Maintaining genetic stability is of paramount importance for the survival of cells and organisms. Double-strand DNA breaks (DSBs) are the most lethal DNA lesion threatening genomic stability, and cells have evolved a variety of mechanisms for their repair. While some of the repair mechanisms are accurate, others are ?risky? and can further destabilize the genome, leading to cancer and other diseases in humans. The molecular events that draw the intermediates of otherwise accurate repair pathways into a ?maelstrom? of destabilizing DNA repair mechanisms, where these intermediates are then processed through risky DNA repair pathways, remain unexplored. The goal of our research is to understand how DSB repair is channeled into the deleterious repair pathways, with particular emphasis on three DSB repair phenomena: 1) break-induced replication (BIR), an unusual type of long-tract repair DNA synthesis that promotes bursts of genetic instabilities; 2) microhomology-mediated BIR (MMBIR), a replicative pathway involving multiple template switching events at positions of microhomologies that yields complex genomic rearrangements; and 3) the transformation of long single-strand DNA intermediates of DSB repair into ?toxic? joint molecules promoting cell death. As a starting point, we are using our dependable and powerful system in yeast, where a single DSB is initiated by a site- specific HO endonuclease; we have demonstrated that all three of the repair events of interest can be used to repair the lesion in this system. The knowledge obtained using this system ? the repair mechanisms, intermediates, participating proteins, and mutation patterns ? is used to inform the experimental design of studies that will evaluate these pathways in other yeast and mammalian systems. Conceptually, the long-term goals are the same across projects and involve three primary areas of inquiry. First, using sensitive genetic approaches, proteins and DNA motifs whose presence affect the funneling of the repair intermediates into the ?maelstrom? of destabilizing repair mechanisms will be identified. Second, a combination of in vivo and in vitro approaches will be used to model and investigate the cell's decision points to understand the circumstances (structures, kinetics, participating proteins, etc.) that draw intermediates into high-risk and/or toxic repair pathways. Third, the patterns of mutations and chromosomal rearrangements that result from the deleterious repair pathways will be evaluated, and computational approaches will be used to apply these findings to human genome databases. To this end, MMBIRFinder, new software developed from previous research, will be used to detect complex genetic changes that cannot be found by currently available algorithms. Overall, this research program will bring improved clarity regarding the mechanisms of DNA repair intermediate processing, which will uncover factors that influence the regulation of dangerous repair pathways and result in destabilization of the genome in eukaryotes.
of complex genetic changes to many human diseases, including cancer and many neurological and developmental syndromes, is well established. Emerging evidence increasingly underscores a critical role for mis-channeled DNA repair events, where alternative, deleterious repair pathways are used, in generating these genome rearrangements. The proposed work will unravel the molecular events driving repair of DNA breaks into a maelstrom of destabilizing mechanisms that result in disease-promoting genomic changes, and it will identify potential points of therapeutic intervention for diseases rooted in genome instability.
