The Role of <i>Drosophila</i> CtIP in Homology-Directed Repair of DNA Double-Strand Breaks

• Main Authors: Ian Yannuzzi, Margaret A. Butler, Joel Fernandez, Jeannine R. LaRocque
• Format: Article
• Language: English
• Published: MDPI AG 2021-09-01
• Series: Genes
• Subjects: CtIP; double-strand break repair; homologous recombination; non-homologous end-joining; single-strand annealing; <i>Drosophila</i>
• Online Access: https://www.mdpi.com/2073-4425/12/9/1430

DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a <i>DmCtIP</i>^Δ allele in <i>Drosophila melanogaster</i>. Using the DSB repair reporter assay direct repeat of <i>white</i> (DR-<i>white</i>), a two-fold decrease in HR in <i>DmCtIP</i>^Δ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the <i>DmCtIP</i>^Δ/Δ mutants compared to heterozygote controls. Through these analyses, we affirmed the importance of end resection on DSB repair pathway choice in multicellular systems, described the function of DmCtIP in short and extensive DNA end resection, and determined the impact of end resection on GCT length during HR.