TRANSPOSITION AND DNA REPAIR IN DROSOPHILA

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Goals: The repair of double-stranded DNA breaks in Drosophila mitotic cells will be analyzed genetically. Such breaks can be repaired by two main classes of mechanisms: recombinational and end-joining. These classes differ significantly in the kinds of repair products that are produced. Within each class, multiple individual pathways are thought to exist. The goal is to perform a genetic dissection of these mechanisms. For each major pathway, the genetic consequences and the structure of the repair products will be determined. Examination of these parameters in a series of mutations at loci involved in the repair processes will reveal how the pathways differ and the type of repair that is affected. Approach: Excision of P transposable elements provides a useful way to produce double-strand breaks at specific, known, locations in the genome. These excisions can be regulated by P transposase. In addition, two site-specific endonucleases, HO and I-SceI, have been shown to function in Drosophila. These three methods will be used to generate specific double-strand breaks in vivo under well-defined genetic conditions. A series of tests using these site-specific breaks will be applied in different genetic backgrounds to provide a detailed description of the consequences of double-strand break repair. Different pathways for repair will be distinguished by mutating genes thought to be involved with one or more mechanism. Seven genetic loci involved with repair were selected to include a variety of such functions. Two recently-developed methods will be used to obtain mutations: the first generates P insertions in or near the targeted genes, and the second uses these insertions to obtain flanking deletions which knock out the genes. Medical significance: DNA repair is recognized as crucial to maintaining the stability of the genome, especially in species with complex genomes. Human mutations in many DNA repair genes, including the human homologs of several of the loci in the present study, cause predisposition to cancer. A better understanding of these repair processes can come from studies using Drosophila, which, like humans, has a relatively complex genome and multicellular organization, thus requiring a high level of genomic stability. At the same time, Drosophila gives researchers the genetic tools needed to analyze the multi-pathway repair system.