Figure 2.

Smug1 inactivation ablates hmU excision activity and gives resistance to hmUdR. (A) Analysis of base excision activity in brain and liver nuclear extracts from wild-type (WT), Ung^−/− and Smug1^−/− mice assayed on a 3′-fluorescently labelled double-stranded oligodeoxyribonucleotide substrate containing a centrally placed hmU:G mispair (left), as well as on a single-stranded 3′-fluorescently labelled substrate containing a single centrally placed U as an UNG activity control. Excision creates an abasic site that is subject to cleavage by APE1; cleavage products of the 3′-fluorescently labelled oligonucleotides were visualized after polyacrylamide gel electrophoresis. Similar results regarding a loss of hmU-excision activity were obtained on assaying multiple different tissues from Smug1^−/− mice of different ages. (B) Effects of a 48-h culture in the presence of various concentrations of hmUdR on the viability of embryo fibroblast cell-lines derived from mice of different genotypes. Survival is expressed relative to that obtained in the absence of hmUdR. The results are shown for triplicate assays performed on two independently derived cell-lines for each indicated genotype.