DNA repair by alternative end-joining in Drosophila

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When breaks occur in DNA, cells activate repair mechanisms to join the ends back together. Usually DNA repair is a precise process that serves to preserve the integrity of the genome. Surprisingly though, some types of repair used for joining broken DNA ends are very prone to errors. How and why this occurs is not clear. This research seeks to discover what makes end-joining repair so error-prone and what DNA sequence contexts might favor such imprecision. The results will provide crucial information that could prove useful for researchers who want to intentionally prevent or promote inaccurate repair, such as when conducting genome editing manipulations. Furthermore, the research will provide undergraduate and graduate-level scientists with transferable skills in molecular biology, genomics, computational modeling, and effective mentoring techniques. Finally, the project will engage a network of current and former lab members as public ambassadors for basic science research.Alternative end-joining is an error-prone mechanism of DNA double-strand break repair, which leads to small deletions and insertions at the break site. Accumulating evidence suggests that these DNA changes may result from the iterative action of the specialized translesion DNA polymerase, theta, with various accessory proteins. However, little is known about how these proteins interact with DNA sequences in the vicinity of the breaks and what role the DNA sequences themselves might play. One hypothesis is that the DNA on one side of the break forms transient secondary structures, such as short loops and hairpins, which drive production of new single-stranded DNA. The nascent strands then pair with microhomologous sequences on the other side of the break and serve as templates for completing the repair, thereby leading to a variety of repair products. This hypothesis will be tested in the fruit fly, Drosophila melanogaster, an organism that relies heavily on alternative end-joining repair and is uniquely amenable to studying how different tissue types or developmental stages might influence this error-prone process. A systematic combination of genetic manipulation, high throughput amplicon sequencing, and computational modeling will be used to characterize different types of secondary-structure forming sequences and the various classes of repair junctions they produce as a result of alternative end-joining. Finally, a possible role of alternative end-joining in repair of breaks induced by the genome editing tool, CRISPR-Cas9, will be assessed; the results of this experiment could provide an avenue for extending the basic understanding of this type of DNA repair to improving current practices for genome editing.