The Interaction of DNA Double-Strand Break Repair and CAG/CTG Trinucleotide Repeat Instability in Drosophila melanogaster.
Blackmer, Jane E.
- Trinucleotide repeats are the underlying molecular cause of many neurodegenerative disorders. A common feature of these repeats is their length dependence. When the repeat number is below a certain threshold, there are no symptoms of disease. Once expanded past a certain threshold, however, these repeats cause pathology on the DNA, RNA, and protein level. Repeats can expand in both somatic tissue ... read moreand in the germline. Expansions occurring in the germline lead to genetic anticipation: when a larger number of repeats are passed on to each successive generation, causing an earlier age of onset of the disease as well as a more severe phenotype. Models for repeat instability focus on the ability of these GC-rich repeats to form secondary structures, especially when the DNA is single stranded. These models therefore describe instability as occurring during replication, transcription, and repair of trinucleotide repeats. Studies in yeast show increased instability of CAG repeats during double-strand break repair via homologous recombination. Therefore, the goal for this experiment was to create and characterize a novel assay in which a double-strand break is created in the male germline of Drosophila melanogaster and repaired via homologous recombination through a region of CAG repeats. We created several constructs through molecular cloning in order to study repeat instability with a varying number of repeats, injected these constructs into Drosophila embryos, and created stocks. We performed the first iteration of the assay in a stock containing 71 CAG repeats before the induction of a double-strand break and repair. Results of the assay showed that the majority of repair events were end joining, with a small percentage of repair events being completed recombination, and an even smaller number of events being aborting recombination that completed repair via end joining. Experiments are ongoing to determine the stability of the repeats across generations and after repair.read less