Paillard S, Strauss F
Institut Jacques Monod, Paris, France.
Nucleic Acids Res. 1991 Oct 25;19(20):5619-24. doi: 10.1093/nar/19.20.5619.
Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes. Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.
Ku蛋白是一种相对丰富的DNA结合蛋白，最初是在一名硬皮病-多发性肌炎重叠综合征患者中作为自身抗原被检测到的（因此得名“Ku”）。它是由两条分子量分别为85,000和72,000的多肽链组成的异二聚体，其特征在于在体外与DNA片段的末端结合，并易位形成规则的多聚体复合物，每30个碱基对的DNA结合一个蛋白。我们使用从培养的猴细胞中提取的蛋白质，研究了Ku蛋白与DNA在体外相互作用的机制。我们发现DNA末端的精确结构对结合并不重要，因为Ku蛋白可以与发夹环和单核小体结合。结合的蛋白持续结合也不需要DNA末端，因为用线性DNA形成的复合物可以被DNA连接酶环化。复合物的解离似乎也需要DNA末端，因为即使在2M NaCl存在的情况下，连接酶封闭的环状复合物也被发现极其稳定。我们还发现Ku分子沿着DNA滑动，对特定序列没有优先结合。因此，Ku蛋白的行为就像串在DNA链上的珠子，这种结合机制使我们能够对该蛋白在细胞核中的功能提出一个新的假设。
