Song K, Jung Y, Jung D, Lee I
Department of Biochemistry, University of Ulsan College of Medicine, Seoul 138-736, Korea.
J Biol Chem. 2001 Mar 16;276(11):8321-7. doi: 10.1074/jbc.M008779200. Epub 2000 Dec 8.
Ku is involved in the metabolism of DNA ends, DNA repair, and the maintenance of telomeres. It consists of a heterodimer of 70- and 80-kDa subunits. Recently we have demonstrated that Ku70 interacted with TRF2, a mammalian telomere-binding protein. Using the same yeast two-hybrid screening system, we now show that Ku70 also interacts with heterochromatin protein 1alpha (HP1alpha), a protein known to be associated with telomeres as well as heterochromatin. HP1 is a suppressor of the position effect variegation in Drosophila and acts as a transcriptional suppressor in mammalian cells. The interaction with Ku70 in the two-hybrid system was confirmed by a glutathione S-transferase pull-down study using bacterial recombinant proteins in vitro. The interaction was also reproduced in vivo in HeLa cells, where endogenous Ku70 coimmunoprecipitated with HP1alpha. This interaction was more effective in acidic pH and weakened considerably as the pH of the reaction buffer was elevated up to 7.5. Ku80 did not interact with HP1alpha directly. The interaction domains of Ku70 and HP1alpha included the Leu-Ser repeat (amino acids 200-385) and the chromo shadow domain, respectively. Ku70 was largely colocalized with transfected HP1alpha but not with a C-terminal deletion mutant, HP1alpha(Delta)C. In contrast to HP1alpha, Ku70 did not repress transcriptional activity of the reporter gene when tethered to DNA after transfection to mammalian cells. The implication of this interaction is discussed.
Ku参与DNA末端的代谢、DNA修复以及端粒的维持。它由一个70 kDa和一个80 kDa亚基组成的异源二聚体。最近我们证明了Ku70与TRF2相互作用，TRF2是一种哺乳动物端粒结合蛋白。利用相同的酵母双杂交筛选系统，我们现在表明Ku70还与异染色质蛋白1α（HP1α）相互作用，HP1α是一种已知与端粒以及异染色质相关的蛋白。HP1是果蝇位置效应斑驳的抑制因子，在哺乳动物细胞中作为转录抑制因子发挥作用。通过使用细菌重组蛋白的体外谷胱甘肽S-转移酶下拉实验，证实了在双杂交系统中Ku70与HP1α的相互作用。在HeLa细胞中也在体内重现了这种相互作用，内源性Ku70与HP1α共免疫沉淀。这种相互作用在酸性pH下更有效，随着反应缓冲液的pH升高到7.5，相互作用显著减弱。Ku80不直接与HP1α相互作用。Ku70和HP1α的相互作用结构域分别包括亮氨酸-丝氨酸重复序列（氨基酸200 - 385）和染色质影域。Ku70在很大程度上与转染的HP1α共定位，但不与C末端缺失突变体HP1α(Delta)C共定位。与HP1α相反，转染到哺乳动物细胞后，当Ku70与DNA相连时，它不会抑制报告基因的转录活性。讨论了这种相互作用的意义。
