Abstract

The selection of the DNA double-strand breaks (DSBs) repair pathway is decisive for genetic stability/instability. We proposed that it acts according to two successive steps: 1-canonical non-homologous end-joining (C-NHEJ) versus single-strand DNA (ssDNA) resection; 2- on ssDNA, gene conversion (GC) versus non-conservative single-strand annealing (SSA) or alternative end-joining (A-EJ).\n\nUsing intramolecular substrates, we systematically analysed the equilibrium between the different DSB repair pathways. We show that ablation of RAD51 stimulated both SSA and A-EJ but did not stimulate C-NHEJ, validating the two-step model. Moreover, we found that two ATP-mutant dominant-negative forms of RAD51 that stimulated non-conservative repair, failed to load into damaged chromatin, clarifying the role of ATP in RAD51-mediated HR, also. In contrast, another dominant-negative form of RAD51, which retains its DNA binding capacities, repressed SSA and A-EJ, revealing two separable functions of RAD51 i.e. GC and non-conservative repair inhibition. In vitro assays show that the binding of RAD51 on both complementary ssDNA is required to block both spontaneous and RAD52-induced strand annealing. Therefore, RAD51 represses non-conservative repair (SSA and A-EJ), by inhibiting the annealing step through ssDNA occupancy, independently of the catalytic strand-exchange activity required for GC.