Increased initial oxidative DNA damage to the promoters of antioxidant genes in FA cells. (A) Repair kinetics of oxidative damage to GPX1 promoter. FA-A cells or gene-corrected cells were treated with H₂O₂ for 2 hours and released for the indicated time intervals followed by genomic DNA or RNA isolation. Samples were then subjected to (left) DNA-damage assay or (right) RT-PCR. (B) Repair kinetics of oxidative damage to GSTP1 promoter. Samples described in panel A were then subjected to (left) DNA-damage assay or (right) RT-PCR. Percentage of intact DNA is the ratio of PCR products after Fpg cleavage to those present in uncleaved DNA. (C) Increased initial oxidative DNA damage in FA-A cells. Cells described in panel A were used for ChIP using an Ab against 8-oxodG and PCR using primers specific for (left) GPX1 or (right) GSTP1 promoter. Representative images (top) and quantifications (bottom) are shown. Results are means ± SD of 3 independent experiments. (D) Repair efficiency as determined by host cell-reactivation assay. The pSSG-promoter reporter vector containing promoter regions of antioxidant gene GCLC, GPX1, GSTP1, or TXNRD1, as well as control gene GAPDH or β-tubulin, were treated with 100μM H₂O₂ for 1 hour in vitro and then transfected into normal, FA-A, or gene-corrected fibroblasts followed by determination of luciferase activity. Results are means ± SD of 3 independent experiments. (E) Repair kinetics of oxidative damage to naked promoter DNA. Genomic DNA was isolated from cells described in panel D followed by Fpg cleavage and qPCR using primers specific for the cloned GPX1 promoter. The level of intact DNA represents the efficiency of repair.