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

LINE-1诱导的正常和衰老细胞DNA损伤 项目摘要 基因组结构变异（SV），包括插入和缺失（indel），使基因组的功能发生显著变化。 对人类多样性和疾病的贡献。已经提出了插入缺失和SV的许多原因， 包括复制滑动、重组、不平等交换和DNA双链修复不完善， 搁浅断裂。这是一个原理证明的建议，实验表明，LINE-1（L1） 反转录转座子也可以显著刺激基因组不稳定性，例如伴随衰老的不稳定性， 超过典型反转录插入的程度。反转录转座子是移动的DNA元件， 通过使用RNA中间体的“复制和粘贴”机制复制自身。L1至少包含 17%的人类DNA虽然它被认为是约100仍然有能力在任何人类逆转录转座 个人，更多的是转录。L1反转录转位也是插入 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的威胁。L1的第二开放阅读框（ORF 2） 编码能够在DNA中产生双链断裂的核酸内切酶活性。有理由认为 这些断裂的一部分是由细胞的非同源末端连接（NHEJ）DNA修复的。 修复途径，其易于产生错误和缺失。因此，L1 s表达升高， 它们编码的核酸内切酶活性，例如在衰老、癌症进展、胚胎发生和在 大脑的某些部分可能会增加细胞DNA损伤。利用下一代测序技术，我们将测试 这一假设在培养细胞和转基因小鼠。在目标1中，转化的细胞系的基因组和 将检查早期传代和衰老的原代人成纤维细胞的DNA损伤增加 在转染活性L1构建体之后。在目标2中，将对来自年轻小鼠和老年小鼠的后代的组织进行组织培养。 检查在诱导L1插入后产生的不标记L1插入的基因组突变， 四环素反应性L1转基因。如果得到支持，该假设有可能改善我们的 了解不仅伴随衰老，而且伴随癌症和各种疾病的遗传变化 与DNA修复失败有关的疾病。

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.