Three dimensional visualization of UV-induced DNA damage and repair enzymes in human fibroblasts.

254 nm紫外光对DNA的损伤主要是环丁烷嘧啶二聚体（CPD）和（6-4）光产物（6-4PP）。这两种光损伤在正常人类细胞中通过核苷酸切除修复（NER）来修复。本研究旨在三维演示紫外线诱导的人细胞核DNA损伤及其修复。我们首先研究了CPD和6-4PP的修复动力学，使用酶联免疫吸附试验（ELISA）与损伤特异性单克隆抗体在正常人和着色性干皮病互补组C（XP-C）细胞。我们还使用抗5-溴-2 '-脱氧尿苷抗体的定量免疫荧光方法检查了修复DNA合成（程序外DNA合成，UDS）的动力学。我们证实了正常人细胞的正常修复和XP-C细胞的受损修复。然后，使用激光扫描共聚焦显微镜，我们成功地在三维可视化的核定位的CPD，6-4PPs和UDS在个人细胞。光损伤或UDS在不同修复时间的典型三维图像很好地反映了通过ELISA或免疫荧光获得的修复动力学。重要的发现是，在照射后2小时，发现未修复的6-4PPs的点状，而不是弥漫性扩散，核定位。同样，在第一和第二个3 h修复阶段，观察到UDS的核定位。这些结果表明，在正常人类细胞中，6-4PPs和CPD都是从细胞核中非随机修复的。为了理解为什么光损伤是从细胞核中非随机修复的，我们使用激光扫描共聚焦显微镜研究了NER酶的核分布。我们发现六种NER酶（XPB、XPC、XPF、XPG、RPA、PCNA）在正常人细胞核中有不同的分布模式。我们现在正在进行比较修复酶和DNA损伤之间的核分布模式。

Two major 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 (NER). This study was performed to aim at 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 (ELISA) with damage-specific monoclonal antibodies in normal human and xeroderma pigmentosum complementation group C (XP-C) cells. We also examined the kinetics of repair DNA synthesis (unscheduled DNA synthesis, UDS) 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 XP-C cells. Then, using laser scanning confocal microscopy, we succeeded in three-dimensional visualization of the nuclear localization of CPDs, 6-4PPs and UDS in individual human cells. The typical three-dimensional images of photolesions or UDS at various repair times reflected well the repair kinetics obtained by ELISA or immunofluorescence. The important finding is that the punctate, not diffusely spread, nuclear localization of unrepaired 6-4PPs was found at 2 h after irradiation. Similarly, the focal nuclear localization of UDS was observed both during the first and the second 3h-repair penods. These results suggest that both 6-4PPs and CPDs are non-randomly repaired from nuclei in normal human cells.To understand why photolesions are non-randomly repaired from nuclei, we investigated the nuclear distribution of NER enzymes using laser scanning confocal microscopy. We found that six NER enzymes (XPB, XPC, XPF, XPG, RPA, PCNA) showed distinctive distribution patterns in normal human cell nuclei. We are now underway to compare the nuclear distribution patterns between repair enzyme and DNA damage.