Xu Xin, Vaithiyalingam Sivaraja, Glick Gloria G, Mordes Daniel A, Chazin Walter J, Cortez David
Department of Biochemistry, Vanderbilt University School of Medicine, 613 Light Hall, 23rd at Pierce Avenue, Nashville, TN 37232, USA.
Mol Cell Biol. 2008 Dec;28(24):7345-53. doi: 10.1128/MCB.01079-08. Epub 2008 Oct 20.
ATR kinase activation requires the recruitment of the ATR-ATRIP and RAD9-HUS1-RAD1 (9-1-1) checkpoint complexes to sites of DNA damage or replication stress. Replication protein A (RPA) bound to single-stranded DNA is at least part of the molecular recognition element that recruits these checkpoint complexes. We have found that the basic cleft of the RPA70 N-terminal oligonucleotide-oligosaccharide fold (OB-fold) domain is a key determinant of checkpoint activation. This protein-protein interaction surface is able to bind several checkpoint proteins, including ATRIP, RAD9, and MRE11. RAD9 binding to RPA is mediated by an acidic peptide within the C-terminal RAD9 tail that has sequence similarity to the primary RPA-binding surface in the checkpoint recruitment domain (CRD) of ATRIP. Mutation of the RAD9 CRD impairs its localization to sites of DNA damage or replication stress without perturbing its ability to form the 9-1-1 complex or bind the ATR activator TopBP1. Disruption of the RAD9-RPA interaction also impairs ATR signaling to CHK1 and causes hypersensitivity to both DNA damage and replication stress. Thus, the basic cleft of the RPA70 N-terminal OB-fold domain binds multiple checkpoint proteins, including RAD9, to promote ATR signaling.
ATR激酶激活需要将ATR-ATRIP和RAD9-HUS1-RAD1（9-1-1）检查点复合物募集到DNA损伤或复制应激位点。与单链DNA结合的复制蛋白A（RPA）至少是募集这些检查点复合物的分子识别元件的一部分。我们发现，RPA70 N端寡核苷酸-寡糖折叠（OB折叠）结构域的碱性裂隙是检查点激活的关键决定因素。这个蛋白质-蛋白质相互作用表面能够结合几种检查点蛋白，包括ATRIP、RAD9和MRE11。RAD9与RPA的结合是由C端RAD9尾巴内的一个酸性肽介导的，该肽与ATRIP检查点募集结构域（CRD）中的主要RPA结合表面具有序列相似性。RAD9 CRD的突变会损害其在DNA损伤或复制应激位点的定位，而不会干扰其形成9-1-1复合物或结合ATR激活剂TopBP1的能力。RAD9-RPA相互作用的破坏也会损害ATR向CHK1的信号传导，并导致对DNA损伤和复制应激的超敏反应。因此，RPA70 N端OB折叠结构域的碱性裂隙结合多种检查点蛋白，包括RAD9，以促进ATR信号传导。
