DNA-dependent protein kinase: DNA binding and activation in the absence of Ku
DNA-dependent protein kinase: DNA binding and activation in the absence of Ku
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DOI：
10.1073/pnas.95.2.525
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发表时间：
1998-01-20
影响因子：
11.1
通讯作者：
Chu, G
中科院分区：
文献类型：
--
作者：
Hammarsten, O;Chu, G
在哺乳动物细胞中，双链断裂修复和V（D）J重组需要DNA依赖性蛋白激酶（DNA-PK），一种由DNA激活的丝氨酸/苏氨酸激酶。DNA-PK由一个460-kDa亚基（p460）和一个异二聚体亚基（Ku）组成，p460含有一个推定的激酶结构域，Ku与双链DNA末端结合。为了进一步研究这一点，将p460和Ku分别纯化至均一，令人惊讶的是，p460能够在没有Ku的情况下与DNA结合。p460与双链DNA末端的结合是盐不稳定的，并且可以被单链或超螺旋DNA破坏，在低盐条件下，p460与DNA末端结合，激酶被激活。在抑制p460结合的高盐条件下，激酶的激活需要Ku的加入。显著地，当DNA长度减少到22 bp时，Ku与p460竞争DNA结合并抑制激酶活性，这些数据表明，p460是一种独立的激酶，通过与双磷酸酶直接相互作用而被激活。Ku的作用是稳定p460与DNA末端的结合。
In mammalian cells, double-strand break repair and V(D)J recombination require DNA-dependent protein kinase (DNA-PK), a serine/threonine kinase that is activated by DNA. DNA-PK consists of a 460-kDa subunit (p460) that contains a putative kinase domain and a heterodimeric subunit (Ku) that binds to double-stranded DNA ends. Previous reports suggested that the activation of DNA-PK requires the binding of Ku to DNA, To investigate this further, p460 and Ku were purified separately to homogeneity, Surprisingly, p460 was capable of binding to DNA in the absence of Ku, The binding of p460 to double-stranded DNA ends was salt-labile and could be disrupted by single-stranded or supercoiled DNA, properties distinct from the binding of Ku to DNA, Under low salt conditions, which permitted the binding of p460 to DNA ends, the kinase was activated. Under higher salt conditions, which inhibited the binding of p460, activation of the kinase required the addition of Ku, Significantly, when the length of DNA decreased to 22 bp, Ku competed with p460 for DNA binding and inhibited kinase activity, These data demonstrate that p460 is a self-contained kinase that is activated by direct interaction with double-stranded DNA and that the role of Ku is to stabilize the binding of p460 to DNA ends.