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

在254 nm紫外光下产生的DNA损伤主要有环丁烷嘧啶二聚体（CPD）和（6-4）光产物（6-4PP）。在正常的人类细胞中，这两种光损伤都是通过核苷酸切除修复（NER）修复的。本研究旨在三维展示紫外线诱导的人细胞核DNA损伤及其修复。我们首先使用酶联免疫吸附试验（ELISA）和损伤特异性单克隆抗体在正常人和色素干皮病补体组C （XP-C）细胞中研究了CPD和6-4PP的修复动力学。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测了修复DNA合成（非预定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，我们成功地实现了个体细胞中CPDs、6-4PPs和UDS的核定位的三维可视化。不同修复时间光蚀或UDS的典型三维图像很好地反映了ELISA或免疫荧光法获得的修复动力学。重要的发现是，在照射后2小时，未修复的6-4PPs出现点状而非弥漫性的核定位。同样，在第一个和第二个3h-修复期，UDS的局灶核定位也被观察到。这些结果表明，6-4PPs和CPDs都是由正常人类细胞的细胞核非随机修复的。为了理解为什么光损伤是由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6种NER酶（XPB、XPC、XPF、XPG、RPA、PCNA）在正常人细胞核中具有独特的分布模式。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.