Figure 7.

Generation and characterization of UNG isoform-specific human cell line clones. (A) N-terminal amino acid sequence of human UNG1 and UNG2. UNG1-specific residues (amino acid 1–35) are marked in blue. Arginine (R) residues in bold and target site for proteolytic processing by MPP (mitochondrial processing peptidase) are indicated. UNG2-specific residues (amino acid 1–44) are yellow, and include the PIP-box in red. The common RPA-binding site (green) and the globular catalytic domain (UNG CD) are indicated. (B) Representative CRISPR/Cas9 UNG isoform-specific knockout clones generated in three human cell lines (L428, HAP1, U373). Clones were screened by western blot analysis and UDG activity assays on whole cell extracts. High molecular weight ³H-U:A nick-translated DNA was used as substrate. The bars represent mean activity of three independent L428 clones. Significantly reduced UDG activity compared to WT (UNG Int2) is indicated with *P< 0.05 or **P< 0.005. (C) Genomic uracil levels in untreated and 5FdU-treated UNG isoform-specific knockout clones. Cells were seeded (0.2 × 10⁻⁶ cells/ml) 24 h before the addition of 1 μM 5FdU. Cells were harvested 24 h post treatment. dU was quantified by LC–MS/MS. Bars represent the mean value of six to eight different clones in each group (indicated). Red dotted line indicates dU level in UNG-treated DNA (detection limit). Significantly increased dU levels in the UNG1+2 knockout group compared to the UNG2 knockouts are indicated with P-values.