Characterization of LINE-1 reverse transcriptase activity

项目摘要 这项研究计划的目的是表征逆转录酶的酶活性。 (RT)人类长散布元件1(线-1，L1RT)。Line-1序列占总序列的17% 人类基因组，而L1 RT活性约占我们基因组的40%，因为它还 导致短穿插元素(如Alu)和其他逆转录元素的扩散。L1 RT活动也是 艾卡迪被认为是黄斑变性等多种人类疾病的幕后推手。 古蒂埃综合征和系统性红斑狼疮。LINE-1的表达和逆转座是 常见于多种癌症，包括90%-100%的乳腺癌、结肠癌和食道癌，使 了解与公共卫生研究相关的L1RT的基本生化活性。 逆转录是LINE-1通过一个过程在我们的基因组中自我繁殖的关键 被称为靶基因启动的逆转录(TPRT)。L1RT由第二开放阅读框编码 L1的(ORF2)，驻留在被称为ORF2p的蛋白质中。在这个提案中，我展示了我已经分离出ORF2p RT结构域，并且在体外具有RT活性。我打算使用这个蛋白质结构域，以及全长 ORF2p和L1 RNPs，以全面表征L1RT的生化和酶特性。在我的第一次 具体目的，我将描述它的过程性，这是RT是否停止和暂停的指示，或者 它是否继续运行，直到到达模板的末尾。我还将测量L1 RT的保真度， 定义它从RNA模板复制DNA的准确性。最后，我给出了初步测序数据 表明L1 RT具有出人意料的能力，可以在没有引物的情况下开始处理RNA模板 建议进行严格的实验来研究这一特性。在我的第二个具体目标中，我关注的是当 L1 RT到达其模板的末端，这可能对于了解细胞如何修复 换位。我提供的初步数据表明，L1RT在其cDNA末端增加了额外的核苷酸，并 建议进行研究以充分描述这一活动的特征。此外，我还将研究L1 RT转换为A的能力 一个模板末端的不同模板以及LINE-1RT是否可以使用修饰的RNA作为模板， 例如在信使核糖核酸疫苗中发现的假尿苷或自然产生的N6-甲基腺苷 修改。 这项工作将促进我们对LINE-1如何在单元格内运行以及LINE-1如何运行的理解 完成其生命周期。这项拟议的研究将对癌症生物学和先天免疫产生影响。 L1 RT被越来越多地认为是DNA损伤剂和模式识别触发因素的信号转导 分别是受体。最后，这项研究将深入了解LINE-1类逆行元件的进化 在所有的生命王国中都能找到。

Project Summary The objective of this research proposal is to characterize the enzymatic activities of the reverse transcriptase (RT) of the human long interspersed element 1 (LINE-1, L1 RT). LINE-1 sequences constitute 17% of the human genome, and L1 RT activity is responsible for approximately 40% of our genome because it has also led to the proliferation of short interspersed elements (e.g., Alu), and other retroelements. L1 RT activity is also implicated as a driving force behind a variety of human diseases such as macular degeneration, Aicardi- Goutières syndrome, and systemic lupus erythematosus. LINE-1 expression and retrotransposition are commonplace in numerous cancers, including 90-100% of breast, colon, and esophageal cancers, making understanding the basic biochemical activity of L1 RT relevant to public health research. Reverse transcription is key to the ability of LINE-1 to self-propagate in our genome, through a process known as target-primed reverse transcription (TPRT). L1 RT is encoded by the second open reading frame (ORF2) of L1, residing in the protein known as ORF2p. In this proposal, I show that I have isolated the ORF2p RT domain, and that this has RT activity in vitro. I intend to use this protein domain, as well as full-length ORF2p, and L1 RNPs to fully characterize the biochemical and enzymatic properties of L1 RT. In my first Specific Aim, I will characterize its processivity, which is an indication of whether the RT stops and pauses, or whether it keeps going until it reaches the end of a template. I will also measure the fidelity of L1 RT, which defines how faithfully it copies DNA from an RNA template. Finally, I present preliminary sequencing data showing that L1 RT has the unexpected ability to begin processing an RNA template without a primer and propose rigorous experiments to study this feature. In my second Specific Aim, I focus on what happens when L1 RT reaches the end of its template, which may be crucial to understanding how cells repair intermediates of transposition. I present preliminary data showing that L1 RT adds extra nucleotides to the end of its cDNA and propose studies to fully characterize this activity. Additionally, I will study the ability of L1 RT to switch to a different template at the end of one template and whether LINE-1 RT can use modified RNA as a template, such as the pseudouridine found in mRNA vaccines or the naturally occurring N6-methyladenosine modification. This work will advance our understanding of how LINE-1 operates inside cells and how LINE-1 completes its life cycle. The proposed research will have implications for cancer biology and innate immune signaling where L1 RT is increasingly recognized as a DNA damaging agent and trigger for pattern recognition receptors, respectively. Finally, this research will give insight into the evolution of LINE-1-like retroelements that are found across all kingdoms of life.