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

正常细胞和衰老细胞中不依赖逆转录转座的 LINE 1 诱导的 DNA 损伤

基本信息

批准号：
9766167
负责人：
JOHN Lawrence GOODIER
金额：
$ 20.47万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9766167
关键词：
Adult Age Aging Algorithm Design Architecture Bioinformatics Biological Brain Cataloging Catalogs Cell Aging Cell Extracts Cells Chromatin Chromosomes Codon Nucleotides Comprehensive Cancer Center Consensus Cultured Cells DNA DNA Damage DNA Double Strand Break DNA Insertion Elements DNA Repair DNA Repair Pathway DNA lesion Data Analyses Deoxyribonuclease I Dermal Disease Elements Embryonic Development Event Evolution Failure Female Fibroblasts Frequencies Future Generations Genetic Crossing Over Genetic Recombination Genome Genomic DNA Genomic Instability Genomics Harvest Human Human Cell Line Human Genome In Vitro Individual Insertion Mutation Investigation L1 Elements Length Lesion Letters Malignant Neoplasms Mentors Mus Mutagens Mutation Newborn Infant Nonhomologous DNA End Joining Nuclear Open Reading Frames Paste substance Predisposition Process Pseudogenes RNA RNA-Directed DNA Polymerase Research Research Personnel Retrotransposition Retrotransposon Sequence Analysis Site Structure Testing Tetanus Helper Peptide Tetracyclines Tissues Transfection Transformed Cell Line Transgenes Transgenic Mice Variant Work aged algorithmic methodologies endonuclease experience genome integrity genome sequencing homologous recombination human DNA improved in vivo insertion/deletion mutation next generation sequencing normal aging novel offspring pregnant pup repaired senescence structural genomics transposon/insertion element tumor progression vector control whole genome

项目摘要

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.

正常细胞和衰老细胞中不依赖逆转录转座的 LINE-1 诱导的 DNA 损伤项目概要基因组结构变异 (SV)，包括插入和删除 (indel)，对对人类多样性和疾病的贡献。已经提出了导致插入缺失和SV的多种原因，包括复制滑移、重组、不等交换和 DNA 双链不完美修复搁浅的休息。这是一个原理证明提案，旨在通过实验证明 LINE-1 (L1) 逆转录转座子还可能显着刺激基因组不稳定性，例如伴随衰老的基因组不稳定性，从而导致基因组不稳定性。程度超过了典型的逆转录转座插入。逆转录转座子是可移动的 DNA 元件，使用 RNA 中间体通过“复制和粘贴”机制复制自身。 L1 至少包括人类DNA的17%。虽然人们认为任何人类中约有 100 种仍具有逆转录转座能力个人，还有更多被转录。 L1 逆转录转座也负责插入超过一百万个非自主 Alu 逆转录转座子和数千个经过处理的假基因。 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 中产生双链断裂的核酸内切酶活性。建议合理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，容易产生错误和缺失。因此，L1s 和它们编码的核酸内切酶活性，例如在衰老、癌症进展、胚胎发生过程中以及在大脑的某些部分可能会增加细胞 DNA 损伤。使用下一代测序，我们将测试这一假设在培养细胞和转基因小鼠中得到了证实。在目标 1 中，转化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是否增加了 DNA 损伤转染活性 L1 构建体后。在目标 2 中，来自年轻和年老小鼠后代的组织将被检查未标记 L1 插入的基因组突变，这些突变是在诱导四环素响应性 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、还伴随癌症和各种疾病的基因变化与 DNA 修复失败相关的情况。