DNA-dependent protein kinase suppresses double-strand break-induced and spontaneous homologous recombination
DNA-dependent protein kinase suppresses double-strand break-induced and spontaneous homologous recombination
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DOI：
10.1073/pnas.052545899
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发表时间：
2002-03-19
影响因子：
11.1
通讯作者：
Nickoloff, JA
中科院分区：
文献类型：
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作者：
Allen, C;Kurimasa, A;Nickoloff, JA
DNA依赖蛋白激酶(DNA-PK)由Ku70、Ku80和催化亚单位(DNA-PKcs)组成，参与非同源末端连接(NHEJ)修复双链断裂(DSB)。NHEJ中涉及的某些蛋白质也通过同源重组(HR)参与DSB修复。为了测试DNA-PKcs对DSB诱导的HR的影响，我们将携带I-SCEL识别序列的neo直接重复HR底物整合到DNA-PKcs缺陷的中国仓鼠卵巢(V3)细胞中。DNA-PKcs缺陷与人DNA-PKcs基因互补。DNA-PKcs互补后，DSB诱发的HR频率降低1.5~3倍。在互补和非互补菌株中，所有的产物都是通过基因转化产生的，没有相关的交叉，平均转化路径长度相似。在互补细胞中抑制DSB诱导的HR可能反映了NHEJ的恢复，这与HR和NHEJ在DSB修复过程中的竞争一致。有趣的是，DNA-PKcs互补后，自发性心率降低了1.6-3.5倍。DNA-PKcs可能通过抑制自发性DSB的NHEJ抑制自发性HR，可能是在复制分叉停滞或受阻时。由于复制蛋白A(RPA)同时参与复制和HR，并被DNA-PKcs磷酸化，DNA-PKcs对自发性HR的抑制可能反映了DNA-PKcs对复制依赖性HR的调节，可能是通过RPA的磷酸化。
DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is involved in repairing double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ). Certain proteins involved in NHEJ are also involved in DSB repair by homologous recombination (HR). To test the effects of DNA-PKcs on DSB-induced HR, we integrated neo direct repeat HR substrates carrying the I-Scel recognition sequence into DNA-PKcs-defective Chinese hamster ovary (V3) cells. The DNA-PKcs defect was complemented with a human DNA-PKcs cDNA. DSB-induced HR frequencies were 1.5- to 3-fold lower with DNA-PKcs complementation. In complemented and uncomplemented strains, all products arose by gene conversion without associated crossover, and average conversion tract lengths were similar. Suppression of DSB-induced HR in complemented cells probably reflects restoration of NHEJ, consistent with competition between HR and NHEJ during DSB repair. Interestingly, spontaneous HR rates were 1.6-to >3.5-fold lower with DNA-PKcs complementation. DNA-PKcs may suppress spontaneous HR through NHEJ of spontaneous DSBs, perhaps at stalled or blocked replication forks. Because replication protein A (RPA) is involved in both replication and HR, and is phosphorylated by DNA-PKcs, it is possible that the suppression of spontaneous HR by DNA-PKcs reflects regulation of replication-dependent HR by DNA-PKcs, perhaps by means of phosphorylation of RPA.