Retrotransposition independent LINE 1-induced DNA damage in normal and aging cells

逆转录转座非依赖的Line-1诱导正常和衰老细胞的DNA损伤 项目总结 基因组结构变异(SVS)，包括插入和缺失(INDELs)，使显著 对人类多样性和疾病的贡献。已经提出了许多导致INDELS和SVS的原因， 包括复制滑移、重组、不等交换以及DNA双链的不完善修复。 搁浅的休息。这是一项原则性建议的证明，旨在通过实验证明LINE-1(L1) 反转录转座子也可能显著刺激基因组的不稳定性，例如伴随衰老而来的基因组不稳定性。 超过标准的转位插入的程度。反转录转座子是一种可移动的DNA元件， 通过使用RNA中间体的“复制和粘贴”机制进行自我复制。L1至少包括 17%的人类DNA。虽然人们认为大约有100人仍然有能力在任何人类身上进行逆转位 个别的，更多的被转录。L1逆转录转座也是插入 超过一百万个非自主的Alu反转录转座子和数千个加工过的假基因。 L1对人类基因组构成持续威胁。L1的第二个开放阅读框(ORF2) 编码能够在DNA中造成双链断裂的内切酶活性。这是合理的建议 这些断裂中的一部分被细胞的非同源末端连接(NHEJ)DNA修复 容易产生错误和缺失的修复途径。因此，L1s和L1s的表达上调 它们编码的内切酶活性，例如在衰老、癌症进展、胚胎发育和 大脑的某些部分可能会增加细胞DNA损伤。使用下一代测序，我们将测试 这一假说出现在培养细胞和转基因小鼠身上。在目标1中，转化的细胞系和 早期传代和衰老的原代人类成纤维细胞将接受DNA损伤增加的检查 在转染活性L1构建体之后。在目标2中，来自幼鼠和老年鼠后代的组织将被 检查未被L1插入标记的基因组突变，这些突变是在诱导 四环素敏感的L1转基因。如果得到支持，这一假设有可能改善我们的 对基因变化的理解不仅伴随着衰老，也伴随着癌症和各种疾病 与DNA修复失败相关的情况。

Retrotransposition-independent LINE-1-induced DNA damage in normal and aging cells PROJECT SUMMARY Genomic structural variants (SVs), including insertions and deletions (indels), make a significant contribution to human diversity and disease. Numerous causes for indels and SVs have been proposed, including replication slippage, recombination, unequal crossing over, and imperfect repair of DNA double- stranded breaks. This is a proof of principle proposal to show experimentally that LINE-1 (L1) retrotransposons may also significantly stimulate genomic instability, such as that accompanying aging, to a degree that exceeds canonical retrotransposition insertions. Retrotransposons are mobile DNA elements that duplicate themselves by a "copy and paste" mechanism using an RNA intermediate. L1s comprise at least 17% of human DNA. While it is thought about 100 remain competent for retrotransposition in any human individual, many more are transcribed. L1 retrotransposition has also been responsible for the insertion of over a million non-autonomous Alu retrotransposons and thousands of processed pseudogenes. L1s pose an ongoing threat to the human genome. The second open reading frame (ORF2) of the L1 encodes endonuclease activity capable of making double-stranded breaks in DNA. It is reasonable to suggest that some proportion of these breaks are repaired by the cell's non-homologous end-joining (NHEJ) DNA repair pathway which is prone to generation of errors and deletions. Thus, elevated expression of L1s and their encoded endonuclease activity, such as occurs during aging, cancer progression, embryogenesis, and in some parts of the brain may increase cellular DNA damage. Using next generation sequencing, we will test this hypothesis in cultured cells and transgenic mice. In Aim 1, the genomes of a transformed cell line and early passage and senescing primary human fibroblasts will be examined for increased DNA damage following transfection of an active L1 construct. In Aim 2, tissues from progeny of young and aged mice will be examined for genomic mutations not marked by an L1 insertion that are generated following the induction of a tetracycline-responsive L1 transgene. If supported, the hypothesis has the potential to improve our understanding of genetic change that accompanies not only aging, but also cancer and various disease conditions associated with failure of DNA repair.