Figure 1
UDG activity determines the levels of uracil and AP sites in DNA. (a) UDG activity assay in vitro. Oligonucleotide duplexes containing U:G were incubated with cell extracts (5–10 μg) from UDG+/+, DLD1flag, and UDG −/− cells at 37°C for 1 h. Reaction products were resolved by electrophoresis through denaturing 20% polyacrylamide gels. (b) Incorporated uracil detected in UDG+/+ and UDG−/− cells by HPLC/MS/MS analysis. Cells were treated with pemetrexed (10 μM) for 6, 24, 48, and 72 h. Cells were harvested and 40 μg of extracted DNA were in vitro reacted with purified UDG (10 U) for 2 h. (c) Cells were treated with 5-FU (10 μM) for 6, 24, 48, and 72 h. Uracil was quantified in the reaction product by LC-MS analysis. (d) AP site formed by pemetrexed in UDG+/+ and UDG−/− cells. Cells were treated with pemetrexed (0–400 nM) for 24 h. DNA was extracted and AP sites measured by ARP reagent. (e) AP site detected in DNA of UDG−/− cells after reacted with purified UDG in vitro. Cells were treated with pemetrexed (0–100 nM) for 24 h and 40 μg DNA extracted from cells was in vitro reacted with purified UDG (10 U) for 2 h and AP sites were measured using ARP. Results are representative of three independent experiments
