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Title: Recovery of Alternative End-Joining Repair Products From Drosophila Embryos
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.  more » « less
Award ID(s):
1716039
NSF-PAR ID:
10094962
Author(s) / Creator(s):
; ;
Date Published:
Journal Name:
Methods in enzymology
Volume:
601
ISSN:
0076-6879
Page Range / eLocation ID:
91-110
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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