Recovery of Alternative End-Joining Repair Products From Drosophila Embryos

Abstract

In this chapter, we describe a method for the recovery and analysis of alternative end-joining (alt-EJ) DNA double-strand break repair junctions following I-SceI cutting in Drosophila melanogaster embryos. Alt-EJ can be defined as a set of Ku70/80 and DNA ligase 4-independent end-joining processes that are typically mutagenic, producing deletions, insertions, and chromosomal rearrangements more frequently than higher-fidelity repair pathways such as classical nonhomologous end joining or homologous recombination. Alt-EJ has been observed to be upregulated in HR-deficient tumors and is essential for the survival and proliferation of these cells. Alt-EJ shares many initial processing steps with homologous recombination, specifically end resection; therefore, studying alt-EJ repair junctions can provide useful insight into aborted HR repair. Here, we describe the injection of plasmid constructs with specific cut sites into Drosophila embryos and the subsequent recovery of alt-EJ repair products. We also describe different analytical approaches using this system and how amplicon sequencing can be used to provide mechanistic information about alt-EJ.

Keywords: Alternative end joining; Amplicon sequencing; Double-strand break repair; Drosophila melanogaster; Embryo; Homologous recombination; Microhomology; Microinjection.