The goal of this proposal is to increase our understanding of the mechanisms that normally regulate homologous recombination in mammalian cells. Such an aim seems relevant in light of the often- made observation that cancer cells exhibit abberant chromosomal rearrangements. To study homologous recombination, mouse Ltk cell lines will be transfected with DNA substrates that contain two defective Herpes thymidine kinase (tk) gene sequences. Recombination events that reconstruct a functional tk gene will be monitored by the appearance of tk-positive cells. This work will refine earlier measurements of the homology dependence of "normal" recombination as well as explore the possibility that DNA lesions or sequences that promote DNA strand breakage can alter the rate or relax the homology requirements of recombination. This proposal also includes studies of "gene targeting," or homologous recombination between a transfected molecule and a specific chromosomal target sequence. Gene targeting offers a powerful tool for studying gene function and potentially a methodology for eventual gene therapy. By studying gene targeting events involving a selectable chromosomal marker, they propose to measure the homology dependence as well as make an assessment of the rate-limiting step of targeted recombination. Experiments will also be done to test the hypotheses that inhibiting random integration of DNA into a mammalian genome will improve targeting efficiency.***
