Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1 - PubMed (original) (raw)

Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1

Tina Thorslund et al. EMBO J. 2007.

Abstract

Germline mutations in BRCA2 predispose to hereditary breast cancers. BRCA2 protein regulates recombinational repair by interaction with RAD51 via a series of degenerate BRC repeat motifs encoded by exon 11 (BRCA2(996-2113)), and an unrelated C-terminal domain (BRCA2(3265-3330)). BRCA2 is also required for meiotic recombination. Here, we show that human BRCA2 binds the meiosis-specific recombinase DMC1 and define the primary DMC1 interaction site to a 26 amino-acid region (BRCA2(2386-2411)). This region is highly conserved in BRCA2 proteins from a variety of mammalian species, but is absent in BRCA2 from Arabidopsis thaliana, Caenorhabditis elegans, and other eukaryotes. We demonstrate the critical importance of Phe2406, Pro2408, and Pro2409 at the conserved motif (2404)KVFVPPFK(2411). This interaction domain, defined as the PhePP motif, promotes specific interactions between BRCA2 and DMC1, but not with RAD51. Thus, the RAD51 and DMC1 interaction domains on BRCA2 are distinct from each other, allowing coordinated interactions of the two recombinases with BRCA2 at meiosis. These results lead us to suggest that BRCA2 is a universal regulator of RAD51/DMC1 recombinase actions.

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Figures

Figure 1

DMC1 interacts with BRCA2 in human cells. Human 293T cells were either mock-transfected (input, lane a) or transfected with Myc-DMC1 (input, lane b). Post-transfection, cells were harvested, lysed, and protein complexes were immunoprecipitated with polyclonal BRCA2 antibodies or nonspecific rabbit IgG, as indicated. The immunoprecipitates were then analysed by Western blotting for BRCA2 (upper panel), Myc-DMC1 (middle panel), or RAD51 (lower panel).

Figure 2

Interactions of BRCA2 with RAD51 and DMC1. (A) Schematic representation of human BRCA2 protein divided into nine GST-tagged fusion proteins. BRC repeats 1–8 (dark blue), the helical domain (green), OB folds (yellow), and nuclear localisation signals (black) are indicated. The boundaries of the nine fragments are as follows: B2-1 is BRCA21–454; B2-2 is BRCA2420–906; B2-3 is BRCA2885–1362 and contains BRC1 and BRC2; B2-4 is BRCA21338–1781 and contains BRC3, BRC4, and BRC5; B2-5 is BRCA21744–2115 and contains BRC6, BRC7, and BRC8; B2-6 is BRCA22106–2472; B2-7 is BRCA22438–2824 and contains the helical domain and one OB fold; B2-8 is BRCA22780–3197 and contains two OB folds; B2-9 is BRCA23189–3418 and contains two nuclear localisation signals. (B) Interactions of recombinant DMC1 or RAD51 with the nine GST-tagged BRCA2 fusion proteins. Lysates of E. coli cells expressing the indicated GST fusions were mixed with glutathione beads, and purified human RAD51 or DMC1 was subsequently added. After extensive washing, complexes bound to beads were analysed by Western probing for DMC1 (upper panel), RAD51 (middle panel), or the BRCA2 fragments (lower panel).

Figure 3

Identification of a DMC1 interaction domain in an internal region of BRCA2. (A) Schematic representation of BRCA2 B2-6, subdivided into five smaller GST fusion proteins. B2-6.1 is BRCA22106–2190; B2-6.2 is BRCA22106–2218; B2-6.3 is BRCA22190–2260; B2-6.4 is BRCA22218–2340; B2-6.5 is BRCA22340–2472. (B) The indicated GST fusions were expressed in E. coli as described in Figure 2 legend and their ability to bind purified DMC1 was analysed as described. (C) Competition assay to analyse interactions between DMC1 or RAD51 and B2-6.5 fusion protein. B2-6.5 was mixed with DMC1 (200 ng) and increasing amounts of RAD51 (0, 200, 400, or 2 μg, respectively). Proteins bound to glutathione beads were analysed by Western blotting.

