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

Ian Yannuzzi,Joel Fernandez,Jeannine R Larocque,Margaret A Butler

doi:10.3390/genes12091430

Ian Yannuzzi, Joel Fernandez + Show 2 more

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https://doi.org/10.3390/genes12091430

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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 DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/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 DmCtIPΔ/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.

Highlights

Maintaining genome integrity is vital to ensuring proper cellular functions and the successful propagation of genetic material
The DR-white assay allows for assessing the usage of intrachromosomal homologous recombination (HR), single-strand annealing (SSA), or non-homologous end-joining (NHEJ)/no double-strand breaks (DSBs)/intersister HR in the repair of a site-specific DSB as described previously (Figure 1A) [26]
We found a ~50% decrease of noncrossover HR events, from 23.2 ± 1.8% in the DmCtIP∆/+ heterozygote controls to 12.5 ± 1.3% in the DmCtIP∆/∆ mutants (p < 0.0001, Student’s t-test) (Figure 1C)

Summary

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. 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]. Exogenous sources include UV radiation, ionizing radiation, and chemical reagents. There are multiple pathways to maintain genome integrity: non-homologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA)

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