Morrish Tammy A, Garcia-Perez José Luis, Stamato Thomas D, Taccioli Guillermo E, Sekiguchi JoAnn, Moran John V
Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618, USA.
Nature. 2007 Mar 8;446(7132):208-12. doi: 10.1038/nature05560.
Long interspersed element-1 (LINE-1 or L1) elements are abundant, non-long-terminal-repeat (non-LTR) retrotransposons that comprise approximately 17% of human DNA. The average human genome contains approximately 80-100 retrotransposition-competent L1s (ref. 2), and they mobilize by a process that uses both the L1 endonuclease and reverse transcriptase, termed target-site primed reverse transcription. We have previously reported an efficient, endonuclease-independent L1 retrotransposition pathway (EN(i)) in certain Chinese hamster ovary (CHO) cell lines that are defective in the non-homologous end-joining (NHEJ) pathway of DNA double-strand-break repair. Here we have characterized EN(i) retrotransposition events generated in V3 CHO cells, which are deficient in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and have both dysfunctional telomeres and an NHEJ defect. Notably, approximately 30% of EN(i) retrotransposition events insert in an orientation-specific manner adjacent to a perfect telomere repeat (5'-TTAGGG-3'). Similar insertions were not detected among EN(i) retrotransposition events generated in controls or in XR-1 CHO cells deficient for XRCC4, an NHEJ factor that is required for DNA ligation but has no known function in telomere maintenance. Furthermore, transient expression of a dominant-negative allele of human TRF2 (also called TERF2) in XRCC4-deficient XR-1 cells, which disrupts telomere capping, enables telomere-associated EN(i) retrotransposition events. These data indicate that L1s containing a disabled endonuclease can use dysfunctional telomeres as an integration substrate. The findings highlight similarities between the mechanism of EN(i) retrotransposition and the action of telomerase, because both processes can use a 3' OH for priming reverse transcription at either internal DNA lesions or chromosome ends. Thus, we propose that EN(i) retrotransposition is an ancestral mechanism of RNA-mediated DNA repair associated with non-LTR retrotransposons that may have been used before the acquisition of an endonuclease domain.
长散在重复序列1（LINE-1或L1）元件是丰富的非长末端重复（non-LTR）逆转座子，约占人类DNA的17%。人类基因组平均包含约80-100个具有逆转座活性的L1（参考文献2），它们通过一种同时使用L1内切酶和逆转录酶的过程进行移动，称为靶位点引发的逆转录。我们之前报道过，在某些DNA双链断裂修复的非同源末端连接（NHEJ）途径存在缺陷的中国仓鼠卵巢（CHO）细胞系中，存在一种高效的、不依赖内切酶的L1逆转座途径（EN(i)）。在此，我们对V3 CHO细胞中产生的EN(i)逆转座事件进行了表征，这些细胞缺乏DNA依赖性蛋白激酶催化亚基（DNA-PKcs）活性，具有功能失调的端粒和NHEJ缺陷。值得注意的是，约30%的EN(i)逆转座事件以定向特异性方式插入到与完美端粒重复序列（5'-TTAGGG-3'）相邻的位置。在对照细胞或缺乏XRCC4的XR-1 CHO细胞（XRCC4是DNA连接所需的NHEJ因子，但在端粒维持中无已知功能）中产生的EN(i)逆转座事件中未检测到类似的插入。此外，在缺乏XRCC4的XR-1细胞中瞬时表达人TRF2（也称为TERF2）的显性负等位基因，该等位基因会破坏端粒帽，从而使端粒相关的EN(i)逆转座事件得以发生。这些数据表明，含有失活内切酶的L1可以将功能失调的端粒用作整合底物。这些发现突出了EN(i)逆转座机制与端粒酶作用之间的相似性，因为这两个过程都可以利用3' OH在内部DNA损伤或染色体末端引发逆转录。因此，我们提出EN(i)逆转座是一种与非LTR逆转座子相关的RNA介导的DNA修复的原始机制，可能在获得内切酶结构域之前就已被使用。
