Lee Joon, Kumagai Akiko, Dunphy William G
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
J Biol Chem. 2007 Sep 21;282(38):28036-44. doi: 10.1074/jbc.M704635200. Epub 2007 Jul 18.
TopBP1 serves as an activator of the ATR-ATRIP complex in response to the presence of incompletely replicated or damaged DNA. This process involves binding of ATR to the ATR-activating domain of TopBP1, which is located between BRCT domains VI and VII. TopBP1 displays increased binding to ATR-ATRIP in Xenopus egg extracts containing checkpoint-inducing DNA templates. We show that an N-terminal region of TopBP1 containing BRCT repeats I-II is essential for this checkpoint-stimulated binding of TopBP1 to ATR-ATRIP. The BRCT I-II region of TopBP1 also binds specifically to the Rad9-Hus1-Rad1 (9-1-1) complex in Xenopus egg extracts. This binding occurs via the C-terminal domain of Rad9 and depends upon phosphorylation of its Ser-373 residue. Egg extracts containing either a mutant of TopBP1 lacking the BRCT I-II repeats or a mutant of Rad9 with an alanine substitution at Ser-373 are defective in checkpoint regulation. Furthermore, an isolated C-terminal fragment from Rad9 is an effective inhibitor of checkpoint signaling in egg extracts. These findings suggest that interaction of the 9-1-1 complex with the BRCT I-II region of TopBP1 is necessary for binding of ATR-ATRIP to the ATR-activating domain of TopBP1 and the ensuing activation of ATR.
在存在未完全复制或受损DNA的情况下，TopBP1作为ATR-ATRIP复合物的激活剂。这一过程涉及ATR与TopBP1的ATR激活结构域结合，该结构域位于BRCT结构域VI和VII之间。在含有诱导检查点的DNA模板的非洲爪蟾卵提取物中，TopBP1与ATR-ATRIP的结合增加。我们发现，TopBP1包含BRCT重复序列I-II的N端区域对于这种检查点刺激的TopBP1与ATR-ATRIP的结合至关重要。TopBP1的BRCT I-II区域在非洲爪蟾卵提取物中也特异性结合Rad9-Hus1-Rad1（9-1-1）复合物。这种结合通过Rad9的C端结构域发生，并依赖于其Ser-373残基的磷酸化。含有缺乏BRCT I-II重复序列的TopBP1突变体或Ser-373处有丙氨酸替代的Rad9突变体的卵提取物在检查点调节方面存在缺陷。此外，从Rad9分离的C端片段是卵提取物中检查点信号传导的有效抑制剂。这些发现表明，9-1-1复合物与TopBP1的BRCT I-II区域的相互作用对于ATR-ATRIP与TopBP1的ATR激活结构域的结合以及随后的ATR激活是必要的。
