Blunt T, Gell D, Fox M, Taccioli G E, Lehmann A R, Jackson S P, Jeggo P A
Medical Research Council Cell Mutation Unit, University of Sussex, Brighton, United Kingdom.
Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10285-90. doi: 10.1073/pnas.93.19.10285.
DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identified the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxyl-terminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse.
DNA依赖性蛋白激酶（DNA-PK）由一个异源二聚体蛋白（Ku）和一个大型催化亚基（DNA-PKcs）组成。Ku蛋白具有双链DNA末端结合活性，可将该复合物招募至DNA末端。尽管DNA-PKcs具有丝氨酸/苏氨酸蛋白激酶活性，但它属于磷脂酰肌醇3激酶超家族。DNA-PK在DNA双链断裂修复和V（D）J重组中发挥作用，最近的证据表明小鼠scid细胞的DNA-PKcs存在缺陷。在本研究中，我们克隆了啮齿动物细胞中DNA-PKcs羧基末端区域的cDNA，并确定了人类细胞中存在两种不同剪接的产物。我们发现DNA-PKcs定位于与小鼠scid基因相同的染色体区域。scid细胞中DNA-PKcs转录本的水平约为野生型水平，而同样存在DNA-PKcs缺陷的V-3细胞系中，转录本水平则非常低。对scid和野生型小鼠细胞羧基末端区域的序列比较，使我们能够在小鼠scid细胞基因的高度保守区域内鉴定出一个无义突变。这是scid小鼠中DNA-PKcs失活突变的一个有力候选者。
