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首页> 外文会议>ACS Symposium Series 904; American Chemical Society National Meeting; 20030323-27; New Orleans,LA(US) >Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level
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Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level

机译：DNA双链断裂修复在单分子水平上的可视化

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摘要

Exposure to low doses of ionizing radiation is universal. The genotoxic effects of radiation are attributable, in large part, to DNA double-strand breaks (DSBs). This manuscript describes the development of reagents for visualizing individual DSBs, that is, with sensitivity at the single-molecule level. It also describes the use of these reagents to determine the pathway used for repair of rare DSBs induced at low radiation doses. To visualize DSBs in situ in living cells, we prepared antibodies to γ-H2AX, a modified histone isoform that accumulates at sites of unrepaired DSBs. We compared results with antibodies obtained using different technologies. Initially, we screened a phage display library to identify single chain antibody variable fragments (scFvs) that were specific for the characteristic C-terminal phosphopeptide of γ-H2AX. Although highly selective for this phosphopeptide in vitro, the antibodies were not able to detect γ-H2AX foci in situ, in irradiated cells. We found that conventional antibodies were superior to the scFvs for this purpose. Immunostaining with either affinity-purified rabbit antibodies or a commercial monoclonal antibody revealed distinct γ-H2AX foci in numbers consistent with the number of DSBs expected at a given dose. Separately, we developed a high-affinity scFv directed against a key DSB repair protein, the DNA-dependent protein kinase catalytic subunit. We introduced this scFv into human SK-MEL-28 melanoma cells by microinjection. The presence of the scFv caused radiation-induced γ-H2AX foci to persist to a much greater extent than in control cells. Similar results were obtained at 150 cGy and 10 cGy doses of gamma radiation. These studies provide the first direct evidence that the DNA-dependent protein kinase is required for the repair of rare DSBs induced at low radiation doses.
机译：普遍接受低剂量的电离辐射。辐射的遗传毒性作用在很大程度上归因于DNA双链断裂（DSB）。该手稿描述了用于可视化单个DSB的试剂的开发，也就是说，具有单分子水平的灵敏度。它还描述了使用这些试剂来确定修复低辐射剂量诱导的稀有DSB的途径。为了可视化活细胞中的DSB，我们制备了针对γ-H2AX的抗体，γ-H2AX是一种修饰的组蛋白同种型，聚集在未修复的DSB的位点。我们将结果与使用不同技术获得的抗体进行了比较。最初，我们筛选了噬菌体展示文库，以鉴定对γ-H2AX的特征性C末端磷酸肽具有特异性的单链抗体可变片段（scFvs）。尽管在体外对这种磷酸肽具有高度选择性，但抗体无法在受辐照的细胞中原位检测γ-H2AX病灶。我们发现，为此目的，常规抗体优于scFv。用亲和纯化的兔抗体或市售单克隆抗体进行的免疫染色均显示出明显的γ-H2AX病灶，其数量与给定剂量下预期的DSB数量一致。另外，我们开发了针对关键DSB修复蛋白（DNA依赖性蛋白激酶催化亚基）的高亲和力scFv。我们通过显微注射将此scFv引入人SK-MEL-28黑色素瘤细胞。与对照细胞相比，scFv的存在导致辐射诱导的γ-H2AX病灶持续存在的程度更大。在150 cGy和10 cGy剂量的γ辐射下获得了相似的结果。这些研究提供了第一个直接证据，即低辐射剂量诱导的稀有DSB修复需要DNA依赖性蛋白激酶。

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