- Authors: So A. et al.
- Year: 2022
- Journal: Nucleic Acids Res 50 2651-2666
- Applications: in vitro / DNA, siRNA / INTERFERin, jetPEI
- Cell types:
- Name: DIvA
- Name: GC.92
Known as: lambda92
- Name: RG37
Description: Human SV-40 immortalized fibroblast cells
Meganuclease I-SceI was expressed by transient transfection of the pCMV-HA-I-SceI expression plasmid (40) with jetPEI (Polyplus-transfection) according to the manufacturer's instructions (Polyplus transfection, New York, NY, USA, #101-40N). The expression of HA-tagged I-SceI was verified by western blotting under each condition. For silencing experiments, 25,000 cells were seeded 1 day before transfection; these experiments were performed using INTERFERin following the manufacturer's instructions (Polyplus Transfection, New York, NY, USA, #409-50) with 20 nM of one of the following siRNAs: Control (5′-AUGAACGUGAAUUGCUCAA-3′), RAD51-1 (Dharmacon, Lafayette, CO, USA, cat# L003530-00-0010), RAD51-2 (5′-GAAGCUAUGUUCGCCAUUA-3′), RAD51-3 (3′-UTR, 5′GACUGCCAGGAUAAAGCUU-3′), RAD51-4 (3′UTR , 5′-GUGCUGCAGCCUAAUGAGA-3′), BRCA2 (5′-GCUGAUCUUCAUGUCAUAA-3′) or RAD52 (5′-CCAACGCACAACAGGAAAC-3′), all of which except those ordered from Dharmacon were synthesized by Eurofins (Ebersberg, Germany). Seventy-two hours later, the cells were transfected with the pCMV-HA-I-SceI expression plasmid.
Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells. Silencing RAD51 or BRCA2 stimulated both SSA and A-EJ, but not C-NHEJ, validating the two-step model. Three different RAD51 dominant-negative forms (DN-RAD51s) repressed GC and stimulated SSA/A-EJ. However, a fourth DN-RAD51 repressed SSA/A-EJ, although it efficiently represses GC. In living cells, the three DN-RAD51s that stimulate SSA/A-EJ failed to load efficiently onto damaged chromatin and inhibited the binding of endogenous RAD51, while the fourth DN-RAD51, which inhibits SSA/A-EJ, efficiently loads on damaged chromatin. Therefore, the binding of RAD51 to DNA, rather than its ability to promote GC, is required for SSA/A-EJ inhibition by RAD51. We showed that RAD51 did not limit resection of endonuclease-induced DSBs, but prevented spontaneous and RAD52-induced annealing of complementary ssDNA in vitro. Therefore, RAD51 controls the selection of the DSB repair pathway, protecting genome integrity from nonconservative DSB repair through ssDNA occupancy, independently of the promotion of CG.