LINE-1 (long interspersed repeated sequence one, 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. All of these dynamic events can lead to disease; in fact, LINE-1 insertional mutagenesis has been found to be responsible for hemophilia and muscular dystrophy, as well as breast and colon cancer in humans. 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, movement and rearrangement of L1 sequences could be instrumental in the generation of genetic diseases, birth defects and cancer. LINE-1 retrotransposition begins with transcription of a full-length, sense-strand L1 RNA and requires two L1-encoded polypeptides. These proteins probably also catalyze the reverse transcription and integration of SINEs (short interspersed repeated sequences) and processed pseudogenes, thereby amplifying the effects of LINE-1 in mammalian genome dynamics. 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. Specifically, the studies proposed here are designed to: 1) elucidate the role of the L1-encoded ORF1 protein during retrotransposition by investigating the nucleic acid and protein-protein interaction activities of wild-type and mutant proteins in detail; 2) identify cellular proteins that interact with ORF1p then determine whether they facilitate and/or inhibit L1 retrotransposition, and; 3) determine whether translation of ORF2 and/or ORF1 involves cap-independent mechanisms for initiation that contribute to the control of L1 retrotransposition.
