genes G C A T T A C G G C A T Article The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks Ian Yannuzzi 1 , Margaret A. Butler 2 , Joel Fernandez 2 and Jeannine R. LaRocque 2, *   Citation: Yannuzzi, I.; Butler, M.A.; Fernandez, J.; LaRocque, J.R. The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks. Genes 2021, 12, 1430. https://doi.org/10.3390/ genes12091430 Academic Editor: Przemyslaw Szafranski Received: 20 August 2021 Accepted: 14 September 2021 Published: 16 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Biology Department, Georgetown College, Georgetown University, Washington, DC 20057, USA; iy46@georgetown.edu 2 Georgetown University Medical Center, Department of Human Science, Georgetown University, Washington, DC 20057, USA; mb2123@georgetown.edu (M.A.B.); Joel.Fernandez@georgetown.edu (J.F.) * Correspondence: Jan.LaRocque@georgetown.edu Abstract: 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 DmCtIP Δ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIP 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 DmCtIP 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. Keywords: CtIP; double-strand break repair; homologous recombination; non-homologous end- joining; single-strand annealing; Drosophila; end resection 1. Introduction Maintaining genome integrity is vital to ensuring proper cellular functions and the successful propagation of genetic material. This integrity relies on the efficient and accurate repair of DNA damage. A DNA double-strand break (DSB) is a distinctly cytotoxic type of DNA damage, where both strands of the DNA double helix are broken. DSBs can result from endogenous or exogenous sources. Endogenous sources include by-products of cellular processes (e.g., reactive oxygen species, single-strand breaks converted to DSBs during replication) or programmed DSBs (e.g., during meiosis and V(D)J recombination); these endogenous sources of DSBs account for about 50 DSBs per cell division [1]. Exoge- nous sources include UV radiation, ionizing radiation, and chemical reagents. Without proper repair of DSBs, cell death, chromosomal rearrangements, or carcinogenesis can oc- cur [2,3]. Thus, there are multiple pathways to maintain genome integrity: non-homologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). In NHEJ, the two DSB ends are recognized, processed, and ligated together—often resulting in the loss or addition of nucleotides at the break site. In contrast to NHEJ, homology-directed repair (HDR) requires homologous sequences to repair DSBs. HDR is Genes 2021, 12, 1430. https://doi.org/10.3390/genes12091430 https://www.mdpi.com/journal/genes