The technique of micropore UV irradiation combined with fluorescent antibody labeling is very powerful for examining whether a protein of interest is recruited to the sites of UV-induced DNA damage. Micropore UV irradiation induces UV-damage at localized areas of nuclei using a polycarbonate isopore membrane filter. The polycarbonate blocks UV radiation, and cells are exposed only through the 5 m pores of the filter. 0.5 h after micropore UV irradiation, cells were fixed and Immunofluorescent double staining for DNA damage and repair protein were performed.
Cells were doubly stained for XPB and for CPD 0.5 h after local UV irradiation. In normal MSU-1 cells, XPB foci overlapped with the corresponding CPD foci, indicating that XPB is quickly recruited to the sites of DNA damage for repair. In contrast, no or less bright XPB foci at the DNA damage sites were observed in repair deficient TTD cell lines.
Genomic DNA is purified from UV-damaged cells and denatured DNA is coated on wells of 96-well plate. The binding of TDM-2 or 64M-2 to DNA damage is detected by sequential treatment with biotinylated 2nd antibody and streptavidin-peroxidase. Then, the absorbance of colored products derived from OPD is measured at 492 nm.
Normal human cells repair 90% of the initial 6-4PP within 3 h after UV irradiation, while they remove 50% of the initial CPD within 24 h. Both damage are repaired by the same nucleotide excision repair (NER) pathway, but 6-4PP forms bigger distortion in DNA than CPD does, resulting in much more efficient repair. In contrast, repair deficient XP-C cells can not repair both damage at all.