Nonhomologous-end-joining factors regulate DNA repair fidelity during Sleeping Beauty element transposition in mammalian cells
Nonhomologous-end-joining factors regulate DNA repair fidelity during Sleeping Beauty element transposition in mammalian cells
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DOI：
10.1128/mcb.23.23.8505-8518.2003
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发表时间：
2003-12-01
影响因子：
5.3
通讯作者：
Kay, MA
中科院分区：
文献类型：
--
作者：
Yant, SR;Kay, MA
在此，我们报告的DNA依赖性蛋白激酶（DNA-PK）调节DNA损伤引入睡美人（SB）元件切除和重新插入在哺乳动物细胞。使用基于质粒和染色体的迁移率测定，我们分析了在DNA结合亚基DNA-PK（Ku）或其催化亚基（DNA-PKcs）缺陷的细胞中转座酶诱导的双链DNA断裂的修复。我们发现SB元件切除后留下的游离3'突出端通过主要的Ku依赖性非同源末端连接途径有效且准确地加工。在没有Ku的情况下，断裂的DNA分子的重新连接导致在供体位点的广泛末端降解，并大大增加了与异位模板重组的频率。因此，主要的DNA-PK-依赖性DNA损伤反应在更容易出错的修复途径上占主导地位，从而促进哺乳动物细胞中转座子动员期间的高保真DNA修复。尽管转座因子在转座酶介导的切除后没有被有效地环化，但DNA-PK缺陷支持比野生型对照中发现的更频繁的转座酶介导的元件插入。我们的结论是，基于其调节切除位点连接多样性和转座子插入频率的能力，DNA-PK在哺乳动物的转座重组过程中起着重要的保护作用。
Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNA-PKcs). We found that the free 3' overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals.