Investigating double-stranded DNA gap repair mechanisms utilizing APOBEC3A mutagenic activity in different-sized gaps in Drosophila melanogaster

Double-stranded DNA gaps represent a major threat to genomic stability, yet how they are repaired remains poorly understood. Among the pathways involved in gap repair is homologous recombination (HR), a high-fidelity DNA repair pathway that restores broken or missing DNA sequences by copying information from an intact homologous template. One aspect of HR that is not well understood is whether repair DNA synthesis proceeds via a static extended D-loop or a migrating-bubble mechanism. These two models can produce identical repair products but differ in when and where single-stranded DNA (ssDNA) is exposed during gap repair. This distinction is important because ssDNA is more vulnerable to damage and mutagenesis than double-stranded DNA, making its distribution potentially informative about the underlying repair mechanism. In addition to the synthesis mechanism itself, an important question is how far resection extends beyond the engineered gap. This study investigated how double-stranded DNA gaps of different lengths are repaired at the endogenous ebony locus in Drosophila melanogaster. We used APOBEC3A (A3A), a cytidine deaminase that selectively mutates ssDNA, as a molecular marker of transient ssDNA exposure during gap repair. A3A was expressed in the germline during Cas9-induced gap repair, and repair products were analyzed using long-read sequencing. In the ebony assay, A3A-associated mutations were recovered both in newly synthesized DNA and on the chromosome used as a repair template. Mutations also extended beyond the engineered gaps into flanking regions, indicating repair-associated processing outside the donor-defined interval. These findings support the conclusion that gap repair at ebony involves nonuniform and spatially restricted ssDNA exposure during repair synthesis and has important implications for genome engineering strategies that rely on homology-directed repair.