The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks

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; Drosophila; double-strand break repair; end resection; homologous recombination; non-homologous end-joining; single-strand annealing.

Figure 1

DR-white assay suggests defects in homologous recombination in DmCtIP^Δ/Δ mutants. (A) The direct repeat of white (DR-white) assay contains two non-functional white repeats: Sce.white (I-SceI recognition site and premature STOP codon) and iwhite (truncated donor sequence). DR-white is integrated into the genome at Chromosome 2 at a known attP landing site using the attB sequence (blue) and followed using the yellow (y⁺) transgene. Embryos and larvae containing DR-white and the heat-shock inducible I-SceI transgene are heat shocked, and a DSB is formed at the I-SceI site. Repair events are observed by crossing single males to y w tester females. The resulting progeny are representative of single double-strand break (DSB) repair events from the premeiotic male germline. Depending on the repair pathway, one of three phenotypes will result. (i) White-eyed progeny (y⁺ w⁻) are indicative of no DSB, intersister homologous recombination (HR), or non-homologous end joining (NHEJ). NHEJ with processing can be determined through molecular analysis. (ii) Red-eyed progeny (y⁺ w⁺) indicate repair by intrachromosomal HR with the iwhite sequence as the donor, restoring the function of the white gene. (iii) Yellow-bodied, white-eyed progeny (y⁻ w⁻) indicate repair by single-strand annealing (SSA), a mitotic crossover event (indistinguishable from SSA), or an abnormal repair event that inhibits y⁺ expression, such as a deletion into the y⁺ transgene. (B) PCR amplification across the CRISPR/Cas9-mediated deletion of DmCtIP. Primers produce 1934 bp product in wildtype (+/+) and 283 bp product in DmCtIP^Δ/Δ mutants (Δ/Δ) (C) I-SceI-induced DSB repair events in a D. melanogaster C-terminal Binding Protein 1 Interacting Protein (DmCtIP^Δ/Δ) mutant background (red; n = 28 germlines, 1520 total flies scored) compared to DmCtIP^Δ/+ heterozygote controls (blue; n = 28 germlines, 1289 total flies scored). Results shown are averages ± standard error of the mean (S.E.M.) of individual male germline events. * p < 0.05, **** p < 0.0001, and ***** p < 0.00001 by unpaired Student’s t-test.

Figure 2

DmCtIP^Δ/Δ mutants are deficient in SSA repair in both short and long end-resection. The P{wIw} SSA assays contain an I-SceI recognition sequence inserted between a non-functional (white box) and a functional (red box) copy of the white gene (full-length gene sequence except for 5′UTR). (A) The complete P{wIw} SSA assay contains a non-functional white gene (whiteΔ(1–52); white box) due to deletions of the first 52 bp, including the ATG start codon. Following I-SceI cleavage, end resection of ~3.6 kb and single-strand annealing, a single non-functional copy of white results (w⁻) due to loss of the ATG site. (B) The “short” P{wIwΔAvrII} SSA assay contains a non-functional white gene (whiteΔ (AvrII); white box) due to deletions of the first 52 bp (including ATG) and the last 3.0 kb (3′ end and 3′UTR). Following I-SceI cleavage, end resection of ~550 bp and single-strand annealing, a single non-functional copy of white results (w⁻) due to loss of the ATG site. (C) Embryos containing the respective P{wIw} constructs and a heat-shock inducible I-SceI transgene are heat shocked, creating a site-specific DSB, and males are crossed to y w tester females to score individual premeiotic germline repair events. I-SceI-induced DSB repair events from the complete P{wIw} assay in a DmCtIP^Δ/Δ mutant background (red; n = 23 germlines, 1708 total flies scored) compared to DmCtIP^Δ/+ heterozygote controls (blue; n = 25 germlines, 1852 total flies scored). DSB repair events from the P{wIwΔAvrII} assay in a DmCtIP^Δ/Δ mutant background (red; n = 22 germlines, 1361 total flies scored) compared to DmCtIP^Δ/+ heterozygote controls (blue; n = 26 germlines, 1957 total flies scored). Results shown are averages ±S.E.M. of individual male germline events. ^‡ p < 10⁻¹⁰; ***** p < 0.00001 by unpaired Student’s t-test.

Figure 3

DR-white.mu assay shows defect in homologous recombination in DmCtIP^Δ/Δ mutants. (A) The direct repeat of white with mutations (DR-white.mu) assay contains 28 silent polymorphisms (vertical lines) on the iwhite.mu donor sequence. In intrachromosomal HR events, the polymorphisms converted from the iwhite.mu donor sequence vary (question marks) and can be determined through PCR amplification and sequencing to determine minimal gene conversion tract (GCT) lengths. (B) I-SceI-induced DSB repair events in DmCtIP^Δ/Δ mutant background (red; n = 14 germlines, total of 631 flies scored) compared to DmCtIP^Δ/+ heterozygote controls (blue; n = 26 germlines, total of 1487 flies scored). Results shown are means ± S.E.M. of individual male germline events. *** p < 0.001 by unpaired Student’s t-test.

Figure 4

DmCtIP does not impact gene conversion tract (GCT) lengths. HR repair events result in GCTs in both DmCtIP^Δ/Δ mutants (red, n = 33) and DmCtIP^Δ/+ heterozygous controls (blue, n = 30). SNPs along the length of the donor sequence are indicated at top (vertical lines). The zero mark represents the DSB site. The last converted SNP is shown and plotted as minimal gene conversion tracts lengths. Average minimal GCT lengths ± S.E.M. are provided for each genotype (p = 0.69, Student’s t-test).