Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes
Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes
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DOI：
10.1126/science.1083430
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发表时间：
2003-06-06
期刊：
影响因子：
56.9
通讯作者：
Elledge, SJ
中科院分区：
文献类型：
--
作者：
Zou, L;Elledge, SJ
ATR（共济失调-毛细血管扩张突变和Rad 3相关）ATRIP（ATR相互作用蛋白）蛋白激酶复合物的功能对于细胞对复制应激和DNA损伤的反应至关重要。在这里，我们表明，复制蛋白A（RPA），与单链DNA（ssDNA）相关联的蛋白质复合物，需要招募ATR的DNA损伤位点和ATR介导的Chk 1在人类细胞中的激活。在体外，RPA刺激ATRIP与ssDNA的结合。ATRIP与RPA包被的ssDNA的结合使得ATR-ATRIP复合物能够与DNA缔合并刺激与DNA结合的Rad 17蛋白的磷酸化。此外，ATRIP的芽殖酵母同源物Ddc 2以RPA依赖的方式被特异性地募集到双链DNA断裂。RPA的检查点缺陷突变体rfa 1-t11在体内和体外都不能将Ddc 2募集到ssDNA中。我们的数据表明，RPA包被的ssDNA是在招募ATR-ATRIP复合物的DNA损伤位点的关键结构，并促进其识别磷酸化底物和启动检查点信号。
The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related) ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.