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, 9 (1), 4554

The Post-Synaptic Function of Brca2

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The Post-Synaptic Function of Brca2

Charles X Wang et al. Sci Rep.

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
RAD51 promotes D-loop dissociation. (a) Scheme of D-loop dissociation by RAD51. The asterisk in this and following schemes indicates 32P-label on ssDNA. (b) Addition of RAD51 (1 µM) to the RAD51-generated D-loops on pUCFBR DNA (6.25 µM, nt) causes their dissociation. As controls, either RAD51 (1 µM) premixed with heterologous pUC19 scDNA (43.75 µM, nt) or RAD51 storage buffer were added to the reactions at the indicated time point. These and all subsequent D-loops were analyzed by autoradiography of 1% agarose gels following gel-electrophoresis and expressed as a percentage of the total homologous plasmid DNA (limiting factor). “0” time point on the graph corresponds to the initiation of D-loop formation. (c) The data from (b) represented as a graph. The vertical arrow marks addition of RAD51, RAD51 + pUC19, or buffer. Error bars indicate standard error of the mean (SEM); the experiments were repeated at least three times.
Figure 2
Figure 2
Effect of the RAD51 K133A and K133R mutations on D-loop dissociation. (a) Experimental scheme. (b) The kinetics of D-loop dissociation by RAD51, or the RAD51 K133A or K133R mutants. The addition of RAD51 (0.5 µM) or RAD51K133R (0.5 µM), but not RAD51K133A (0.5 µM), causes dissociation of RAD51-generated D-loops. As a control, the RAD51 storage buffer was used. D-loop dissociation was initiated at “4 min” time point since the beginning of D-loop formation. (c) Graphical representation of the data from panel (b). Error bars indicate SEM; the experiments were repeated at least three times.
Figure 3
Figure 3
BRCA2 prevents D-loop dissociation by RAD51. (a) Experimental scheme of post-synaptic addition of BRCA2/BRC4 and RAD51. (b) Effect of BRCA2 on D-loop dissociation by RAD51. BRCA2 and RAD51 in the indicated concentrations were pre-incubated prior to addition to the pre-formed D-loops. (c) Data from (b) represented graphically. (d) Effect of BRC4 on D-loop dissociation by RAD51. BRC4 in the indicated concentrations was pre-incubated with RAD51 prior to addition to the pre-formed D-loops. Lane 1 (“No Rnx”) represents protein-free control. Lanes 2 and 9 represent D-loops not subjected to excess RAD51. (e) Graphical representation of data from (d). Error bars indicate SEM and the experiments were repeated at least three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 4
Figure 4
BRC4 has differential effects on D-loop yield depending upon initial RAD51 concentration. (a) Schematic depicting order of addition for BRC4 and RAD51 proteins to the 32P-ssDNA. BRC4 and RAD51 were either added sequentially (open symbols) or pre-incubated (closed symbols) before addition to the 32P- ssDNA. RAD51 was either at 1 µM (circles) or 1.5 µM (squares) concentration. (b) Effect of pre-synaptic combination of RAD51 and BRC4 on the D-loop yield. Representative gel images are shown in Fig. S7. Error bars indicate SEM and the experiments were repeated at least three times.
Figure 5
Figure 5
BRCA2 controls homologous recombination by regulating D-loop dissociation. An excess of RAD51 may cause D-loop dissociation, whereas BRCA2 modulates dissociation of D-loops via direct interaction with RAD51 through its BRC repeats. Inlet, the balance between RAD51 and BRCA2 at the site of DSB repair may determine the outcome of HR.

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