Do nucleotide excision repair proteins play a role in sensing DNA damage induced by UV.

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We have developed a novel method that uses a microfilter mask to produce ultraviolet (UV) DNA lesions in localized areas of the cell nucleus. This technique allows us to visualize localized DNA repair in situ using immunologic probes. Two major types of DNA photoproducts [cyclobutane pyrimidine dimers and (6-4) photoproducts] were indeed detected in several foci per nucleus in normal human fibroblasts. They were repaired at those localized sites with different speeds, indicating that DNA photoproducts remain in relatively fixed subnuclear positions during repair. A nucleotide excision repair (NER) protein, proliferating cell nuclear antigen (PCNA), was recruited to the subnuclear sites of DNA damage within 30 min after UV exposure. The level of PCNA varied with DNA repair activity and diminished within 24 h. In contrast, almost no PCNA fluorescence was observed within 3 h in xeroderma pigmentosum (XP) fibroblasts, which could not repair both types of photolesions. These results demonstrate that this technique is useful for visualizing normal NER process in vivo. Interestingly however, in XP cells, PCNA appeared at UV damage sites after a delay and persisted as late as 72 h after UV exposure. This result suggests that this technique is also valuable for examining an incomplete or stalled NER process caused by the lack of one functional NER protein. Thus, the present technique provides a powerful approach to understanding the temporal and spatial interactions between DNA damage and damage-binding proteins in vivo.