Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena 07745, Germany.
Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena 07745, Germany; Faculty of Biology and Pharmacy, Friedrich Schiller University Jena, Jena 07743, Germany.
Cell Rep. 2014 Jan 16;6(1):182-95. doi: 10.1016/j.celrep.2013.12.018. Epub 2014 Jan 2.
The MRN complex (Mre11/Rad50/Nbs1) is important in double-strand break (DSB) recognition, end resection, replication fork stabilization, and ATM and ATR activation. Complete deletion of MRN is incompatible with cell and organism life, presumably due to replication-born DSBs; however, the underlying mechanism remains unknown. We devised a noninvasive high-content assay, termed high-content microscopy-assisted cell-cycle phenotyping (hiMAC), to investigate the fate of cells lacking Nbs1. Surprisingly, deletion of Nbs1 does not kill cells during replication. The primary lesions in Nbs1-deleted cells are replication intermediates that result from defective resolution rather than fork destabilization. These lesions are converted to DSBs in the subsequent G2 phase, which subsequently activate Chk1, delay G2 progression, and lead to chromosome instability. Nbs1-deleted cells establish a DSB equilibrium that permits cell cycling but activates p53, causing G1 and G2 arrest, and cell death. Thus, we identify a physiological role of Nbs1 in the resolution of stalled replication forks.
MRN 复合物（Mre11/Rad50/Nbs1）在双链断裂（DSB）识别、末端切除、复制叉稳定以及 ATM 和 ATR 的激活中起重要作用。MRN 的完全缺失与细胞和生物体的生命不相容，可能是由于复制产生的 DSB；然而，其潜在机制尚不清楚。我们设计了一种非侵入性的高通量分析方法，称为高内涵显微镜辅助细胞周期表型分析（hiMAC），以研究缺乏 Nbs1 的细胞的命运。令人惊讶的是，Nbs1 缺失并不会在复制过程中杀死细胞。Nbs1 缺失细胞中的主要损伤是复制中间体，这是由于缺陷的解决而不是叉不稳定造成的。这些损伤在随后的 G2 期转化为 DSB，随后激活 Chk1，延迟 G2 进程，并导致染色体不稳定。Nbs1 缺失细胞建立 DSB 平衡，允许细胞循环，但激活 p53，导致 G1 和 G2 期阻滞和细胞死亡。因此，我们确定了 Nbs1 在解决停滞复制叉方面的生理作用。
