In Situ Visualization of Ultraviolet-Light-Induced DNA Damage Repair in Locally Irradiated Human Fibroblasts

Abstract

We have developed a novel method that uses a microfilter mask to produce ultraviolet-induced 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 at different speeds, indicating that DNA photoproducts remain in relatively fixed nuclear positions during repair. A nucleotide excision repair protein, proliferating cell nuclear antigen, was recruited to the sites of DNA damage within 30 min after ultraviolet exposure. The level of proliferating cell nuclear antigen varied with DNA repair activity and diminished within 24 h. In contrast, almost no proliferating cell nuclear antigen fluorescence was observed within 3 h in xeroderma pigmentosum fibroblasts, which could not repair either type of photolesion. These results demonstrate that this technique is useful for visualizing the normal nucleotide excision repair process in vivo. Interestingly, however, in xeroderma pigmentosum cells, proliferating cell nuclear antigen appeared at ultraviolet damage sites after a delay and persisted as late as 72 h after ultraviolet exposure. This result suggests that this technique is also valuable for examining an incomplete or stalled nucleotide excision repair process caused by the lack of a single functional nucleotide excision repair protein. Thus, the technique provides a powerful approach to understanding the temporal and spatial interactions between DNA damage and damage-binding proteins in vivo.

The Spacer Region of XPG Mediates Recruitment to Nucleotide Excision Repair Complexes and Determines Substrate Specificity

“…The sequential recruitment of the NER factors to sites of UV damage can be visualized by using local irradiation of individual cells followed by staining with antibodies directed against the NER proteins of interest (13,53). We made use of this technique to determine whether the spacer region of XPG is needed for the recruitment of XPG to sites of UV damage.…”

Comparison of diarsenic oxide and tetraarsenic oxide on anticancer effects: Relation to the apoptosis molecular pathway

“…PCNA is known to associate with DNA repair processes. Thus, the level of PCNA is known to correlate with DNA repair activity (27). In particular, As 4 O 6 inhibited PCNA and Bcl-X L expression.…”

PCNA-dependent accumulation of CDKN1A into nuclear foci after ionizing irradiation

“…Table I UV micropore irradiation was based on the method described previously [39]. The medium was removed, the cells were rinsed twice with PBS and immediately after covered by polycarbonate foils containing pores of 3 µm diameter in size.…”

“…To further investigate the role of CDKN1A recruitment to DNA damaged sites, we compared nucleotide excision repair (NER)-proficient AG1522C cells and NER-deficient XPA (Xeroderma Pigmentosum complementation group A) cells exposed to 100 J/cm 2 UV-light under a microfilter-mask, carried out essentially as described before [39].…”