Two-Stage Synapsis of DNA Ends during Non-homologous End Joining.
Two-Stage Synapsis of DNA Ends during Non-homologous End Joining.
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DOI：
10.1016/j.molcel.2016.02.010
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发表时间：
2016-03-17
期刊：
影响因子：
16
通讯作者：
Loparo JJ
中科院分区：
文献类型：
--
作者：
Graham TG;Walter JC;Loparo JJ
DNA双链断裂（DSBs）的修复对基因组的稳定性至关重要。人类细胞中最常见的DSB修复机制是非同源末端连接（NHEJ），通过直接连接将断裂的DNA末端重新连接起来。目前尚不清楚NHEJ机制的组成部分如何组装连接DNA末端的突触复合体。在这里，我们使用脊椎动物无细胞提取物中的单分子成像来显示DNA末端的突触发生在至少两个阶段，这两个阶段由不同的NHEJ因子控制。DNA末端最初连接在一个远程复合体中，其形成需要Ku70/80异源二聚体和DNA依赖性蛋白激酶催化亚基（DNA- pkcs）。这需要XLF （XRCC4-LIG4的非催化功能）和DNA-PK活性。这些结果揭示了控制DNA末端对齐的突触复合体的结构转变。我们的方法提供了一种在单分子分辨率下研究生理DNA双链断裂修复的方法。
Repair of DNA double-strand breaks (DSBs) is essential for genomic stability. The most common DSB repair mechanism in human cells, non-homologous end joining (NHEJ), rejoins broken DNA ends by direct ligation. It remains unclear how components of the NHEJ machinery assemble a synaptic complex that bridges DNA ends. Here, we use single-molecule imaging in a vertebrate cell-free extract to show that synapsis of DNA ends occurs in at least two stages that are controlled by different NHEJ factors. DNA ends are initially tethered in a long-range complex whose formation requires the Ku70/80 heterodimer and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The ends are then closely aligned, which requires XLF, a non-catalytic function of XRCC4-LIG4, and DNA-PK activity. These results reveal a structural transition in the synaptic complex that governs alignment of DNA ends. Our approach provides a means of studying physiological DNA double-strand break repair at single-molecule resolution.