Calsou P, Frit P, Humbert O, Muller C, Chen D J, Salles B
Institut de Pharmacologie et de Biologie Structurale, CNRS UPR 9062, 205 route de Narbonne, F-31077 Toulouse Cedex, France.
J Biol Chem. 1999 Mar 19;274(12):7848-56. doi: 10.1074/jbc.274.12.7848.
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.
DNA依赖蛋白激酶（DNA-PK）是哺乳动物细胞双链断裂修复所必需的。DNA-PK包含异源二聚体Ku和一个460 kDa的丝氨酸/苏氨酸激酶催化亚基（p460）。Ku在体外与DNA末端或DNA螺旋中的其他间断处结合，并能够通过一个不依赖ATP的过程进入DNA分子。从体外实验可以清楚地看出，Ku刺激p460募集到DNA上，并激活对附近DNA结合蛋白底物的激酶活性。在此，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，虽然在没有p460亚基的情况下Ku可以从自由末端进入DNA，但当整个Ku/p460组装在DNA末端时，激酶活性是Ku沿DNA螺旋转运所必需的。当激酶活性受损时，包括Ku和p460的DNA-PK在DNA末端被阻断，并通过DNA聚合、降解或连接阻止其加工。DNA-PK催化活性对Ku进入DNA的控制可能代表了对DNA末端DNA交易的重要调控。
