Figure 5. Absence of excision repair of CPDs in telomeres.

(A) WI38 human diploid fibroblasts were irradiated with minimally-lethal doses of UVC (10 J/m²) and returned to the incubator for varying lengths of time before harvesting (0–48 h). The DNA was then isolated, the IPoD technique was used to isolate damaged DNA, photolyase was used to remove CPD, and PCR was performed on region of interest: Telomere, mitochondrial CYTB gene (mtDNA), 28S ribosomal DNA, or p53. For each time point, the integrated intensity of the band or lane containing the PCR–amplified IP pulldown fraction is normalized against the unamplified input DNA for that time point. The amount of DNA in the fraction pulled down by antibody to CPD decreases with time in the p53 and 28S genes, reflecting normal excision repair, but not in telomeres or mtDNA (CYTB gene). (B) A similar experiment was performed in primary human skin fibroblasts UV-irradiated at 20 J/m². In this experiment, CPD were removed from IPoD-immunoprecipitated CPD containing DNA using DNA photolyase before PCR amplification and repair in the two telomeric DNA strands was analyzed together and separately. Each experiment was performed in triplicate. P values are derived from the two-tailed heteroscadastic Student's t-test.