Structure-function analysis of TOPBP1's role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system.
Structure-function analysis of TOPBP1's role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system.
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DOI：
10.1038/s41598-020-80626-1
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发表时间：
2021-01-11
影响因子：
4.6
通讯作者：
Michael WM
中科院分区：
文献类型：
--
作者：
Montales K;Kim A;Ruis K;Michael WM
蛋白激酶ATR在DNA双链断裂位点被激活，在促进DNA末端切除和调节细胞周期进程中起重要作用。TOPBP1是一种含有多个BRCT重复序列的蛋白质，可在DSB处激活ATR。在这里，我们已经开发了一个实验工具，DMAX系统，研究TOPBP1介导的ATR信号的生化机制。DMAX将简单的线性dsDNA分子与爪蟾卵提取物结合，并导致生理相关的DSB诱导的ATR活化。我们发现，DNA的5000个核苷酸，在飞摩尔浓度，有效地激活ATR在这个系统中。通过结合TOPBP 1点突变体的免疫耗尽和加回，我们使用DMAX来确定TOPBP 1的九个BRCT结构域中的哪些是将TOPBP 1募集到DSB所需的，以及哪些结构域是ATR介导的CHK 1磷酸化所需的。我们发现，BRCT1和BRCT7是重要的招聘和BRCT5功能下游的招聘，以促进ATR介导的磷酸化CHK1。我们还表明，BRCT7起着第二个作用，独立的招聘，在促进ATR信号。这些发现为ATR生物学提供了新的研究工具和新的见解。
The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1’s nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.
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影响因子：
16
作者：
Gong, Zihua;Kim, Ja-Eun;Leung, Charles Chung Yun;Glover, J. N. Mark;Chen, Junjie
通讯作者：
Chen, Junjie
影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
Di Virgilio, Michela;Ying, Carol Y.;Gautier, Jean
通讯作者：
Gautier, Jean
影响因子：
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作者：
Lupardus, PJ;Cimprich, KA
通讯作者：
Cimprich, KA
影响因子：
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作者：
Lee, JH;Paull, TT
通讯作者：
Paull, TT