The goals of the proposed research are to determine the mechanisms and genetic consequences of DNA damage-induced recombination in mammalian cells. Genetic recombination is a ubiquitous and fundamental cellular process that is involved, for example, in gene regulation, development, and the genetic changes associated with cellular transformation and progression to the tumorigenic state. Recombination is stimulated by agents that damage DNA including UV light, ionizing radiation, and chemicals. These DNA damaging agents are also known to cause cellular transformation and cancer. Recombination is also stimulated by transcription. The proposed studies focus on several aspects of damage- induced recombination, including the effects of substrate structure, transcriptional activity, and cellular DNA repair capacity on recombination rates and mechanisms. Recombination substrates will be engineered to facilitate fine-resolution analysis of many recombinant products. DNA damage will be created with nucleases that produce specific double-strand breaks (DSBs), UV light, and SV40 T antigen. DSBs will be used to stimulate extrachromosomal recombination between neomycin genes (neo) carrying phenotypically silent restriction fragment length polymorphisms (RFLPs), creating mismatches in heteroduplex DNA for studies of mismatch repair efficiencies and biases. Mismatches will include all possible single-base mismatches and various loops of different lengths and structures (i.e., palindromic and nonpalindromic). Mismatch repair has important roles in recombination and in the maintenance of genetic integrity. Recent evidence has linked a defect in mismatch repair with an inherited form of colon cancer. Spontaneous, UV- induced, and DSB-induced intrachromosomal recombination will be studied with integrated, transcriptionally regulated neo heteroalleles in hamster and human cells. Recombination substrates will be flanked by known restriction sites and neo alleles will contain silent RFLP mutations to facilitate the rescue and fine-resolution mapping of recombinant products. Gene conversion tract positions, directionality and structures, and crossover points in spontaneous and damage-induced products will be determined. SV40 T antigen causes gross structural damage to chromosomes that is dependent on its binding the p53 tumor suppressor protein. Studies of T antigen-induced recombination at a specific locus will provide information about the mechanism of T antigen-induced chromosome damage. Mutant cell lines deficient in repair of UV damage (and with altered recombinational responses) will be used to explore the recombinational roles of the defective gene products. Studies of the mechanisms of UV-induced recombination and UV repair in human cells derived from patients with the UV-sensitive, cancer-prone syndrome xeroderma pigmentosum, will provide insight into the molecular defects in these cells. Together, the proposed studies will further our understanding of the initiation, mechanisms, and genetic consequences of spontaneous and DNA damage-induced recombination in mammalian cells and clarify the relationships among recombination, DNA damage and repair, and carcinogenesis.
