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. ? ? ?
