Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair
Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair
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DOI：
10.1128/mcb.23.16.5836-5848.2003
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发表时间：
2003-08-01
影响因子：
5.3
通讯作者：
Meek, K
中科院分区：
文献类型：
--
作者：
Ding, Q;Reddy, YVR;Meek, K
DNA依赖蛋白激酶(DNA-PK)通过识别和结合DNA断裂，在非同源DNA末端连接(NHEJ)中发挥重要作用。我们已经证明，在体外，纯化的DNA-PK经历了自动磷酸化，导致了激酶复合体的活性丧失和解体。因此，我们认为DNA-PK催化亚基(DNA-PKcs)的自动磷酸化可能在DNA修复的后续步骤中起关键作用。最近，我们在DNA-PKcs中定义了七个自磷酸化位点。其中6个紧密聚集在4,127个残基蛋白的38个残基内。在这里，我们表明，虽然主簇中任何单个位点的磷酸化对DNA-PK在体内的功能并不关键，但几个位点的突变会取消DNA-PK在NHEJ中发挥作用的能力。这并不是由于DNA-PK活性的普遍缺陷，因为对突变蛋白的研究表明，其激酶活性和与DNA结合的Ku形成络合物的能力基本保持不变。然而，对罕见的编码关节和末端的分析表明，在突变的DNA-PKcs介导的关节中，核溶解末端处理显著减少。因此，我们认为主要簇内的自动磷酸化介导了DNA-PK复合体的构象变化，这对DNA末端处理是关键的。然而，这些位点上的自动磷酸化可能不足以进行激酶的分解。
The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.