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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells
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DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells

机译：DNA损伤调节活细胞核内Brca2的迁移

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摘要

How the biochemical reactions that lead to the repair of DNA damage are controlled by the diffusion and availability of protein reactants within the nucleoplasm is poorly understood. Here, we use gene targeting to replace Brca2 (a cancer suppressor protein essential for DNA repair) with a functional enhanced green fluorescent protein (EGFP)-tagged form, followed by fluorescence correlation spectroscopy to measure Brca2-EGFP diffusion in the nucleoplasm of living cells exposed to DNA breakage. Before damage, nucleoplasmic Brca2 molecules exhibit complex states of mobility, with long dwell times within a sub-fL observation volume, indicative of restricted motion. DNA damage significantly enhances the mobility of Brca2 molecules in the S/G2 phases of the cell cycle, via signaling through damage-activated protein kinases. Brca2 mobilization is accompanied by increased binding within the nucleoplasm to its cargo, the Rad51 recombinase, measured by fluorescence cross-correlation spectroscopy. Together, these results suggest that DNA breakage triggers the redistribution of soluble nucleoplasmic Brca2 molecules from a state of restricted diffusion, into a mobile fraction available for Rad51 binding. Our findings identify signal-regulated changes in nucleoplasmic protein diffusion as a means to control biochemical reactions in the cell nucleus.
机译：人们很少了解如何通过核反应物中蛋白质反应物的扩散和利用来控制导致DNA损伤修复的生化反应。在这里，我们使用基因定位技术以功能增强的绿色荧光蛋白（EGFP）标记形式取代Brca2（DNA修复所必需的癌症抑制蛋白），然后通过荧光相关光谱法测量Brca2-EGFP在活细胞核中的扩散暴露于DNA断裂。在损坏之前，核质的Brca2分子表现出复杂的迁移状态，在低于fL的观察体积内具有较长的停留时间，表明运动受限。 DNA损伤通过损伤激活蛋白激酶的信号传导，大大增强了Brca2分子在细胞周期S / G2期的迁移率。 Brca2动员伴随着核内与其货物Rad51重组酶的结合增加，通过荧光互相关光谱法测量。总之，这些结果表明，DNA断裂触发了可溶性核质Brca2分子从受限扩散状态到可用于Rad51结合的可移动部分的重新分布。我们的发现确定了信号调节核质蛋白扩散的变化，作为控制细胞核中生化反应的一种手段。

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    Anand D. Jeyasekharan; Nabieh Ayoub; Robert Mahen; Jonas Ries; Alessandro Esposito; Eeson Rajendra; Hiroyoshi Hattori; Rajan P. Kulkarni; Ashok R. Venkitaraman;

  • 作者单位

    The Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Cambridge CB2 OXZ, United Kingdom;

    The Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Cambridge CB2 OXZ, United Kingdom;

    The Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Cambridge CB2 OXZ, United Kingdom;

    Eidgenoessiche Technische Hochschule Zurich, Laboratory of Physical Chemistry, CH-8093 Zurich, Switzerland;

    The Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Cambridge CB2 OXZ, United Kingdom;

    The Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Cambridge CB2 OXZ, United Kingdom;

    The Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Cambridge CB2 OXZ, United Kingdom;

    The Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Cambridge CB2 OXZ, United Kingdom;

    The Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Cambridge CB2 OXZ, United Kingdom;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    DNA damage response; protein dynamics; fluorescence spectroscopy; single-molecule imaging;

    机译：DNA损伤反应;蛋白质动力学;荧光光谱;单分子成像;

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