Gene targeting involves homologous recombination between an introduced DNA and a chromosomal locus. The efficiency of this process is generally quite low, and it appears to be limited by the status of the chromosomal target. The purpose of this proposal is to test reagents that promise to elevate the frequency of successful gene targeting by specifically cleaving the target DNA. We are working with chimeric nucleases that combine a DNA-binding domain comprised of zinc fingers with a nonspecific DNA-cleavage domain. Because the DNA sequences recognized by zinc fingers can be altered by changing a few key amino acids in the protein, these chimeras have the potential to direct cleavage to a wide range of target sites. Although the proposed experiments are restricted to Drosophila, this gene targeting method is very general and should be applicable to the generation of animal models of human disease and ultimately to human gene therapy. The capabilities of the chimeric nucleases for stimulating gene targeting will be tested in the fruit fly, Drosophila melanogaster. A specific target has been chosen in the yellow (y) gene, and nucleases that recognize that sequence have been constructed. To test the ability of these enzymes to find and cleave their target in the Drosophila genome, expression of the nucleases will be induced in larvae, and the emerging adults screened for new y mutations that have occurred by inaccurate repair of the break. Then a linear donor DNA will be provided to introduce a specific mutation at the break. Various methods of delivering the chimeric nucleases and donor DNA will be compared to find the protocol with the best combination of convenience and efficiency. Parameters of the system will be investigated, including the homology requirements, the effects of the size and location of the introduced mutation, and the effects of disabling DNA repair genes that contribute to homologous or nonhomologous recombination pathways. To test the generality of the method, it will be applied to a second Drosophila locus, the rosy (ry) gene.