Amplification of Risk Caused by Mis-Routing of DNA Double-Strand Break Repair

Indiana University Indianapolis, Indianapolis IN

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Abstract

DESCRIPTION (provided by applicant): Double-strand DNA breaks (DSBs) are dangerous for human health because imprecise or faulty repair often leads to mutations and chromosome aberrations causing genetic diseases and cancer. The long-term goal of the investigator is to develop ways to minimize genomic instability resulting from DSBs. It is essential for this purpose to establish how DSB repair is executed and regulated, and how it leads to genome destabilization. The aim of this project is to unravel a number of molecular mechanisms capable of amplifying the consequences of DSBs in the model organism Saccharomyces cerevisiae. Firstly, this proposal is focused on chromatid fusions, which amplify the destabilizing effect of a single DSB by channeling it into breakage-fusion- bridge (BFB) cycles that create a series of rearrangement-prone secondary DSBs. Preliminary data allowed the investigator to propose that chromatid fusions can be stimulated by DSBs by allowing inter-molecular single-strand annealing (SSA) between inverted DNA repeats (IRs). Genetic methods and physical analyses of molecular intermediates are proposed to investigate this, as well as other homology-driven pathways of chromatid fusions that are currently poorly understood. Second, this proposal will unravel the mechanisms that allow broken chromosomes to acquire telomeres. Preliminary data suggested that break-induced replication (BIR) is the primary mechanism by which chromosomes undergoing BFBs are stabilized, which makes BIR the primary source of BFB-associated GCRs such as deletions, amplifications, and translocations. This research will specifically investigate the formation of translocations, which is the most deleterious outcome of BIR. Finally, results from genetic studies led to the hypothesis that interruption of BIR or other aberrant processing of BIR intermediates results in new chromosomal breakages that lead to cascades of DNA instability similar to the non-reciprocal translocations (NRTs) pathway known to amplify the number of rearrangements that result from an initial DSB in mammals. Thus, this proposal represents the first yeast model capable of simulating mammalian NRTs and is intended to unravel the molecular mechanisms of this process. In addition, the effects of genetic and environmental factors on channeling BIR repair into the GCR-producing pathways will be investigated. In summary, this research will elucidate the molecular mechanisms by which DSB repair can result in genomic consequences more destructive than the initial breakage. It is proposed that chromatid fusions, BIR, and NRTs are three such processes capable of amplifying the risks caused by a DSB due primarily to triggering BFB cycles. Further, experiments are proposed to test whether the magnification of damage that results from these genome-destabilizing DSB repair processes could be further amplified by cellular exposure to various environmental factors. To this end, experiments are planned to test the effects of various DNA damaging agents, including anti-cancer drugs, to investigate whether these agents might increase the frequency of high-risk repair processes or otherwise alter their outcomes. PUBLIC HEALTH RELEVANCE: This research is aimed to unravel the molecular mechanisms that lead to genomic destabilization by channeling double-strand DNA breaks into chromosomal rearrangements. Because genetic aberrations are a hallmark of cancer cells, this research will further our understanding of the etiology of some cancers.