Abstract
Homologous Recombination (HR) is a high-fidelity process with a range of biologic functions from generation of genetic diversity to repair of DNA double-strand breaks (DSBs). In mammalian cells, BRCA2 facilitates the polymerization of RAD51 onto ssDNA to form a presynaptic nucleoprotein filament. This filament can then strand invade a homologous dsDNA to form the displacement loop (D-loop) structure leading to the eventual DSB repair. Here, we have found that RAD51 in stoichiometric excess over ssDNA can cause D-loop disassembly in vitro; furthermore, we show that this RAD51 activity is countered by BRCA2. These results demonstrate that BRCA2 may have a previously unexpected activity: regulation of HR at a post-synaptic stage by modulating RAD51-mediated D-loop dissociation. Our in vitro results suggest a mechanistic underpinning of homeostasis between RAD51 and BRCA2, which is an important factor of HR in mammalian cells.
Highlights
Double-strand breaks (DSBs) represent the most lethal type of DNA damage occurring in the cell approximately 50 times per cell cycle[1]
Our current results show that RAD51 can actively dissociate displacement loop (D-loop) through unwinding of dsDNA
We show that BRCA2 may have a post-synaptic function in Homologous Recombination (HR) by modulating D-loop dissociation
Summary
Double-strand breaks (DSBs) represent the most lethal type of DNA damage occurring in the cell approximately 50 times per cell cycle[1]. BRCA2 helps to stabilize the RAD51-ssDNA filament by inhibiting the RAD51 ATPase activity[4,5]. The ssDNA dissociates from the D-loop and anneals to the second resected end of the DSB. This D-loop dissociation represents an important step during HR, which prevents formation of crossovers in mitotically dividing cells or reduces their formation to the optimal level during meiosis[10,11]. Maintaining the optimal RAD51/BRCA2 balance may be important for the formation of the active RAD51-ssDNA filament during presynaptic stage and for the proper timing of D-loop dissociation at the post-synaptic stage of DNA strand exchange
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