Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)
Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)
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DOI：
10.1073/pnas.0406466102
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发表时间：
2005-02-01
影响因子：
11.1
通讯作者：
Meek, K
中科院分区：
文献类型：
--
作者：
Convery, E;Shin, EK;Meek, K
在所有真核生物中存在两种主要的DNA双链断裂修复途径，非同源DNA末端连接（NHEJ）和同源重组（HR）。虽然这两种途径都可以在整个细胞周期中发挥作用，但NHEJ在G（0）/G（1）中占主导地位（当复制的姐妹染色单体不可用时），而HR在S和G中的贡献更大（2）。细胞如何在这两种重要的DNA修复途径之间进行选择在很大程度上是未知的。DNA依赖性蛋白激酶（DNA-PK）对NHEJ至关重要。在这里，我们描述了两个保守的剪接变异体的催化亚基的DNA-PK（DNA-PKcs），主要在非分裂细胞中表达。虽然两者都编码稳定的产物，但都不能逆转DNA-PKcs缺陷细胞中的NHEJ缺陷。事实上，表达DNA-PKcs变体之一的细胞比完全缺乏DNA-PKcs的细胞稍微更放射敏感。我们研究了表达DNA-PKcs变体的细胞是否有任何其他DNA修复缺陷，发现这些细胞对依托泊苷和丝裂霉素C的敏感性比完全不表达DNA-PKcs的细胞要高得多。由于这两种试剂诱导的DNA损伤的修复需要完整的HR，我们测试了DNA-PKcs的NHEJ缺陷变体是否在整合底物中抑制双链断裂诱导的HR。在表达NHEJ缺陷变体的细胞中，HR显著降低。由于剪接变体仅在非分裂细胞中高度表达，因此当姐妹染色单体不能用作精确修复的模板时，静止细胞将被提供一种通过HR抑制修复的机制，从而提高基因组稳定性。
Two major DNA double-strand break repair pathways exist in all eukaryotes, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Although both pathways can function throughout the cell cycle, NHEJ predominates in G(0)/G(1) (when a replicated sister chromatid is unavailable), whereas HR makes a more substantial contribution in S and G(2). How a cell chooses between these two important DNA repair pathways is largely unknown. DNA-dependent protein kinase (DNA-PK) is critical for NHEJ. Here, we describe two conserved splice variants of a catalytic subunit of DNA-PK (DNA-PKcs) that are expressed predominately in nondividing cells. Although both encode stable products, neither reverses the NHEJ defects in DNA-PKcs-deficient cells. In fact, cells expressing one of the DNA-PKcs variants are slightly more radiosensitive than cells completely deficient in DNA-PKcs. We investigated whether cells expressing the DNA-PKcs variants had any other DNA repair deficits and found that these cells are considerably more sensitive to both etoposide and mitomycin C than cells that express no DNA-PKcs at all. Because repair of DNA damage induced by these two agents requires intact HR, we tested whether the NHEJ-defective variants of DNA-PKcs inhibit double-strand break-induced HR in an integrated substrate. In cells expressing the NHEJ-defective variants, HR was markedly reduced. Because the splice variants are expressed highly only in nondividing cells, quiescent cells would be afforded a mechanism to inhibit repair by means of HR when sister chromatids are not available as templates for accurate repair with low risk of genome rearrangement, thereby enhancing genome stability.