Nucleotide Excision Repair Removes Thymidine Analog 5-Ethynyl-2'-deoxyuridine From the Human Genome

5-Ethynyl-2'-deoxyuridine (EdU) is one of the several thymidine analogs that are used in labeling DNA in cells and in DNA combing (DNA fiber analysis) to study DNA replication1-5. We wished to use these analogs to investigate the repair complex (DNA-protein) of nucleotide excision repair. UV irradiated human cell lines were incubated with EdU, BrdU, IdU, CldU, F-ara-EdU or AmdU to incorporate these analogs into the 26-27 nucleotide long excision repair products. Unexpectedly, we found that EdU even in the absence of DNA damage was released in the form of 26-27nt-long oligomers. We conclude that EdU is incorporated into replicating DNA and is recognized as damage by the nucleotide excision repair system and removed from the genome followed by repair synthesis and re-incorporation into DNA, thus creating futile cycle that leads to cell death. This property of EdU, which was not seen with any of the other thymidine analogs, may be taken advantage of in treating cancer, in particular brain cancers because EdU crosses blood-brain barrier. Overall design: XR-seq experiment was done as previously reported with modifications. In brief, Hela cells were treated with 10 µM EdU and harvested after 6h, 12h and 24h. Cell pellets from two 150 mm culture plates were suspended and lysed in 1mL cold buffer A ((25 mM HEPES at pH7.9, 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, 12.5% glycerol, 0.5% NP-40). The primary EdU excision products were isolated by immunoprecipitation with TFIIH antibody followed by ligation of 5' and 3' adapters. After adaptor ligation, excision oligomers containing EdU were further purified by immunoprecipitation with anti-BrdU antibody that also reacts with EdU. Purified EdU excision oligomers were directly subjected to PCR amplification using 50- and 63-nt-long primers that introduce specific barcodes compatible with the Illumina TruSeq small RNA kit, without damage reversal step as this is done with CPD or (6-4)PP photoproducts. The PCR products containing excised oligonucleotides were ~145 base pairs (bp) in length and resolved on 10% sequencing gel. EdU excision oligomers from different time point were gel purified, pooled and sequenced on the NextSeq-P3 platform at the University of North Carolina–Chapel Hill High-Throughput Sequencing Facility.