Mechanisms of human LINE-1 retrotransposition and DNA repair

LINE-1元件(L1)约占人类基因组的17%，不仅通过自身的扩展，而且通过动员非L1序列，在哺乳动物基因组的形成中发挥着重要作用。尽管这组活跃的、可移动的元件对哺乳动物基因组的结构、组织和稳定性以及由它们的作用引起的疾病有明显的影响，但在世界范围内，只有少数实验室研究L1逆转座子的生物学。我们想要阐明人类LINE-1逆转录转座的机制，并确定参与这一基因组形成过程的组件。L1整合的过程可分为“靶启动逆转录”(TPRT)，它描述了L1复制、整合和负链合成以及迄今尚不了解的正链合成的起始步骤。我们的第一个目标是确定L1编码的基因产物和宿主因子，这些基因产物和宿主因子参与了新的L1拷贝插入基因组，并阐明了L1整合的机制。我们将试图通过分析预先存在于人类细胞基因组中的L1和Alu插入的靶点复制来揭示L1逆转录转座的正链合成机制的细节。由于L1特异性内切酶活性引起的DNA损伤是作为L1整合过程的一部分由宿主修复的，我们想要确定宿主S修复机制中参与与L1逆转座子相关的双链断裂修复的那些因素。如果宿主S的脱氧核糖核酸修复机制受损，L1活性就会导致染色体损伤。此外，有初步迹象表明L1在肿瘤细胞中的表达上调。因此，我们的第二个目标是系统地表征L1功能元件在癌细胞系中的表达，并评估L1逆转录转座对潜在致癌事件的大规模基因组重排的影响。

LINE-1 elements (L1) represent about 17% of the human genome and play a major part in shaping the mammalian genome, not only through their own expansion but also through the mobilization of non-L1 sequences. Despite the clearly demonstrated impact this group of active, mobile elements has on structure, organization and stability of mammalian genomes and the diseases that are caused by their action, only few laboratories investigate the biology of L1 retrotransposition worldwide. We want to elucidate the mechanisms of human LINE-1 retrotransposition and identify the components that are involved in this genome shaping process. The process of L1 integration can be devided into "Target-Primed Reverse Transcription" (TPRT), which describes the initiating step of L1 replication, integration and minus-strand synthesis, and plus-strand synthesis that is not understood so far. Our first goal is to determine L1-encoded gene products and host factors that are involved in the insertion of a new L1 copy into the genome and shed light on the mechanism of L1 integration. We will attempt to reveal details about the mechanism of plus-strand synthesis of L1 retrotransposition by analysis of target site duplications from L1- and Alu insertions, preexisting in the genome of human cells. Since DNA damage caused by L1-specific endonuclease activity is repaired by the host as part of the L1 integration process, we want to identify those factors of the host´s repair mechanisms that take part in Double-Strand Break repair linked to L1 retrotransposition. If the host´s DNA repair mechanism is impaired, L1 activity is causing chromosomal damage. Furthermore, first indications exist for an upregulation of L1 expression in tumor cells. Therefore, it is our second goal to systematically characterize the expression of functional L1 elements in cancer cell lines and evaluate the effect of L1 retrotransposition on large-scale genome rearrangements underlying cancerogenic events.