Do nucleotide excision repair proteins play a role in sensing DNA damage induced by UV.

核苷酸切除修复蛋白在感知紫外线引起的 DNA 损伤中发挥作用吗？

基本信息

批准号：
11680554
负责人：
MORI Toshio
金额：
$ 1.66万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680554/
关键词：
UV cyclobutane pyrimidine dimer (6-4) photoproduct xeroderma pigmentosum (XP)PCNA nucleotide excision repair DNA damage local UV irradiation シクロブタン型ダイマーミクロブタン型ダイマー共焦点レーザー顕微鏡

项目摘要

We have developed a novel method that uses a microfilter mask to produce ultraviolet (UV) DNA lesions in localized areas of the cell nucleus. This technique allows us to visualize localized DNA repair in situ using immunologic probes. Two major types of DNA photoproducts [cyclobutane pyrimidine dimers and (6-4) photoproducts] were indeed detected in several foci per nucleus in normal human fibroblasts. They were repaired at those localized sites with different speeds, indicating that DNA photoproducts remain in relatively fixed subnuclear positions during repair. A nucleotide excision repair (NER) protein, proliferating cell nuclear antigen (PCNA), was recruited to the subnuclear sites of DNA damage within 30 min after UV exposure. The level of PCNA varied with DNA repair activity and diminished within 24 h. In contrast, almost no PCNA fluorescence was observed within 3 h in xeroderma pigmentosum (XP) fibroblasts, which could not repair both types of photolesions. These results demonstrate that this technique is useful for visualizing normal NER process in vivo. Interestingly however, in XP cells, PCNA appeared at UV damage sites after a delay and persisted as late as 72 h after UV exposure. This result suggests that this technique is also valuable for examining an incomplete or stalled NER process caused by the lack of one functional NER protein. Thus, the present technique provides a powerful approach to understanding the temporal and spatial interactions between DNA damage and damage-binding proteins in vivo.

我们已经开发了一种新型方法，该方法使用微滤罩掩模在细胞核局部区域产生紫外线（UV）DNA病变。这种技术使我们能够使用免疫探针可视化局部DNA修复。在正常人成纤维细胞中，确实检测到两种主要类型的DNA光产物[环丁烷嘧啶二聚体和（6-4）光产物]。它们在那些具有不同速度的局部位点进行了修复，表明在修复过程中，DNA光产物仍处于相对固定的亚核位置。核苷酸切除修复（NER）蛋白，增殖细胞核抗原（PCNA）在紫外线暴露后30分钟内募集到DNA损伤的亚核位点。 PCNA的水平随DNA修复活性而变化，并在24小时内降低。相比之下，在三甲状腺色素（XP）成纤维细胞中，几乎没有观察到3小时内观察到PCNA荧光，该成纤维细胞无法修复两种类型的光子。这些结果表明，该技术可用于在体内可视化正常的NER过程。但是，有趣的是，在XP细胞中，PCNA在延迟后出现在紫外线损伤部位，并在紫外线暴露后持续到72小时。该结果表明，该技术对于检查缺乏一种功能性NER蛋白而引起的不完整或停滞的NER过程也很有价值。因此，目前的技术提供了一种强大的方法，可以理解体内DNA损伤与损伤结合蛋白之间的时间和空间相互作用。