Division of Biology, California Institute of Technology, Pasadena, CA, USA.
Cell Cycle. 2011 Feb 1;10(3):469-80. doi: 10.4161/cc.10.3.14711.
In the presence of double-stranded DNA breaks (DSBs), the activation of ATR is achieved by the ability of ATM to phosphorylate TopBP1 on serine 1131, which leads to an enhancement of the interaction between ATR and TopBP1. In Xenopus egg extracts, the Mre11-Rad50-Nbs1 (MRN) complex is additionally required to bridge ATM and TopBP1 together. In this report, we show that CtIP, which is recruited to DSB-containing chromatin, interacts with both TopBP1 and Nbs1 in a damage-dependent manner. An N-terminal region containing the first two BRCT repeats of TopBP1 is essential for the interaction with CtIP. Furthermore, two distinct regions in the N-terminus of CtIP participate in establishing the association between CtIP and TopBP1. The first region includes two adjacent putative ATM/ATR phosphorylation sites on serines 273 and 275. Secondly, binding is diminished when an MRN-binding region spanning residues 25-48 is deleted, indicative of a role for the MRN complex in mediating this interaction. This was further evidenced by a decrease in the interaction between CtIP and TopBP1 in Nbs1-depleted extracts and a reciprocal decrease in the binding of Nbs1 to TopBP1 in the absence of CtIP, suggestive of the formation of a complex containing CtIP, TopBP1, and the MRN complex. When CtIP is immunodepleted from egg extracts, the activation of the response to DSBs is compromised and the levels of ATR, TopBP1, and Nbs1 on damaged chromatin are reduced. Thus, CtIP interacts with TopBP1 in a damage-stimulated, MRN-dependent manner during the activation of ATR in response to DSBs.
在双链 DNA 断裂 (DSB) 的情况下，ATR 的激活是通过 ATM 磷酸化 TopBP1 丝氨酸 1131 来实现的，这导致 ATR 和 TopBP1 之间的相互作用增强。在非洲爪蟾卵提取物中，Mre11-Rad50-Nbs1 (MRN) 复合物还需要桥接 ATM 和 TopBP1 在一起。在本报告中，我们表明，CtIP 被招募到含有 DSB 的染色质中，以损伤依赖性方式与 TopBP1 和 Nbs1 相互作用。TopBP1 的第一个 BRCT 重复的前两个包含 N 端区域对于与 CtIP 的相互作用是必需的。此外，CtIP 的 N 端有两个不同的区域参与建立 CtIP 和 TopBP1 之间的关联。第一个区域包括丝氨酸 273 和 275 上两个相邻的假定 ATM/ATR 磷酸化位点。其次，当跨越残基 25-48 的 MRN 结合区域缺失时，结合减少，表明 MRN 复合物在介导这种相互作用中起作用。这进一步通过 CtIP 和 TopBP1 之间的相互作用在 Nbs1 耗尽提取物中减少以及在没有 CtIP 的情况下 Nbs1 与 TopBP1 的结合减少得到证明，提示形成包含 CtIP、TopBP1 和 MRN 复合物的复合物。当 CtIP 从卵提取物中免疫耗尽时，DSB 反应的激活受损，受损染色质上的 ATR、TopBP1 和 Nbs1 的水平降低。因此，CtIP 以损伤刺激、MRN 依赖的方式与 TopBP1 相互作用，以激活ATR 对 DSB 的反应。
