Lukas Claudia, Melander Fredrik, Stucki Manuel, Falck Jacob, Bekker-Jensen Simon, Goldberg Michal, Lerenthal Yaniv, Jackson Stephen P, Bartek Jiri, Lukas Jiri
Danish Cancer Society, Institute of Cancer Biology, Copenhagen, Denmark.
EMBO J. 2004 Jul 7;23(13):2674-83. doi: 10.1038/sj.emboj.7600269. Epub 2004 Jun 17.
Mdc1/NFBD1 controls cellular responses to DNA damage, in part via interacting with the Mre11-Rad50-Nbs1 complex that is involved in the recognition, signalling, and repair of DNA double-strand breaks (DSBs). Here, we show that in live human cells, the transient interaction of Nbs1 with DSBs and its phosphorylation by ATM are Mdc1-independent. However, ablation of Mdc1 by siRNA or mutation of the Nbs1's FHA domain required for Mdc1 binding reduced the affinity of Nbs1 for DSB-flanking chromatin and caused aberrant pan-nuclear dispersal of Nbs1. This occurred despite normal phosphorylation of H2AX, indicating that lack of Mdc1 does not impair this DSB-induced chromatin change, but rather precludes the sustained engagement of Nbs1 with these regions. Mdc1 (but not Nbs1) became partially immobilized to chromatin after DSB generation, and siRNA-mediated depletion of H2AX prevented such relocalization of Mdc1 and uncoupled Nbs1 from DSB-flanking chromatin. Our data suggest that Mdc1 functions as an H2AX-dependent interaction platform enabling a switch from transient, Mdc1-independent recruitment of Nbs1 to DSBs towards sustained, Mdc1-dependent interactions with the surrounding chromosomal microenvironment.
Mdc1/NFBD1部分通过与参与DNA双链断裂（DSB）识别、信号传导和修复的Mre11-Rad50-Nbs1复合物相互作用来控制细胞对DNA损伤的反应。在此，我们表明在活的人类细胞中，Nbs1与DSB的瞬时相互作用及其被ATM磷酸化不依赖于Mdc1。然而，通过siRNA敲除Mdc1或突变Mdc1结合所需的Nbs1的FHA结构域会降低Nbs1对DSB侧翼染色质的亲和力，并导致Nbs1异常的全核分散。尽管H2AX正常磷酸化，但仍会出现这种情况，这表明缺乏Mdc1不会损害这种DSB诱导的染色质变化，而是排除了Nbs1与这些区域的持续结合。DSB产生后，Mdc1（而非Nbs1）会部分固定在染色质上，并且siRNA介导的H2AX缺失会阻止Mdc1的这种重新定位，并使Nbs1与DSB侧翼染色质解偶联。我们的数据表明，Mdc1作为一个依赖于H2AX的相互作用平台，能够实现从Nbs1不依赖于Mdc1的瞬时募集到DSB向与周围染色体微环境的持续、依赖于Mdc1的相互作用的转变。
