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Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level

机译：在单分子水平上可视化DNA双链断裂修复

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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 y-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 Y-H2AX. Although highly selective for this phosphopeptide in vitro, the antibodies were not able to detect Y-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 y-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 Y-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：暴露于低剂量的电离辐射是普遍的。辐射的基因毒性作用是由于，在很大程度上，DNA双链断裂（DSB的）。这个手稿描述了用于可视化DNA双链断裂个体，即，与在单分子水平灵敏度的试剂的发展。它也描述了使用这些试剂来确定用于在低剂量辐射诱导DNA双链断裂难得的修复途径。为了显现原位DNA双链断裂在活细胞中，我们制作的抗体为y-H2AX，修改组蛋白同种，其累积的未修复DNA双链断裂的网站。我们比较了使用不同技术获得的抗体的结果。最初，我们筛选噬菌体展示文库以鉴定单链抗体可变片段（scFvs），其是特异性的用于Y-H2AX的特性C末端磷酸。虽然高选择性此磷酸体外，抗体无法检测到的Y H2AX焦点原位，在照射细胞。我们发现，与传统的抗体均优于scFv的用于这一目的。与任一亲和纯化的兔抗体或市售单克隆抗体免疫染色显示与预期在给定的剂量DSB数量一致号码不同的y H2AX焦点。另外，我们开发了高亲和力的scFv针对关键DSB修复蛋白，将DNA依赖性蛋白激酶催化亚单位。这次介绍的scFv到通过显微注射人类SK-MEL-28黑色素瘤细胞。 scFv的存在而产生的辐射诱导的Y H2AX焦点坚持到更大的程度比在对照细胞。类似的结果在150 cGy的获得和10 cGy的剂量γ辐射。这些研究提供了第一个直接证据表明，DNA依赖性蛋白激酶需要在低剂量辐射诱导DNA双链断裂难得的修复。

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