Subnuclear localization of Ku protein: Functional association with RNA polymerase II elongation sites
Subnuclear localization of Ku protein: Functional association with RNA polymerase II elongation sites
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DOI：
10.1128/mcb.22.22.8088-8099.2002
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发表时间：
2002-11-01
影响因子：
5.3
通讯作者：
Dynan, WS
中科院分区：
文献类型：
--
作者：
Mo, XM;Dynan, WS
Ku是一种丰富的核蛋白，在DNA双链断裂的修复中具有重要功能。各种观察表明，Ku也与细胞转录机制相互作用，尽管这种相互作用的机制和意义还不清楚。在本研究中，我们研究了Ku在正常生长的人类细胞的亚核分布，通过使用共聚焦显微镜，染色质免疫沉淀和蛋白质免疫沉淀。所有这三种方法都表明Ku与RNA聚合酶11（RNAP II）延伸位点的关联。这种关联独立于DNA依赖性蛋白激酶催化亚基发生，并且具有高度选择性。与RNAP 11的起始同种型或与一般转录起始因子没有可检测的关联。在体外蛋白质-蛋白质相互作用的测定表明，协会Ku与延伸蛋白介导的，在一定程度上，由一个离散的C-末端结构域的Ku 80亚基。这种相互作用与显性负突变体的功能中断抑制转录在体外和体内，并抑制细胞生长。这些结果表明，Ku与转录位点的关联对于维持全局转录水平是重要的。将双链断裂修复蛋白拴系到限定的硫核结构也可以有利于维持基因组稳定性。
Ku is an abundant nuclear protein with an essential function in the repair of DNA double-strand breaks. Various observations suggest that Ku also interacts with the cellular transcription machinery, although the mechanism and significance of this interaction are not well understood. In the present study, we investigated the subnuclear distribution of Ku in normally growing human cells by using confocal microscopy, chromatin immunoprecipitation, and protein immunoprecipitation. All three approaches indicated association of Ku with RNA polymerase 11 (RNAP II) elongation sites. This association occurred independently of the DNA-dependent protein kinase catalytic subunit and was highly selective. There was no detectable association with the initiating isoform of RNAP 11 or with the general transcription initiation factors. In vitro protein-protein interaction assays demonstrated that the association of Ku with elongation proteins is mediated, in part, by a discrete C-terminal domain in the Ku80 subunit. Functional disruption of this interaction with a dominant-negative mutant inhibited transcription in vitro and in vivo and suppressed cell growth. These results suggest that association of Ku with transcription sites is important for maintenance of global transcription levels. Tethering of double-strand break repair proteins to defined sulmuclear structures may also be advantageous in maintenance of genome stability.