| Popis: |
Common fragile sites (CFSs) are regions of DNA exceptionally prone to breakage.While these regions have implications in cancer, the causes of chromosome fragility remain poorly understood. This is partially due to relatively low-resolution cytological mapping of CFSs, and the use of exogenous agents to induce chromosome breakage. In an effort to better understand the causes of fragility, I have developed Drosophila melanogaster as a model for CFS study. In doing so, I have developed approaches to identify CFSs at a high resolution, based on both spontaneous chromosomal events and breakage induced by the inhibition of replication. In the first approach, I used a mutant form of mus309, the ortholog of human BLM helicase, to locate CFSs by visualizing sites of DNA breakage as mitotic crossovers. High rates of breakage correspond to CFSs, and results from my study indicate that there is a significantly non-uniform rate of mitotic crossovers across the left arm of chromosome 2. This work constitutes the first report of specific regions sensitive to endogenous damage in D. melanogaster. Further, the resolution of damage detection can be brought even higher with SNP mapping. My second approach is a novel assay that uses S2 cell culture to detect preferential sites of exogenous DNA integration, a hallmark of CFSs. This allows me to survey the entire genome, while using high- throughput sequencing to obtain a resolution of CFS detection orders of magnitude better than previous studies. I obtained numerous integration events under a variety of conditions, providing evidence of putative fragile regions. I anticipate that the assays I developed will serve as valuable tools for the detection of DNA breakage in future studies. Further, I expect the characterization of the CFSs detected from both of these approaches will lead to a better understanding of the causes of inherent genome instability. |
