The DNA-dependent protein kinase catalytic activity regulates DNA end processing by means of Ku entry into DNA
The DNA-dependent protein kinase catalytic activity regulates DNA end processing by means of Ku entry into DNA
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DOI：
10.1074/jbc.274.12.7848
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发表时间：
1999-03-19
影响因子：
4.8
通讯作者：
Salles, B
中科院分区：
文献类型：
--
作者：
Calsou, P;Frit, P;Salles, B
DNA依赖性蛋白激酶（DNA-PK）是哺乳动物细胞双链断裂修复所必需的。DNA-PK含有异二聚体Ku和460-kDa丝氨酸/苏氨酸激酶催化亚基（p460），Ku在体外结合到DNA末端或DNA螺旋中的其他不连续处，并且能够通过ATP非依赖性过程进入DNA分子。从体外实验中可以清楚地看出，Ku刺激p460向DNA的募集，并激活附近的DNA结合蛋白底物的激酶活性。我们在人核细胞提取物中研究了激酶催化活性对Ku与DNA结合的影响。我们证明，尽管Ku在p460亚基缺失时可以从游离端进入DNA，但当整个Ku/p460组装在DNA末端时，Ku沿DNA螺旋沿着转位需要激酶活性。包括Ku和p460的DNA-PK在DNA末端被阻断，并通过DNA聚合、降解或连接阻止它们的加工。通过DNA-PK催化活性控制Ku进入DNA可能代表了DNA末端的DNA交易的重要调节。
The DNA-dependent protein kinase (DNA-PK) is required for double-strand break repair in mammalian cells. DNA-PK contains the heterodimer Ku and a 460-kDa serine/threonine kinase catalytic subunit (p460), Ku binds in vitro to DNA termini or other discontinuities in the DNA helix and is able to enter the DNA molecule by an ATP-independent process. It is clear from in vitro experiments that Ku stimulates the recruitment to DNA of p460 and activates the kinase activity toward DNA-binding protein substrates in the vicinity. Here, we have examined in human nuclear cell extracts the influence of the kinase catalytic activity on Ku binding to DNA, We demonstrate that, although Ku can enter DNA from free ends in the absence of p460 subunit, the kinase activity is required for Ku translocation along the DNA helix when the whole Ku/p460 assembles on DNA termini, When the kinase activity is impaired, DNA-PK including Ku and p460 is blocked at DNA ends and prevents their processing by either DNA polymerization, degradation, or ligation. The control of Ku entry into DNA by DNA-PK catalytic activity potentially represents an important regulation of DNA transactions at DNA termini.