Evidence for an inositol hexakisphosphate-dependent role for Ku in mammalian nonhomologous end joining that is independent of its role in the DNA-dependent protein kinase.
Evidence for an inositol hexakisphosphate-dependent role for Ku in mammalian nonhomologous end joining that is independent of its role in the DNA-dependent protein kinase.
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DOI：
10.1093/nar/gkn572
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发表时间：
2008-10
影响因子：
14.9
通讯作者：
Hanakahi LA
中科院分区：
文献类型：
--
作者：
Cheung JC;Salerno B;Hanakahi LA
非同源末端连接（NHEJ）是DNA双链断裂（DSB）修复的重要途径，在维持哺乳动物细胞基因组稳定性方面起着关键作用。虽然Ku 70/80（Ku）在NHEJ中作为DNA依赖性蛋白激酶（DNA-PK）的一部分发挥作用，但遗传学证据表明Ku在NHEJ中的作用超出了其参与DNA-PK的范围。肌醇六磷酸（IP 6）以前被发现刺激NHEJ在体外和Ku被确定为IP 6结合因子。通过突变分析，我们确定了一个二分IP 6结合位点Ku和产生的IP 6结合突变体，范围从1.22%至58.48%的野生型结合。值得注意的是，这些Ku IP 6结合突变体在体外参与NHEJ受损，我们观察到IP 6结合和NHEJ之间的正相关性。Ku IP 6结合突变体是与野生型Ku一样结合DNA并激活DNA-PK的功能分离突变体。我们的观察确定了一个迄今未定义的IP 6结合位点Ku和显示，这种相互作用是重要的DSB修复NHEJ在体外。此外，这些数据表明，除了暴露的DNA末端的结合和DNA-PK的活化，Ku异二聚体在哺乳动物NHEJ中起作用，其通过IP 6的结合来调节。
Nonhomologous end-joining (NHEJ) is an important pathway for the repair of DNA double-strand breaks (DSBs) and plays a critical role in maintaining genomic stability in mammalian cells. While Ku70/80 (Ku) functions in NHEJ as part of the DNA-dependent protein kinase (DNA-PK), genetic evidence indicates that the role of Ku in NHEJ goes beyond its participation in DNA-PK. Inositol hexakisphosphate (IP6) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP6-binding factor. Through mutational analysis, we identified a bipartite IP6-binding site in Ku and generated IP6-binding mutants that ranged from 1.22% to 58.48% of wild-type binding. Significantly, these Ku IP6-binding mutants were impaired for participation in NHEJ in vitro and we observed a positive correlation between IP6 binding and NHEJ. Ku IP6-binding mutants were separation-of-function mutants that bound DNA and activated DNA-PK as well as wild-type Ku. Our observations identify a hitherto undefined IP6-binding site in Ku and show that this interaction is important for DSB repair by NHEJ in vitro. Moreover, these data indicate that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian NHEJ that is regulated by binding of IP6.
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