Inhibition of uracil removal by ATA in HAP cells

To determine whether the putative UDG inhibitor inhibits uracil removal in cells. Also, to compare the removal of uracil in wild type and UDG knockout HAP cells Conclusion: ATA inhibits uracil removal in wild type HAP cells. ATA also inhibits uracil removal in UDG KO cells. Without knockout of additional uracil removal pathways, such as SMUG1 glycosylase, we cannot determine the degree to which ATA inhibits backup uracil removal pathways. Notes: HAP cells were seeded into plates and treated with DMSO or with a putative uracil dna glycosylase inhibitor (ATA)). Following treatment, cells were transfected with a plasmid-based reporter that measures uracil removal. The samples labelled "Undamaged" were transfected with plasmids encoding BFP and GFP. The samples labeled "Damaged" were transfected with a plasmid encoding GFP and a uracil-containing plasmid encoding BFP. If uracil is present in the plasmid, wild type BFP transcripts are produced. If uracil is removed. non-fluorescent transcripts are produced. Hence, increased BFP fluorescence in the samples labeled "Damaged" means decreased removal of uracil from DNA. Daily performance tests on the flow cytometer using the manufacturer's performance tracking beads.