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

人体成纤维细胞中紫外线诱导的 DNA 损伤和修复酶的三维可视化。

基本信息

批准号：
09680533
负责人：
MORI Toshio
金额：
$ 1.34万
依托单位：
Nara Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680533/
关键词：
DNA damage nucleotide excision repair XPB XPC XPF XPG PCNA laser scanning confocal microscopy シクロブタン型ダイマー (6-4)型ダイマー不定期DNA合成色素性乾皮症モノクロナール抗体紫外線

项目摘要

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.

254 nm 紫外线产生的两种主要 DNA 损伤是环丁烷嘧啶二聚体 (CPD) 和 (6-4) 光产物 (6-4PP)。正常人体细胞中的两种光损伤均通过核苷酸切除修复（NER）进行修复。这项研究的目的是三维演示紫外线引起的 DNA 损伤及其在人体细胞核中的修复。我们首先使用酶联免疫吸附测定 (ELISA) 和损伤特异性单克隆抗体在正常人和着色性干皮病互补 C 组 (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查了修复 DNA 合成（非计划 DNA 合成，UDS）的动力学。我们证实了正常人体细胞的正常修复和 XP-C 细胞的受损修复。然后，利用激光扫描共焦显微镜，我们成功地对单个人体细胞中 CPD、6-4PP 和 UDS 的核定位进行了三维可视化。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，在辐照后 2 小时，发现了未修复的 6-4PP 的点状而非扩散性核定位。类似地，在第一个和第二个 3 小时修复期间观察到 UDS 的局灶核定位。这些结果表明，在正常人类细胞中，6-4PP 和 CPD 都是从细胞核非随机修复的。为了理解为什么光损伤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）在正常人体细胞核中表现出独特的分布模式。我们现在正在比较修复酶和 DNA 损伤之间的核分布模式。