Figure 4

Yeast two-hybrid analysis of interactions between DMC1 or RAD51 and a series of BRCA2 fragments. Two-hybrid analysis was carried out as described in Methods, and diluted cells were plated on permissive −Leu/−Trp plates (−LT), or selection plates: −Leu/−Trp/−His + 25 mM 3-AT (−LTH+25 mM 3-AT); −Leu/−Trp/−His +100 mM 3-AT (−LTH+100 mM 3-AT); or −Leu/−Trp/−Ura (−LTU). The BRCA2 fragment boundaries are as follows: BRC1 (BRCA2996–1084); BRC2 (BRCA21206–1274); BRC3 (BRCA21415–1483); BRC4 (BRCA21511–1579); BRC5 (BRCA21658–1726); BRC6 (BRCA21831–1899); BRC7 (BRCA21965–2033); BRC8 (BRCA22045–2113); B2-6 (BRCA22106–2472); B2-6.5 (BRCA22340–2472); B2-9 (BRCA23189–3418); TR2 (BRCA23265–3330). A large BRCA2 fragment, BRCA2996–2113, containing all eight BRC repeats was also analysed.

Figure 5

Characterisation of interactions between DMC1 and the C-terminal region of BRCA2. (A) Schematic representation of BRCA2 B2-9, subdivided into three smaller GST fusion proteins. TR1 is BRCA23189–3274 and contains one nuclear localisation signal (black); TR2 is BRCA23265–3330 and contains both nuclear localisation signals; TR3 is BRCA23325–3418. (B) Interaction of DMC1 or RAD51 with the three GST-tagged BRCA2 TR-fragments. Complexes bound to glutathione beads were analysed by Western probing for DMC1, RAD51, and BRCA2 fragments, as described in Figure 2b. (C) Competition between RAD51 and DMC1 for TR2 binding. TR2 was mixed with DMC1 and increasing amounts of RAD51 (lanes a–d), or RAD51 was mixed with increasing amounts of DMC1 (lanes e–h). Complexes bound to glutathione beads were analysed by Western blotting. (D) Schematic representation of the TR2 region of BRCA2. Red circles correspond to putative phosphorylation sites and blue circles to sites that have been phosphorylated in synthetic biotinylated peptides. The S3291 is known to be phosphorylated in vivo. (E) Interactions between RAD51 or DMC1 and the series of phosphopeptides shown in (d). Synthetic biotinylated phosphopeptides (300 ng) were mixed with either DMC1 or RAD51 (60 ng), and complexes bound to Streptavidin beads were analysed by Western blotting. (F) Effect of S3291 phosphorylation on the competition between RAD51 and DMC1 for TR2. TR2 peptide or S3291Phospho-TR2 peptide (300 ng) was mixed with DMC1 (60 ng) and increasing amounts of RAD51 (0, 30, 60, or 180 ng). Complexes bound to Streptavidin beads were analysed by Western blotting. Biotinylated peptides were visualised by HRP-conjugated Streptavidin.

Figure 6

Identification of the DMC1 interacting domain PhePP. (A) Schematic representation of four overlapping peptides corresponding to B2-6.5. IR1 is BRCA22340–2407; IR2 is BRCA22350–2417; IR3 is BRCA22371–2438; IR4 is BRCA22405–2472. (B) Interactions between the biotinylated IR peptides (300 ng) and DMC1 (60 ng) were assayed by streptavidin pull-downs and detected by Western blotting. Biotinylated TR2 was used as a control. (C) Peptide array with overlapping 30 mers of BRCA22350–2417 spotted on a membrane (see text for detail), probed with DMC1, and analysed by Western blotting. Subsequently, the membrane was stained with Ponceau to reveal the exact location of the spotted peptides. (D) Sequence alignment of the PhePP motif BRCA2 from a variety of organisms. At (A): A. thaliana BRCA2 homologue A. Ce: C. elegans. Um: Ustilago maydis. Tc: Trypanosoma cruzi. Amino acids indicated in blue are fully conserved in mammalian species, green indicate conservation of strong groups, whereas purple indicates conservation of weaker groups. (E) Analysis of essential amino acids required for interactions between BRCA22382–2411 and DMC1. The amino-acid substitution peptide array contains 600 30-amino acid long peptides spotted onto a membrane. Each peptide has one amino acid of BRCA22382–2411 replaced with one of the 20 common amino acids (see text for details). The membrane was probed with DMC1 followed by Western blotting.

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Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1 - PubMed (original) (raw)