The main purpose of this proposal is to investigate mechanisms of homeologous recombination in mammalian cells. Recombination between homeologous elements can result in chromosomal rearrangements often leading to the development of cancer, and other human genetic diseases. I will investigate whether DSBs increase the frequency of recombination between homeologous sequences in embryonic stem (ES) cells by using recombination substrates which are diverged direct repeats of the receptor gene, the neomycin phosphotransferase (neo) gene, and also direct repeats of the naturally occurring Alu element, an abundantly repetitive element in the mammalian genome. In addition, I will investigate the mechanism for DSB repair in gene targeting experiments in ES cells which are defective in the mismatch repair (MMR) pathway. Previous experiments in this lab suggest that the repair of DSBs is occurring either by gap repair or by mismatch repair with a bias toward the unbroken strand. These experiments with the MMR-defective ES cells should distinguish between these two possibilities. Understanding molecular mechanisms which may promote homeologous recombination, such as DSBs and a defective MMR pathway, will lead to potential treatment of many human genetic diseases in the future.
