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, 113 (13), 3515-20

BRCA2 Regulates DMC1-mediated Recombination Through the BRC Repeats

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BRCA2 Regulates DMC1-mediated Recombination Through the BRC Repeats

Juan S Martinez et al. Proc Natl Acad Sci U S A.

Abstract

In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1-3 and 6-8. However, BRC1-3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6-8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1-ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPA-ssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.

Keywords: BRCA2; DMC1; RAD51; mediator; meiosis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The BRC repeats bind to DMC1 in vitro. (A) Structural conservation of DMC1 and RAD51. Superposition of DMC1 (1V5W) onto RAD51-BRC4 interface (1N0W) showing the structural conservation of DMC1 and RAD51 at the subunit interface which is shown in the complex with the BRC4 repeat (purple). The conserved Phe of the FxxA motif (module I) and Phe of LFDE motif (module II) are highlighted in orange and red, respectively (sticks). (B) SDS/PAGE gel showing a pull down experiment with each GST-tagged BRC peptide or BRC4 mutant (∆7BRC4) (0.4 µM) and increasing concentrations (0.3, 0.6, 1, 1.3 µM) of DMC1. The asterisk denotes contaminating GST. (C) The data from B was fitted to a single-site binding curve. Error bars represent the SD for three or more independent experiments.
Fig. 2.
Fig. 2.
The BRC repeats enhance joint molecule formation by DMC1. (A) DMC1 (75 nM) and the indicated concentrations of each BRC peptide, Δ7BRC4, were preincubated with a 5′-end 32P-labeled 90-mer ssDNA [oAC203, 0.2 µM nt; (2.4 nM, molecule)] for 10 min at 37 °C and the supercoiled DNA (scDNA), (pUC19, 0.8 nM, molecule) was added last to start the reaction. The mix was incubated at 37 °C for 30 min and the products were resolved on a 1% agarose gel. (B) Quantification of joint molecule formation from A. Error bars, s.d. (n = 3).
Fig. S1.
Fig. S1.
Effects of BRC6 and BRC8 protein concentration on joint molecule formation by DMC1, and of BRC4, BRC7, and Δ7BRC4 on joint molecule formation at a lower concentration of DMC1. (A) Joint molecule reactions were carried out as in Fig. 2A, but at higher concentrations of BRC6; BRC8 serves as a control to show that the decrease is specific to BRC6. (B) Quantification of A. (C) Joint molecule formation was carried out as in Fig. 2, but with 25 nM DMC1 and 75 nM (nt) ssDNA [0.8 nM (molecule)] and pUC19 at 0.25 nM molecule. (D) Quantification of C. Error bars in B and D represent the SD for three and two independent experiments, respectively.
Fig. 3.
Fig. 3.
BRC1, -2, -3, -5, -6, -8, and Δ7BRC4 enhance DMC1 assembly on ssDNA. (A) DMC1 (25 nM) was incubated with the individual BRC repeats for 15 min at 37 °C before addition of 5′-end 32P-labeled ssDNA (oAC203, 0.2 µM nt) and further incubation for 1 h. The complexes were analyzed by PAGE and visualized by autoradiography. (B) Quantification of the EMSA from A. Error bars, s.d. (n = 3).
Fig. S2.
Fig. S2.
The BRC repeats do not bind to ssDNA. DNA binding assay (EMSA) where DMC1 (0.6 µM) or the individual BRC repeats (24 µM) were mixed with 5′-end 32P-labeled ssDNA (dT40, 0.3 µM nt) and incubated for 1 h. The complexes were analyzed by PAGE and visualized by autoradiography.
Fig. 4.
Fig. 4.
Only BRC4 and BRC6, but not BRCA2, marginally alter the ssDNA-dependent ATPase activity of DMC1. DMC1 (3 μM) was incubated with increasing concentrations of GST-BRC peptide or BRCA2, as indicated, before addition of 90-mer ssDNA [oAC203, 9 µM (nt)] and was further incubated for 1 h in the presence of 1 mM MgCl2 and 2 mM ATP. (A and B) Quantification of ATP hydrolysis by DMC1 as a function of BRC peptide. (C) Quantification of ATP hydrolysis by DMC1 as a function of BRCA2 concentration. (Error bars, SD; n > 3.)
Fig. S3.
Fig. S3.
The BRC repeats do not manifest ATPase activity. Autoradiography of a TLC plate showing an ATPase assay where DMC1 (3 μM) or the GST-BRC peptides (24 μM) were individually mixed with 90-mer ssDNA (9 µM nt) and incubated for 1 h in the presence of 1 mM MgCl2 and 2 mM ATP. The quantification of the percentage of Pi produced, and the calculated rate of ATP hydrolysis is indicated below each lane.
