Assessing the Effects of Cis and Trans Factors on Fragility at the Common Fragile Site FRA16D.
Haouzi, Alice Alexandra.
- Common Fragile Sites (CFSs) are regions within chromosomes prone to breakage and characterized by late replication. FRA16D, located within the tumor suppressor gene WWOX, is the second most highly expressed CFS in humans and has been associated with a variety of cancer cell lines. It is our hypothesis that multiple sequences within FRA16D work in a concerted effort to cause expression of the CFS. ... read moreThe first goal of this project was to assess these cis factors through a subtractive approach, using a YAC breakage assay designed to correlate the number of FOAR colonies to the rate of breakage. Deleting F1 and F5, two sequences containing AT repeats, caused a significant decrease in breakage, while deleting P5P5b, a region expected to form two large cruciforms (one of which is AT-rich), did not yield a change in fragility. In contrast, an additive approach was taken to measure the fragility of the F5 region via a direct duplication recombination assay (DDRA). The introduction of F5 with 24 interrupted AT repeats did not yield an increase in breakage, while the introduction of F1 with 23 perfect AT repeats had previously been shown to cause fragility. Taking findings from both approaches suggests that F5 might only cause breakage in the context of FRA16D, acting as a kind of “enabler” in the region. Trans factors affecting breakage were also tested via the DDRA in yeast strains containing a specific cassette with the 34 (AT) repeats of the F1 sequence. The deletion of MUS81 showed a very significant decrease in the rate of FOAR compared to the wild type, a result congruent with Mus81’s role as an endonuclease that causes breaks at CFSs. The deletion of MEC1, an ATR homolog, yielded an increase in FOAR that was milder than expected, based on the well-documented effect of ATR kinases in preventing fragility. This result was explained by finding that MEC1 was not actually deleted and was perhaps being preserved as an extrachromosomal element.read less