RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling
RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling
复制标题
DOI：
10.4161/15384101.2014.967076
复制
发表时间：
2015-01-01
期刊：
影响因子：
4.3
通讯作者：
Sancar, Aziz
中科院分区：
文献类型：
--
作者：
Lindsey-Boltz, Laura A.;Kemp, Michael G.;Sancar, Aziz
ATR-Chk1 信号通路介导细胞对 DNA 损伤和复制应激的反应，由许多在真核生物中保守的核心因子组成。然而，人类和其他高等真核物种拥有参与该信号网络调节的其他因素，但尚未得到广泛研究。在这里，我们展示了RHINO（Rad9、Rad1、Hus1相互作用的核孤儿）通过与9-1-1检查点钳的Rad9和Rad1亚基以及ATR激酶激活剂TopBP1直接相互作用，即使在没有DNA损伤剂治疗的情况下，也可以与人类细胞中的9-1-1检查点钳和TopBP1形成复合物。 RHINO 与 9-1-1 的相互作用具有足够的亲和力，可以在体外纯化稳定的异四聚体 RHINO-Rad9-Hus1-Rad1 复合物。在人类细胞中，在没有 DNA 损伤的情况下，RHINO 的一部分定位于染色质，并且这种关联在紫外线照射后会增强。此外，我们发现 Lac 阻遏蛋白 (LacR)-RHINO 融合蛋白与哺乳动物细胞染色质中 LacO 重复序列的结合会以 Rad9 和 Claspin 依赖性方式诱导 Chk1 磷酸化。最后，RHINO 的缺失部分消除了 UV 照射后的 ATR-Chk1 信号传导，但不影响 9-1-1 钳与 TopBP1 的相互作用或 9-1-1 在染色质上的负载。我们得出结论，RHINO 是哺乳动物细胞中 ATR-Chk1 信号传导的真正调节者。
The ATR-Chk1 signaling pathway mediates cellular responses to DNA damage and replication stress and is composed of a number of core factors that are conserved throughout eukaryotic organisms. However, humans and other higher eukaryotic species possess additional factors that are implicated in the regulation of this signaling network but that have not been extensively studied. Here we show that RHINO (for Rad9, Rad1, Hus1 interacting nuclear orphan) forms complexes with both the 9-1-1 checkpoint clamp and TopBP1 in human cells even in the absence of treatments with DNA damaging agents via direct interactions with the Rad9 and Rad1 subunits of the 9-1-1 checkpoint clamp and with the ATR kinase activator TopBP1. The interaction of RHINO with 9-1-1 was of sufficient affinity to allow for the purification of a stable heterotetrameric RHINO-Rad9-Hus1-Rad1 complex in vitro. In human cells, a portion of RHINO localizes to chromatin in the absence of DNA damage, and this association is enriched following UV irradiation. Furthermore, we find that the tethering of a Lac Repressor (LacR)-RHINO fusion protein to LacO repeats in chromatin of mammalian cells induces Chk1 phosphorylation in a Rad9- and Claspin-dependent manner. Lastly, the loss of RHINO partially abrogates ATR-Chk1 signaling following UV irradiation without impacting the interaction of the 9-1-1 clamp with TopBP1 or the loading of 9-1-1 onto chromatin. We conclude that RHINO is a bona fide regulator of ATR-Chk1 signaling in mammalian cells.