The Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, United Kingdom.
PLoS One. 2013 May 2;8(5):e61893. doi: 10.1371/journal.pone.0061893. Print 2013.
A-type lamins encoded by LMNA form a structural fibrillar meshwork within the mammalian nucleus. How this nuclear organization may influence the execution of biological processes involving DNA transactions remains unclear. Here, we characterize changes in the dynamics and biochemical interactions of lamin A/C after DNA damage. We find that DNA breakage reduces the mobility of nucleoplasmic GFP-lamin A throughout the nucleus as measured by dynamic fluorescence imaging and spectroscopy in living cells, suggestive of incorporation into stable macromolecular complexes, but does not induce the focal accumulation of GFP-lamin A at damage sites. Using a proximity ligation assay and biochemical analyses, we show that lamin A engages chromatin via histone H2AX and its phosphorylated form (γH2AX) induced by DNA damage, and that these interactions are enhanced after DNA damage. Finally, we use three-dimensional time-lapse imaging to show that LMNA inactivation significantly reduces the positional stability of DNA repair foci in living cells. This defect is partially rescued by the stable expression of GFP-lamin A. Thus collectively, our findings suggest that the dynamic structural meshwork formed by A-type lamins anchors sites of DNA repair in mammalian nuclei, providing fresh insight into the control of DNA transactions by nuclear structural organization.
由 LMNA 编码的 A 型核纤层蛋白在哺乳动物核内形成结构纤维状网格。这种核组织如何影响涉及 DNA 交易的生物过程的执行尚不清楚。在这里，我们描述了 DNA 损伤后核纤层 A/C 的动力学和生化相互作用的变化。我们发现，DNA 断裂会降低活细胞中 GFP-核纤层 A 在整个核内的流动性，这可以通过动态荧光成像和光谱法来测量，提示其掺入稳定的大分子复合物，但不会诱导 GFP-核纤层 A 在损伤部位的局灶性积累。使用邻近连接测定和生化分析，我们表明核纤层 A 通过 DNA 损伤诱导的组蛋白 H2AX 及其磷酸化形式 (γH2AX) 与染色质结合，并且这些相互作用在 DNA 损伤后增强。最后，我们使用三维延时成像来显示 LMNA 失活会显著降低活细胞中 DNA 修复焦点的位置稳定性。通过稳定表达 GFP-核纤层 A 可以部分挽救该缺陷。因此，我们的研究结果表明，A 型核纤层蛋白形成的动态结构网格将 DNA 修复位点锚定在哺乳动物核内，为核结构组织对 DNA 交易的控制提供了新的见解。
