West R B, Yaneva M, Lieber M R
Departments of Pathology and of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles, California 90033, USA.
Mol Cell Biol. 1998 Oct;18(10):5908-20. doi: 10.1128/MCB.18.10.5908.
DNA-dependent protein kinase (DNA-PK) is the only eukaryotic protein kinase known to be specifically activated by double-stranded DNA (dsDNA) termini, accounting for its importance in repair of dsDNA breaks and its role in physiologic processes involving dsDNA breaks, such as V(D)J recombination. In this study we conducted kinase and binding analyses using DNA-PK on DNA termini of various lengths in the presence and absence of Ku. We confirmed our previous observations that DNA-PK can bind DNA termini in the absence of Ku, and we determined rate constants for binding. However, in the presence of Ku, DNA-PK can assume either a productive or a nonproductive configuration, depending on the length of the DNA terminus. For dsDNA greater than 26 bp, the productive mode is achieved and Ku increases the affinity of the DNA-PK for the Ku:DNA complex. The change in affinity is achieved by increases in both the kinetic association rate and reduction in the kinetic dissociation rate. For dsDNA smaller than 26 bp, the nonproductive mode, in which DNA-PK is bound to Ku:DNA but is inactive as a kinase, is assumed. Both the productive and nonproductive configurations are likely to be of physiologic importance, depending on the distance of the dsDNA break site to other protein complexes, such as nucleosomes.
DNA依赖性蛋白激酶（DNA-PK）是已知唯一能被双链DNA（dsDNA）末端特异性激活的真核蛋白激酶，这说明了它在dsDNA断裂修复中的重要性以及在涉及dsDNA断裂的生理过程（如V(D)J重组）中的作用。在本研究中，我们在有或没有Ku存在的情况下，使用DNA-PK对各种长度的DNA末端进行了激酶和结合分析。我们证实了之前的观察结果，即DNA-PK在没有Ku的情况下可以结合DNA末端，并确定了结合的速率常数。然而，在有Ku存在的情况下，DNA-PK可以呈现出有效或无效的构型，这取决于DNA末端的长度。对于大于26 bp的dsDNA，会实现有效模式，并且Ku会增加DNA-PK对Ku:DNA复合物的亲和力。亲和力的变化是通过动力学缔合速率的增加和动力学解离速率的降低来实现的。对于小于26 bp的dsDNA，会呈现无效模式，即DNA-PK与Ku:DNA结合但作为激酶无活性。有效和无效构型可能都具有生理重要性，这取决于dsDNA断裂位点与其他蛋白质复合物（如核小体）的距离。
