Comparative Mechanisms of Genomic Instability

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DESCRIPTION (provided by applicant): Chromosomal translocations resulting in inappropriate control of disease-related genes are important causative factors in environmentally induced disorders such as cancer. For example, translocations resulting in overexpression of the BCL-2 and c-MYC genes are the hallmarks of follicular B-cell lymphoma and Burkitt's lymphoma, respectively. DNA double-strand breaks (DSBs) are the first step in the process of chromosomal translocation. However, little is known about the mechanism(s) of the breakages on the translocated genes, why the DSBs tend to locate in certain genomic fragile site "hotspots", and the effects of environmental agents on the genomic instability at these susceptible hotspots. In this application, the intent is to use a comparative genetic approach to determine mechanisms of DNA structure-induced genomic instability. Interestingly, the most common breakpoints in such genes occur near regions that are capable of adopting non-B DNA structures. This group has discovered that H-DNA and Z-DNA-forming sequences near the translocation breakpoint hotspots in the human c-MYC gene induce DSBs, resulting in high levels of genetic instability in mammalian cells. Hence, the objectives of this application are to determine the mechanisms involved in genetic instabilities at breakpoint hotspots associated with disease, and further the development of novel approaches to reduce genetic instability caused by environmental DNA damaging agents. The immediate goals are to test the hypotheses that non-B-DNA structures found in the BCL-2 and c-MYC breakpoint regions are implicated in genetic instability across species, and that DNA damage induced by environmental factors at these hotspots enhance their susceptibility to genomic instability. The following are proposed: 1) to measure non-B DNA-induced genetic instability in different species. The naturally occurring H-DNA or Z-DNA-forming sequences from the human c-MYC and BCL-2 genes will be screened for their mutagenic potentials in a variety of species including yeast, mouse, and human. DNA structure-induced DSBs, illegitimate recombination, or point mutations in cells will be detected by facile blue/white screening; 2) to determine the susceptibility of non-B DNA-sequences to environmental carcinogen-induced DNA damage and mutagenesis. Environmental carcinogens such as irradiation are known to result in more non-B structure formation, and a reduced error-free repair of the damage. Thus, the amount of DNA damage induced and repaired in non-B sequences will be measured, and the level of genetic instability induced by environmental agents at these fragile site "hotspots" determined; and 3) to identify the genes/pathways that are involved in the genetic instability at non-B DNA sequences in the presence and absence of environmental carcinogens. Using a high-throughput screen gene products involved in DNA structure-induced genetic instability will be identified at genomic hotspots to begin to elucidate the pathways involved in genetic instability. Data obtained will give a better understanding of the mechanisms of genomic instability and the impact of environmental agents on these mechanisms. These discoveries should begin to unravel the pathogenesis of diseases that are caused by genomic instability, and ultimately to the development of new approaches for treatment and prevention.