Stage-specific Recombination Factors Differentially Regulate Double-strand Break Repair Fidelity and Influence Genome Stability

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) with high fidelity, yet how defects at distinct steps of the recombination pathway influence gene conversion (GC) outcomes and contribute to genome instability remains poorly defined. Here, we develop Di-GRAPH, a computational framework that integrates coverage profiling and discordant read mapping to systematically analyze mutational signatures, repair pathway choice, and GC architecture. At a genomic scale, Di-GRAPH detects, quantifies and classifies DNA damage-dependent gross chromosomal rearrangements across diverse genomic contexts. Applying Di-GRAPH to mutants defective in resection (exo1Δ), recombination intermediate processing (sgs1Δ, srs2Δ), and strand invasion (rad51Δ), we identify stage-specific GC patterns, mutation-specific repair signatures and distinct patterns of genome-wide rearrangements. Loss of Exo1 minimally affects GC formation but severely compromises genome stability in undamaged cells. Absence of Sgs1 and Srs2 leads to extended and discontinued GCs, accompanied by widespread ectopic recombination and retrotransposon mobilization following DSB repair. Rad51 deficiency results in severely impaired GC outcomes, strong reliance on non-homologous end joining, elevated break-proximal mutagenesis, and extensive genome-wide rearrangements in both DSB survivors and undamaged cells. Coverage and discordant-read signals were strongly correlated across most genomic features, indicating that recombination-associated translocations are frequently coupled to local DNA amplification. Comparative sequence identity analyses reveal distinct homology requirements, with ORFs showing the highest homology dependence, whereas repetitive elements recombine under more relaxed constraints. Together, this integrated analysis reveals that defects at discrete steps of the HR pathway impose distinct and predictable genome-wide instability signatures, providing a mechanistic framework for understanding how recombination fidelity is enforced across the DNA repair process.