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.