Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination
Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination
复制标题
DOI：
10.1093/nar/28.14.2752
复制
发表时间：
2000-07-15
影响因子：
14.9
通讯作者：
Meek, K
中科院分区：
文献类型：
--
作者：
Kienker, LJ;Shin, EK;Meek, K
DNA依赖性蛋白激酶（DNA-PK）在BRA双链断裂修复（DSBR）和V（D）J重组中均被利用，但这种多蛋白复合物参与这些过程的机制尚不清楚。为了评估DNA-PK介导的蛋白磷酸化在DSBR和V（D）J重组中的重要性，我们评估了磷脂酰肌醇3-激酶抑制剂渥曼青霉素对电离辐射诱导的DNA双链断裂修复和V（D）J重组的影响。J重组酶诱导的B细胞系HDR 37，Wortmannin放射增敏的HDR 37，但对V（D）J重组没有影响，尽管DNA-PK活性显著降低。另一方面，对野生型人DNA-PK催化亚基（DNA-PKcs）和激酶结构域突变体的哺乳动物表达载体的研究表明，只有DNA-PKcs的激酶活性形式可以在DNA-PKcs缺陷细胞系（Sf 19）中重建DSBR和V（D）J重组，这意味着DNA-PKcs激酶活性对DSBR和V（D）J重组都是必需的。通过分析重组野生型人DNA-PKcs表达不同的细胞系，这些明显矛盾的结果得到了调和。这些研究证实，最低DNA-PKcs蛋白水平足以支持V（D）J重组，但不足以赋予对电离辐射的抗性。
DNA-dependent protein kinase (DNA-PK) is utilized in both BRA double-strand break repair (DSBR) and V(D)J recombination, but the mechanism by which this multiprotein complex participates in these processes is unknown, To evaluate the importance of DNA-PK-mediated protein phosphorylation in DSBR and V(D)J recombination, we assessed the effects of the phosphatidyl inositol 3-kinase inhibitor wortmannin on the repair of ionizing radiation-induced DNA double-strand breaks and V(D)J recombination in the V(D)J recombinase inducible B cell line HDR37, Wortmannin radiosensitized HDR37, but had no affect on V(D)J recombination despite a marked reduction in DNA-PK activity. On the other hand, studies with mammalian expression vectors for wildtype human DNA-PK catalytic subunit (DNA-PKcs) and a kinase domain mutant demonstrated that only the kinase active form of DNA-PKcs can reconstitute DSBR and V(D)J recombination in a DNA-PKcs-deficient cell line (Sf19), implying that DNA-PKcs kinase activity is essential for both DSBR and V(D)J recombination. These apparently contradictory results were reconciled by analyses of cell lines varying in their expression of recombinant wild-type human DNA-PKcs, These studies establish that minimal DNA-PKcs protein levels are sufficient to support V(D)J recombination, but insufficient to confer resistance to ionizing radiation.