Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse
Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse
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DOI：
10.1073/pnas.93.19.10285
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发表时间：
1996-09-17
影响因子：
11.1
通讯作者：
Jeggo, PA
中科院分区：
文献类型：
--
作者：
Blunt, T;Gell, D;Jeggo, PA
DNA依赖蛋白激酶(DNA-PK)由一个异二聚体蛋白(Ku)和一个大的催化亚基(DNA-PKcs)组成。Ku蛋白具有双链DNA末端结合活性，有助于将复合体招募到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，最近的研究表明小鼠SCID细胞中存在DNA-PKcs缺陷。在这项研究中，我们克隆了啮齿动物细胞DNA-PKcs羧基末端的cDNA，并鉴定了在人类细胞中存在两种不同的剪接产物。我们发现DNA-PKcs定位于与小鼠SCID基因相同的染色体区域，SCID细胞含有接近野生型水平的DNA-PKcs转录产物，而同样缺乏DNA-PKcs的V-3细胞株含有非常低的转录水平。对SCID和野生型小鼠细胞的羧基末端区域进行序列比较，使我们能够在小鼠SCID细胞中鉴定出该基因高度保守区域内的无义突变。这代表了SCID小鼠DNA-PKcs失活突变的有力候选者。
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 identifed 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.