Double-Strand Break Repair Assays Determine Pathway Choice and Structure of Gene Conversion Events inDrosophila melanogaster

Double-strand breaks (DSBs) must be accurately and efficiently repaired to maintain genome integrity. Depending on the organism receiving the break, the genomic location of the DSB, and the cell-cycle phase in which it occurs, a DSB can be repaired by homologous recombination (HR), nonhomologous end-joining (NHEJ), or single-strand annealing (SSA). Two novel DSB repair assays were developed to determine the contributions of these repair pathways and to finely resolve repair event structures in Drosophila melanogaster. Rad51-dependent homologous recombination is the preferred DSB repair pathway in mitotically dividing cells, and the pathway choice between HR and SSA occurs after end resection and before Rad51-dependent strand invasion. HR events are associated with long gene conversion tracts and are both bidirectional and unidirectional, consistent with repair via the synthesis-dependent strand annealing pathway. Additionally, HR between diverged sequences is suppressed in Drosophila, similar to levels reported in human cells. Junction analyses of rare NHEJ events reveal that canonical NHEJ is utilized in this system.