Drosophila, which Lacks Canonical Transcription-Coupled Repair Proteins, Performs Transcription-Coupled Repair

Previous work with the classic T4 Endonuclease V digestion of irradiated Drosophila DNA followed by Southern hybridization led to the conclusion that Drosophila lacked transcription-coupled repair (TCR). This conclusion was reinforced by the Drosophila Genome Project which revealed that Drosophila lacked CSA, CSB, or UVSSA homologs, whose orthologs are present in eukaryotes that carry out TCR ranging from Arabidopsis to humans. A recently developed in vivo excision assay and the Excision Repair-sequencing (XR-seq) method have enabled genome-wide analysis of nucleotide excision repair in various organisms at single nucleotide resolution and strand-specific manner. Using these methods, we have discovered that Drosophila S2 cells carry out robust TCR comparable to that observed in mammalian cells. Our findings provide new insights into the mechanisms of TCR among various species. Overall design: We apply XR-seq in S2-DGRC , the cells were obtained from the Drosophila Genetic Resources Center. For UV treatments, cells were inoculated into R-150 plates and grown to about 25 to 80 percent confluence. Media was gently removed from the semi-adherent monolayer, cells were irradiated with UV-C, and then fresh media containing sterilized, conditioned media was added to cells, and cells were incubated at 27 degrees. Following predetermined repair times, plates were placed on ice and cooled cells were harvested by scraping and rinsing with ice-cold PBS. Cells were pelleted, transferred to Eppendorf tubes, washed with cold PBS, and resuspended in 340 ul of cold TE. samples were immunoprecipitated with anti-CPD antibody, and then ligated to adapters and processed for sequencing (XR-seq). This series includes re-analysis of XR-seq of UV-treated normal human fibroblast cells and Cockayne syndrome cells. GSE76391: GSM1985849 GSM1985850 GSM1985851 GSM1985852 GSM1985853 GSM1985854; GSE67941:GSM1659164 GSM1659165