Isolation of Ku70-binding proteins (KUBs)
Isolation of Ku70-binding proteins (KUBs)
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DOI：
10.1093/nar/27.10.2165
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发表时间：
1999-05-15
影响因子：
14.9
通讯作者：
Boothman, DA
中科院分区：
文献类型：
--
作者：
Yang, CR;Yeh, SY;Boothman, DA
DNA依赖的蛋白激酶(DNA-PK)在通过非同源末端连接重新封闭DNA双链断裂中起着关键作用。除了DNA-PK、XRCC4和DNA连接酶IV外，其他在修复途径中起作用的蛋白(S)尚不清楚；DNA-PK包括一个催化亚基(DNA-PKcs)和一个DNA结合亚基(Ku70和Ku80)。我们用酵母双杂交的方法分离了Ku70结合蛋白(KUB1-KUB4)。序列分析表明，KUB1是载脂蛋白J(ApoJ)，也被称为X射线诱导转录本8(XIP8)，睾酮抑制的前列腺信息2(TRPM-5)和聚集素。KUB2为Ku80，KUB3和KUB4未知，转录本为10kb。免疫共沉淀分析证实apoJ/XIP8或KUB3与Ku70在MCF-7：WS8乳腺癌和IMR-90正常肺成纤维细胞中的相互作用。远距离Western分析证实了apoJ/XIP8与Ku70的相互作用，在MCF-7细胞中稳定过表达apoJ/XIP8：WS8导致Ku70/Ku80 DNA末端结合降低，并被apoJ/XIP8单抗恢复。ApoJ/XIP8在电离辐射抗性/敏感性中的作用正在研究中。
DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80), We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-5) and clusterin. KUB2 is Ku80, KUB3 and KUB4 are unknown, >10 kb transcripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses, Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.