Wu X, Lieber M R
Division of Molecular Oncology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mol Cell Biol. 1996 Sep;16(9):5186-93. doi: 10.1128/MCB.16.9.5186.
DNA ends are generated during double-strand-break repair and recombination. A p70-p86 heterodimer, Ku, accounts for the DNA end binding activity in eukaryotic cell extracts. When one or both subunits of Ku are missing, mammalian cells are deficient in double-strand-break repair and in specialized recombination, such as V(D)J recombination. Little is known of which regions of Ku70 and Ku86 bind to each other to form the heterodimeric complex or of which regions are important for DNA end binding. We have done genetic and biochemical studies to examine the domains within the two subunits important for protein assembly and for DNA end binding. We found that the C-terminal 20-kDa region of Ku70 and the C-terminal 32-kDa region of Ku86 are important for subunit-subunit interaction. For DNA binding, full-length individual subunits are inactive, indicating that heterodimer assembly precedes DNA binding. DNA end binding activity by the heterodimer requires the C-terminal 40-kDa region of Ku70 and the C-terminal 45-kDa region of Ku86. Leucine zipper-like motifs in both subunits that have been suggested as the Ku70-Ku86 interaction domains do not appear to be the sites of such interaction because these are dispensable for both assembly and DNA end binding. On the basis of these studies, we have organized Ku70 into nine sequence regions conserved between Saccharomyces cerevisiae, Drosophila melanogaster, mice, and humans; only the C-terminal three regions are essential for assembly (amino acids [aa] 439 to 609), and the C-terminal four regions appear to be essential for DNA end binding (aa 254 to 609). Within the minimal active fragment of Ku86 necessary for subunit interaction (aa 449 to 732) and DNA binding (aa 334 to 732), a proline-rich region is the only defined motif.
DNA末端在双链断裂修复和重组过程中产生。一种p70 - p86异二聚体Ku，负责真核细胞提取物中的DNA末端结合活性。当Ku的一个或两个亚基缺失时，哺乳动物细胞在双链断裂修复和特殊重组（如V(D)J重组）方面存在缺陷。对于Ku70和Ku86的哪些区域相互结合形成异二聚体复合物，以及哪些区域对DNA末端结合很重要，人们了解甚少。我们进行了遗传学和生物化学研究，以检查两个亚基中对蛋白质组装和DNA末端结合重要的结构域。我们发现，Ku70的C末端20 kDa区域和Ku86的C末端32 kDa区域对亚基 - 亚基相互作用很重要。对于DNA结合，全长的单个亚基是无活性的，这表明异二聚体组装先于DNA结合。异二聚体的DNA末端结合活性需要Ku70的C末端40 kDa区域和Ku86的C末端45 kDa区域。两个亚基中被认为是Ku70 - Ku86相互作用结构域的类似亮氨酸拉链的基序似乎并不是这种相互作用的位点，因为这些基序对于组装和DNA末端结合都是可有可无的。基于这些研究，我们将Ku70分为酿酒酵母、黑腹果蝇、小鼠和人类之间保守的九个序列区域；只有C末端的三个区域对组装是必需 的（氨基酸[aa]439至609），而C末端的四个区域似乎对DNA末端结合是必需的（aa 254至609）。在亚基相互作用（aa 449至732）和DNA结合（aa 334至732）所需的Ku86最小活性片段内，一个富含脯氨酸的区域是唯一确定的基序。
