ddC, dideoxycytidine.