5-FU–Induced DNA Strand Breaks Are Reduced in TDG-Deficient Cells whereas Overall Repair Activity Is Increased.

(A) Complementation of Tdg knockout MEFs with wild-type and catalytically deficient TDG. Stable transfectants of Tdg^−/− MEFs ectopically expressing either TDG variant from the native promoter show TDG levels about the same as endogenous, as detected by western blotting.

(B) Reduced levels of 5-FU–induced DNA strand breaks in cells lacking active TDG. Steady-state levels of DNA single- and double-strand breaks in the cell lines indicated were assessed by the alkaline Comet assay using automated comet tail moment analysis. 5-FU treatment results in a significant tail moment increase in wild-type, but not in Tdg knockout MEFs. The generation of 5-FU–specific DNA strand breaks in Tdg knockout cells is restored by complementation with wild-type Tdg, but not with the catalytically inactive mutant. Shown are box plots with individual tail moments per cell, medians, interquartile ranges (boxes), 2.5%–97.5% percentiles (whiskers) and outliers (dots) of pooled data (600 to 900 cells) obtained from three independent experiments.

(C) 5-FU treatment triggers DNA SSB repair in TDG wild-type and knockout cells. The top panel shows nuclei of Tdg-proficient and -deficient cells stained with a polyclonal anti-XRCC1 antibody (XRCC1_ab) after 5-FU treatment. The statistical analysis of XRCC1 foci per cell across the populations analyzed (n ≥ 100 cells per population) is shown as a scatter plot with medians and the interquartile ranges.

pC, empty vector; pTdg, vector expressing TDG; pTdg_cat, vector expressing a catalytic dead variant.