Genetics and Biochemistry of a Murine Retroposon

描述（由申请人提供）：LINE-1 （Long Interspersed Nuclear Element-1，或L1）是哺乳动物基因组中的主要动力。逆转录将L1的后代沉积在整个基因组中，有时会导致基因破坏，修饰邻近基因的表达，和/或邻近DNA的转导。此外，L1作为分散的重复DNA，为错配序列的同源重组提供了底物，导致基因重复、缺失、染色体易位和潜在的外显子洗牌。人类基因组中由L1的存在和运动引起的任何一个动态事件都可能导致疾病；事实上，LINE-1插入突变已被发现是多种疾病的原因，包括血友病和肌肉萎缩症，以及乳腺癌和结肠癌。因此，了解参与反转录转位的中间体的细节以及用于控制其体内表达和运动的机制是非常重要的。如果L1表达和逆转录的正常控制机制在发育（配子体发生或早期胚胎发生）或体细胞中因环境损害或衰老而紊乱，L1序列的移动和重排可能是导致遗传疾病、出生缺陷和癌症的遗传不稳定的重要组成部分。我们的长期目标是详细了解逆转录过程，包括所涉及的生化中间体，以及它在遗传和进化时间上的控制。L1逆转录始于全长、义链L1 RNA的转录，需要2个L1编码的多肽顺式作用。本研究旨在：1)通过研究突变对逆转录转位活性的影响，以及对分离的ORF1蛋白核酸结合和伴侣活性的影响，进一步阐明l1编码的ORF1蛋白在逆转录转位中的作用；2)确定ORF1与ORF2蛋白相互作用的基础，为ORF2蛋白的结构和功能提供新的生化信息；3)确定2种L1编码蛋白翻译控制的分子基础，并定义与L1RNA从翻译模板功能转变为组装L1反转录机器相关的蛋白质成分。

DESCRIPTION (provided by applicant): LINE-1 (Long Interspersed Nuclear Element-1, or L1) is a major dynamic force in the mammalian genome. Retrotransposition deposits the progeny of L1 throughout the genome, sometimes leading to gene disruption, modified expression of adjacent genes, and/or transduction of neighboring DNA. In addition, L1, as interspersed repetitive DNA, provides a substrate for homologous recombination of mispaired sequences, leading to gene duplication, deletion, chromosome translocation and, potentially, exon shuffling. Any 1 of the dynamic events caused by the presence and movement of L1 in the human genome can lead to disease; in fact, LINE-1 insertional mutagenesis has been found to be responsible for a wide variety of diseases including hemophilia and muscular dystrophy, as well as breast and colon cancer. Thus, it is extremely important to understand the details of the intermediates involved in retrotransposition and the mechanisms used to control their expression and movement in vivo. If the normal control mechanisms of L1 expression and retrotransposition become deranged either during development (gametogenesis or early embryogenesis) or in somatic cells in response to environmental insults or aging, movement and rearrangement of L1 sequences could be an instrumental component of the genetic instability responsible for genetic diseases, birth defects and cancers. Our long-range goal is to understand the retrotransposition process in detail, including the biochemical intermediates involved, as well as its control in genetic and evolutionary time. L1 retrotransposition begins with transcription of full-length, sense-strand L1 RNA and requires the 2 L1-encoded polypeptides acting in cis. The studies proposed here are specifically designed to: 1) Further elucidate the role of the L1-encoded ORF1 protein during retrotransposition by investigating the effects of mutations on retrotransposition activity, and on the nucleic acid binding and chaperone activities of the isolated ORF1 protein; 2) Determine the basis for the interaction between the ORF1 and ORF2 proteins, and provide new biochemical information about the structure and functions of the ORF2 protein; 3) Determine the molecular basis of translational control of the 2 L1-encoded proteins, and define the protein components associated with the L1RNA as it transitions from its function as the translation template to an assembled L1 retrotransposition machine.