Fig. 3.

EdU-substituted DNA is recognized as damage and excised by nucleotide excision repair in vitro. (A) Schematic of substrate synthesis and in vitro excision of EdU by mammalian CFE. 140 bp duplex substrates were synthesized with a ³²P label adjacent to a uniquely located modification. Modifications (red triangles) included a (6, 4) PP, an EdU, or two EdUs. Substrates were synthesized by phosphorylating, annealing, and ligating six component oligonucleotides. Ligation sites are indicated by dots. The central oligonucleotide containing the modification was labeled at the 5′ end with ³²P (yellow dot site). A control UM substrate was also synthesized (not shown). Substrates were incubated with CFE, and unreacted full-length substrate and excised products were purified and resolved with a sequencing gel. (B) Result of a representative experiment. (C) Percentage of excision products relative to substrate was quantified based on three biological replicates. Data are means ± SEMs; n = 3. *P < 0.05, **P < 0.01, two-tailed unpaired t test.