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

254 nm紫外光对DNA的两种主要损伤是环丁烷嘧啶二聚体(CPD)和(6-4)光产物(6-4PP)。这两种光渗漏都是通过核苷酸切除修复(NER)在正常人类细胞中修复的。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。我们首先用损伤特异性单抗的酶联免疫吸附试验(ELISA)研究了CPD和6-4PP在正常人和色素性干皮病互补C组(XP-C)细胞中的修复动力学。我们还用抗5-溴-2‘-脱氧尿苷抗体的定量免疫荧光方法检测了修复DNA合成(非程序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，利用激光扫描共聚焦显微镜，我们成功地对CPDS、6-4PPS和UDS在单个人类细胞中的核定位进行了三维可视化。不同修复时间的光刻或UDS的典型三维图像很好地反映了通过ELISA法或免疫荧光法获得的修复动力学。重要的发现是，未修复的6-4PPS在照射后2小时出现点状、非弥漫性的核定位。同样，在第一次和第二次3h修复期间，都观察到了UDS的局灶性核定位。这些结果表明，在正常人细胞中，6-4pps和CPD都是从细胞核非随机修复的。为了理解为什么光刻是从细胞核非随机修复的，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)在正常人细胞核中具有不同的分布模式。我们现在正在比较修复酶和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.