Figure 6 |. RAD51 strand-exchange activity is required for the protection of centromere HORs in quiescent cells.

(A) Schematic of RAD51 separation-of-function variant functionalities. (B) Experimental schematic for the detection of centromeric breaks upon endogenous RAD51 depletion and expression of FLAG fusion of RAD51 variants in quiescent hTERT-RPE1 cells. (C) Western blot confirming the depletion of endogenous RAD51 (end. RAD51) 48 hours after siRNA treatment, and expression of separation-of-function variants upon doxycycline (DOX) treatment. Lamin-A levels were used as a loading control. (D, E) Quantification and representative images of exo-FISH following depletion of endogenous RAD51 and re-expression of the RAD51 separation-of-function variants, 96 hours after siRNA treatment in serum-starved hTERT-RPE1 cells. Quantification of FISH signals were performed as in Figure 2. Scale bar represents 10 μm. FISH signal intensity is X10,000 arbitrary units (A.U.). At least 30 cells were imaged per experimental condition. The averages of each experimental condition were used to perform a two-sided unpaired t-test (*p<0.05, **p<0.01). Filled and empty circles indicate presence and absence, respectively. See also Figure S7.