Three-Dimensional Visualization of Ultraviolet-Induced DNA Damage and Its Repair in Human Cell Nuclei

The two major forms of DNA damage produced by 254 nm UV light are cyclobutane pyrimidine dimer (CPD) and (6–4) photoproduct (6–4PP). Both photolesions are repaired in normal human cells by nucleotide excision repair; however, little is known about where CPD or 6–4PP are repaired in relation to the various subnuclear structures. This study aimed to produce a three-dimensional demonstration of UV-induced DNA damage and its repair in human cell nuclei. We first investigated the repair kinetics of CPD and 6–4PP using an enzyme-linked immunosorbent assay with damage-specific monoclonal antibodies in normal human and xeroderma pigmentosum complementation group C cells. We also examined the kinetics of repair DNA synthesis (unscheduled DNA synthesis) using a quantitative immunofluorescence method with anti-5-bromo-2’-deoxyuridine antibodies. We confirmed the normal repair in normal human cells and the impaired repair in xeroderma pigmentosum complementation group C cells. Then, using laser scanning confocal microscopy, we succeeded in forming a three-dimensional visualization of the nuclear localization of CPD, 6–4PP, and unscheduled DNA synthesis in individual human cells. The typical three-dimensional images of photolesions or unscheduled DNA synthesis at various repair times reflected the repair kinetics obtained by enzyme-linked immunosorbent assay or immunofluorescence very well. The important finding is that the punctate, not diffusely spread, nuclear localization of unrepaired 6–4PP was found 2 h after irradiation. Similarly, the focal nuclear localization of unscheduled DNA synthesis was observed during both the first and the second 3 h repair periods. The present results suggest that both 6–4PP and CPD are nonrandomly repaired from nuclei in normal human cells