Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination
Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination
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DOI：
10.1073/pnas.94.15.8076
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发表时间：
1997-07-22
影响因子：
11.1
通讯作者：
Alt, FW
中科院分区：
文献类型：
--
作者：
Gu, YS;Jin, SF;Alt, FW
V(D)J重组需要淋巴特异性和普遍表达的酶活性，所有三种已知的普遍表达的V(D)J重组活性还参与DNA双链断裂修复(DSBR)，其中两种是DNA依赖蛋白激酶(DNA-PK)的组成部分，包括Ku80和DNA-PK催化亚基(DNA-PKcs)；第三种是XRCC4，功能未知，Ku70蛋白是DNA-PK的附加成分；Ku70与Ku80形成异源二聚体以产生酶的DNA末端结合组分，为了测试Ku70的可能功能，我们利用基因靶向突变产生了一个缺乏Ku70表达的小鼠胚胎干细胞系，我们发现Ku70(-/-)细胞不产生可检测到的Ku70和非常少的Ku80，这表明它们的水平之间存在直接的相互关系，相应地，这些细胞缺乏与Ku相关的非特异性DNA末端结合活性。值得注意的是，相对于Ku70(+/-)或野生型胚胎干细胞，Ku70(-/-)胚胎干细胞对伽马辐射的敏感性显著增加。此外，Ku70(-/-)细胞缺乏有效地重新连接通过强制表达RAG-1和RAG-2而在瞬时引入的V(D)J重组底物中释放的信号和编码末端的能力。我们的结论是Ku70基因产物参与了DSBR和V(D)J重组，并证实Ku70基因可以归类为X射线交叉互补组6(XRCC6)的成员。讨论了Ku70(-/-)和Ku80(-/-)V(D)J复合缺陷之间的潜在差异。
V(D)J recombination requires both lymphoid-specific and generally expressed enzymatic activities, All three known generally expressed activities involved in V(D)J recombination are also involved in DNA double-strand break repair (DSBR), Two of these are components of the DNA-dependent protein kinase (DNA-PK) and include Ku80 and DNA-PK catalytic subunit (DNA-PKcs); the third, XRCC4, is a protein of unknown function, The Ku70 protein is an additional component of DNA-PK; Ku70 forms a heterodimer with Ku80 to generate the DNA end-binding component of the enzyme, To test putative functions for Ku70, we have used gene-targeted mutation to generate a murine embryonic stem cell line which lacks Ku70 expression, We find that the Ku70(-/-) cells produce no detectable Ku70 and very little Ku80, suggesting a direct interrelationship between their levels, Correspondingly, these cells lack the nonspecific DNA end-binding activity associated with Ku. Significantly, the Ku70(-/-) embryonic stem cells have markedly increased sensitivity to gamma-irradiation relative to Ku70(+/-) or wild-type embryonic stem cells. Furthermore, the Ku70(-/-) cells lack the ability to effectively rejoin signal and coding ends liberated in transiently introduced V(D)J recombination substrates by enforced RAG-1 and RAG-2 expression. We conclude that the Ku70 gene product is involved in DSBR and V(D)J recombination and confirm that the Ku70 gene can Toe classified as a member of the x-ray cross-complementation group 6 (XRCC6). Potential differences between the Ku70(-/-) and Ku80(-/-) V(D)J recombination defects are discussed.