Genetic and Molecular Analyses of Human LINE-1 Retrotransposition

描述（由申请人提供）：长穿插元件-1 （LINE-1或L1）是一种丰富的可移动遗传元件，约占人类DNA的17%。活跃的人类L1s长度约为6kb，它们编码两个蛋白（ORF1p和ORF2p），这两个蛋白对逆转录至关重要。正在进行的LINE-1逆转录事件有助于个体间的遗传变异，并且，在某些情况下，LINE-1插入基因可能导致遗传疾病。在过去的十年中，我的实验室开发了有效的检测方法来监测培养的哺乳动物细胞中的LINE-1逆转录转位。在这里，我们将使用这些检测结合遗传、分子和生化方法来进一步阐明L1逆转录转位的机制。我们还将鉴定影响人类细胞中LINE-1逆转录的宿主蛋白。对L1逆转录过程的基本机制理解将有助于更深入地了解转座因子活性如何促进人类疾病、人类基因组进化和人类多样性。

DESCRIPTION (provided by applicant): Long Interspersed Element-1 (LINE-1 or L1) is an abundant mobile genetic element that comprises approximately 17% of human DNA. Active human L1s are about 6 kb in length and they encode two proteins (ORF1p and ORF2p) that are critical for retrotransposition. Ongoing LINE-1 retrotransposition events contribute to inter-individual genetic variation, and, on occasion, LINE-1 insertions into genes can result in genetic disease. During the past decade, my laboratory has developed efficient assays to monitor LINE-1 retrotransposition in cultured mammalian cells. Here, we will use these assays in conjunction with genetic, molecular, and biochemical approaches to further elucidate the mechanism of L1 retrotransposition. We also will identify host proteins that affect LINE-1 retrotransposition in human cells. A basic mechanistic understanding of the process of L1 retrotransposition will lead to greater insight about how transposable element activity contributes to human disease, human genome evolution, and human diversity. PUBLIC HEALTH RELEVANCE: Long INterspersed Element-1 (LINE-1) is an abundant mobile genetic element that comprises 17% of human DNA. Active LINE-1 elements are able to retrotranspose or "jump," inserting themselves into a new genomic location by a copy and paste mechanism. On occasion, new LINE-1 insertions in the germ line, in early development, or in somatic cells can result in disease-producing mutations. Despite the mutagenic potential of LINE-1 elements, little is known about the molecular mechanism of LINE-1 retrotransposition and even less is known about cellular proteins that influence this process. During the past decade, my laboratory has developed a toolbox to study LINE- 1 retrotransposition in a controlled manner in mammalian cultured cells. These tools have allowed significant progress in this field by my lab and others. In this proposal, we will use genetic, molecular biological, and biochemical approaches to further elucidate the molecular mechanism of LINE-1 retrotransposition. We also will explore how mutations in host proteins involved in host defense and/or DNA repair pathways affect LINE-1 jumping. Through these studies, we will learn more about how this intriguing family of repetitive DNA sequences contributes to the structure and function of the human genome.