Mari Pierre-Olivier, Florea Bogdan I, Persengiev Stephan P, Verkaik Nicole S, Brüggenwirth Hennie T, Modesti Mauro, Giglia-Mari Giuseppina, Bezstarosti Karel, Demmers Jeroen A A, Luider Theo M, Houtsmuller Adriaan B, van Gent Dik C
Department of Biochemistry, Center for Biomics, Erasmus MC, University Medical Center, 3000 CA Rotterdam, The Netherlands.
Proc Natl Acad Sci U S A. 2006 Dec 5;103(49):18597-602. doi: 10.1073/pnas.0609061103. Epub 2006 Nov 21.
DNA double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends were in dynamic equilibrium with Ku70/80 in solution, showing that NHEJ complex assembly is reversible. Accumulation of XRCC4/ligase IV on DSBs depended on the presence of Ku70/80, but not DNA-PK(CS). We detected a direct interaction between Ku70 and XRCC4 that could explain these requirements. Our results suggest that this assembly constitutes the core of the NHEJ reaction and that XRCC4 may serve as a flexible tether between Ku70/80 and ligase IV.
通过非同源末端连接（NHEJ）进行的DNA双链断裂（DSB）修复需要多种蛋白质在DNA末端组装。尽管生化研究已经阐明了NHEJ反应机制的几个方面，但对于活细胞中的NHEJ了解较少，主要是因为无法在DNA损伤处可视化NHEJ修复蛋白。在此，我们提供证据表明，脉冲近红外激光可以产生DSB，而细胞核没有任何可见变化，并且我们表明NHEJ蛋白在照射区域积累。DSB和Ku积累水平随时间降低，表明这种方法使我们能够在体内研究DNA修复动力学。值得注意的是，DNA末端的Ku异二聚体与溶液中的Ku70/80处于动态平衡，表明NHEJ复合体组装是可逆的。DSB上XRCC4/连接酶IV的积累取决于Ku70/80的存在，而不是DNA-PK（CS）。我们检测到Ku70与XRCC4之间存在直接相互作用，这可以解释这些需求。我们的结果表明，这种组装构成了NHEJ反应的核心，并且XRCC4可能作为Ku70/80与连接酶IV之间的灵活纽带。
