Yan Shan, Michael W Matthew
The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
J Cell Biol. 2009 Mar 23;184(6):793-804. doi: 10.1083/jcb.200810185. Epub 2009 Mar 16.
TopBP1 and the Rad9-Rad1-Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR-ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-alpha to sites of replication stress. Furthermore, we show that pol-alpha is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-alpha, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.
TopBP1与Rad9-Rad1-Hus1（9-1-1）复合物在停滞的复制叉处激活共济失调毛细血管扩张症突变基因和Rad3相关蛋白（ATR）激酶。ATR通过其结合伴侣ATR相互作用蛋白（ATRIP）被招募到停滞的复制叉处；然而，目前尚不清楚TopBP1和9-1-1是如何被招募的，以便它们能够与ATR-ATRIP结合并启动信号传导。在本研究中，我们利用非洲爪蟾卵提取物来确定加载9-1-1的条件。我们发现，TopBP1是将9-1-1和DNA聚合酶（pol）-α招募到复制应激位点所必需的。此外，我们还表明，加载Rad9也直接需要pol-α。我们的研究确定了一条由TopBP1控制且包括pol-α的组装途径，该途径介导9-1-1复合物加载到停滞的复制叉上。这些发现阐明了DNA上检查点信号复合物组装的早期事件，并确定TopBP1是复制应激的关键传感器。
