In situ visualization of ultraviolet-light-induced DNA damage repair in locally irradiated human fibroblasts

We have developed a novel method that uses a microfilter mask to produce ul traviolet-induced DNA lesions in localized areas of the cell nucleus. This technique allows us to visualize localized DNA repair in situ using immunol ogic probes. Two major types of DNA photoproducts [cyclobutane pyrimidine d imers and (6-4) photoproducts] were indeed detected in several foci per nuc leus in normal human fibroblasts. They were repaired at those localized sit es at different speeds, indicating that DNA photoproducts remain in relativ ely fixed nuclear positions during repair. A nucleotide excision repair pro tein, proliferating cell nuclear antigen, was recruited to the sites of DNA damage within 30 min after ultraviolet exposure. The level of proliferatin g cell nuclear antigen varied with DNA repair activity and diminished withi n 24 h. In contrast, almost no proliferating cell nuclear antigen fluoresce nce was observed within 3 h in xeroderma pigmentosum fibroblasts, which cou ld not repair either type of photolesion. These results demonstrate that th is technique is useful for visualizing the normal nucleotide excision repai r process in vivo. Interestingly, however, in xeroderma pigmentosum cells, proliferating cell nuclear antigen appeared at ultraviolet damage sites aft er a delay and persisted as late as 72 h after ultraviolet exposure. This r esult suggests that this technique is also valuable for examining an incomp lete or stalled nucleotide excision repair process caused by the lack of a single functional nucleotide excision repair protein. Thus, the technique p rovides a powerful approach to understanding the temporal and spatial inter actions between DNA damage and damage-binding proteins in vivo.