Fig. 5.
Fig. 5.
Only BRC4 reduces DMC1 assembly on dsDNA. (A) dsDNA binding assay (EMSA) where DMC1 (0.6 µM) was mixed with each BRC repeat before addition of 5′-end 32P-labeled dsDNA [dT40•dA40, 3 µM (bp)] and further incubated for 1 h. The complexes were analyzed by PAGE and visualized by autoradiography. (B) Quantification of A. (C) Quantification of EMSA as in A but with 0.3 µM DMC1. (Error bars, SD; n > 3.)
Fig. S4.
Fig. S4.
Binding of DMC1 (0.3 µM) to dsDNA in the presence of the BRC repeats. Shown is the EMSA experiment that is quantified in Fig. 5C.
Fig. 6.
Fig. 6.
Purified BRCA2 stimulates DMC1-dependent DNA pairing. (A) BRCA2 alone (0.5 nM), DMC1 (75 nM) alone, or DMC1 plus the indicated concentrations of GFP-MBP-BRCA2 were preincubated with the 5′-end 32P-labeled 90-mer ssDNA (oAC203, 0.2 µM nt; 2.4 nM molecule) for 10 min at 37 °C, (left to right, respectively); scDNA (pUC19, 0.8 nM molecule) was added last to start the reaction. The mix was incubated at 37 °C for 30 min and the products resolved on 1% agarose gel. (B) Quantification of D-loop formation from A. (Error bars, SD; n = 3.)
Fig. S5.
Fig. S5.
Purified GFP-MBP-BRCA2 complements Brca2-deficient cells and stimulates RAD51-mediated joint molecule formation. (A) BRCA2 tagged with GFP-MBP at the N terminus was purified from human HEK293 cells and analyzed by SDS/PAGE. Lane 1: BRCA2 (0.9 μg) was loaded on a precast 7.5% SDS/PAGE gel and stained with SYPRO Ruby. Lane 2: Western blot of purified BRCA2 protein (0.5 μg) using an antibody specific for the carboxy-terminus of BRCA2 (CA1033, EMD). Mr, size markers. (B) Mitomycin C survival of stably transfected clones of Brca2-deficient hamster cells complemented with human GFP-MBP-tagged BRCA2 (green and blue), the vector containing the GFP-MBP tag (violet and pink), V79 parental cells (Brca2+/+) (orange) and VC8 (Brca2−/−) (gold). (C) RAD51 (75 nM) and the indicated concentrations of BRCA2 were preincubated with a 5′-end 32P-labeled 90-mer ssDNA [2.4 nM (molecule)] for 10 min at 37 °C and scDNA [0.8 nM (molecule)] was added last to start the reaction. The mix was incubated at 37 °C for 30 min, terminated by incubation with Proteinase K, and resolved on a 1% agarose gel. (D) Quantification of C. Error bars in D represent the SD for three independent experiments.
Fig. 7.
Fig. 7.
BRCA2 stimulates DMC1-promoted DNA strand exchange. (A) Scheme of the DNA strand exchange reaction. (B) Gel showing a DNA strand exchange reaction where RPA (25 nM) was first incubated with an ssDNA substrate (167-mer, 4 nM molecule) for 5 min at 37 °C. Then, RAD51 or DMC1 (0.22 µM) and increasing concentrations of 2xMBP-BRCA2 (20–80 nM) were added and incubated for 5 min at 37 °C. A 5′-end 32P-labeled 40 bp duplex DNA (4 nM molecule) complementary to the ssDNA was added last, and the reaction was further incubated for 30 min at 37 °C. (C) Quantification of the DNA strand exchange product formation shown in B. (Error bars, range; n = 2.)
Fig. 8.
Fig. 8.
Proposed model for BRCA2 function in meiotic recombination. (A) Schematic of BRCA2 primary structure showing the BRC repeats that would preferentially bind to free RAD51 (BRC1–5) or to free DMC1 (BRC6–8). (B) Hypothetical scheme showing BRCA2 binding ssDNA, displacing RPA, and delivering a stable nucleus of RAD51 and DMC1 to the ssDNA. The DMC1 nucleus enables growth of the nascent DMC1 filament away from the BRCA2 heteronucleus and concomitant displacement of RPA.
Fig. S6.
Fig. S6.
Comparison of joint molecule formation by DMC1 in Ca+2, in the presence or absence of Mg+2. (A) Autoradiograph showing a D-loop assay. DMC1 at the indicated concentrations was preincubated with a 5′-end 32P-labeled 90-mer ssDNA [oAC203, 3 µM (nt), 33 nM (molecule)] for 10 min at 37 °C, and supercoiled DNA (scDNA) [pUC19, 10.3 nM (molecule)] was added last to start the reaction. The mix was incubated at 37 °C for 30 min, and the products were resolved on a 1% agarose gel. (B) Quantification of D-loop formation from A. Error bars in B represent the SD for two independent experiments.

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