An in vivo analysis of the dynamics of UV-induced DNA damage repair using micropore UV irradiation and specific antibodies

We describe a novel method that uses a microfilter mask to produce ultraviolet (UV)-induced DNA lesions in localized areas of the cell nucleus and subsequently visualize localized DNA repair in situ using immunologic probes. This simple technique allows us to study how nucleotide excision repair (NER) of photolesions is processed in human cells in vivo by analyzing the temporal and spatial interactions between specific DNA damage and NER proteins in the complex nuclear structure. The micropore UV irradiation induces two major types of DNA photoproducts, cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PP), in several foci per nucleus, which are repaired at those localized sites in normal human fibroblasts, indicating that DNA photoproducts remain in relatively fixed subnuclear positions during repair. Thus, NER proteins must be recruited to the subnuclear sites of DNA damage. Indeed, the recruitment of proliferating cell nuclear antigen (PCNA) is observed after micropore UV irradiation. Detergent-insoluble PCNA varies in amount at DNA damage sites according to the activity of NER. In contrast, xeroderma pigmentosum (XP)-A fibroblasts cannot repair CPD or 6-4PP, and PCNA is not recruited to DNA damage sites for repair. The UV microfilter DNA lesion assay is also utilized to investigate the pathogenesis of defective NER-related diseases on the molecular level. Indeed, we found that in photosensitive trichothiodystrophy (TTD)/XP-D cell strains, the capacity of defective TFIIH complex for accumulating at DNA damage sites determines the severity of DNA repair deficiency. The novel technique of micropore UV irradiation combined with fluorescent antibody labeling enables us to analyze the details of normal and impaired NER processes which cannot be examined using in vitro biochemical techniques. Moreover, the micropore UV irradiation can be applicable to the induction of various types of UV damage as well as DNA photoproducts. Thus, this technique may allow us to analyze various cellular responses to UV in vivo.