DNA repair and genetic recombination are important areas of research into fundamental aspects of carcinogenesis. This focus is due to the long appreciated understanding that chromosomal aberrations, loss of heterozygosity and other recombination-mediated genetic changes can both result from and lead to malignant transformation. Recent demonstrations that defective DNA repair underlies a number of different human malignancies has also led to an emphasis on understanding the role of DNA repair in carcinogenesis. In yeast, there are a number of genes (first identified as genes with functions in repair of radiation damage) with dual functions in both DNA repair and recombination. Many mutant alleles of these RAD genes show affects on DNA repair, recombination, and mutagenesis. In mammalian cells, relatively few studies have experimentally addressed the question of whether there are shared pathways for DNA repair and genetic recombination, or how defective mammalian DNA repair genes may alter genetic recombination mechanisms. In view of the significance of genetic rearrangements in malignant transformation and their generation by mitotic recombination, these are central issues in fully understanding the relationships between DNA damage, DNA repair, and the generation of genetic changes potentially leading to cancer. Recent progress in cloning mammalian DNA repair genes, as well as the development of molecular approaches for studying recombination in somatic mammalian cells, has made it possible to address how genes may function both in DNA repair and genetic recombination. Experiments described in this application will focus on mammalian DNA repair genes involved in homologous recombination, using specially constructed CHO cell lines with modified target gene loci for in vivo recombination assays. The hamster homolog of the ERCC4 (XpF) gene will be used to construct targeting vectors to knock-out ERCC4 in CHO cell lines already developed for analysis of recombination mechanisms; targeted correction and direct-repeat APRT recombination assays will be used to characterize recombination phenotypes of ERCC1- and ERCC4-deficient cell lines. The role of the XRCC3 gene, which may be also involved in crosslink repair through homologous recombination mechanisms, will be studied in the ERCC1/XpF recombination pathway, and epistasis experiments will determine its relationship to ERCC1. Additional CHO cell lines will be engineered to contain an I-SceI site in intrachromosomal recombination substrates for the controlled introduction of DSBs, enabling investigation of DSB-induced recombination in these cell systems.
