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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.

我们开发了一种新的方法，使用微滤膜在细胞核的局部区域产生紫外线（UV） DNA损伤。这项技术使我们能够使用免疫探针可视化定位DNA原位修复。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]确实在正常人类成纤维细胞的每个细胞核的几个病灶中检测到。它们在这些局部位点以不同的速度修复，这表明DNA光产物在修复过程中保持在相对固定的亚核位置。一种核苷酸切除修复（NER）蛋白，即增殖细胞核抗原（PCNA），在紫外线照射后30分钟内被招募到DNA损伤的亚核位点。PCNA水平随DNA修复活性的变化而变化，并在24 h内减弱。相比之下，在3 h内，色素干皮病（XP）成纤维细胞几乎没有观察到PCNA荧光，它不能修复这两种类型的光损伤。这些结果表明，该技术对观察体内正常的NER过程是有用的。然而，有趣的是，在XP细胞中，PCNA在紫外线损伤部位延迟出现，并持续到紫外线暴露后72小时。这一结果表明，该技术对于检查由于缺乏一种功能性NER蛋白而导致的不完整或停滞的NER过程也很有价值。因此，目前的技术为了解DNA损伤和损伤结合蛋白在体内的时间和空间相互作用提供了一种强有力的方法。